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WO2003072729A2 - Enzymes - Google Patents

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WO2003072729A2
WO2003072729A2 PCT/US2003/005478 US0305478W WO03072729A2 WO 2003072729 A2 WO2003072729 A2 WO 2003072729A2 US 0305478 W US0305478 W US 0305478W WO 03072729 A2 WO03072729 A2 WO 03072729A2
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seq
polynucleotide
polypeptide
amino acid
sequence
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WO2003072729A3 (fr
Inventor
William W. Sprague
Amy E. Kable
Sally Lee
Jennifer A. Griffin
Shanya D. Becha
Ernestine A. Lee
April J.A. Hafalia
Reena Khare
Brooke M. Emerling
Joseph P. Marquis
Jayalaxmi Ramkumar
Vicki S. Elliott
Thomas W. Richardson
Mariah R. Baughn
Pei Jin
David Chien
Phillip R. Hawkins
Amy D. Wilson
Narinder K. Chawla
Uyen K. Tran
Soo Yeun Lee
Yeganeh Zebarjadian
Xin Jiang
Alan A. Jackson
Umesh G. Bhatia
John D. Burrill
Julie J. Blake
Anne Ho
Wenjin Zheng
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Incyte Corp
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Incyte Corp
Incyte Genomics Inc
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Priority to AU2003216376A priority Critical patent/AU2003216376A1/en
Publication of WO2003072729A2 publication Critical patent/WO2003072729A2/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the invention relates to novel nugleic acids, enzymes encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metabolism, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and cell proliferative disorders, including cancer.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and enzymes.
  • the cellular processes of biogenesis and biodegradation involve a number of key enzyme classes including oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and others.
  • Each class of enzyme comprises many substrate-specific enzymes having precise and well regulated functions. Enzymes facilitate metabolic processes such as glycolysis, the tricarboxylic cycle, and fatty acid metabolism; synthesis or degradation of amino acids, steroids, phospholipids, and alcohols; regulation of cell signaling, proliferation, inflamation, and apoptosis; and through catalyzing critical steps in DNA replication and repair and the process of translation.
  • Oxidoreductases Many pathways of biogenesis and biodegradation require oxidoreductase (dehydrogenase or reductase) activity, coupled to reduction or oxidation of a cofactor.
  • Potential cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E.A. and A.R. Leech (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U. K. pp. 779-793).
  • Reductase activity catalyzes transfer of electrons between substrate(s) and cofactor(s) with concurrent oxidation of the cofactor.
  • Reverse dehydrogenase activity catalyzes the reduction of a cofactor and consequent oxidation of the substrate.
  • Oxidoreductase enzymes are a broad superfamily that catalyze reactions in all cells of organisms, including metabolism of sugar, certain detoxification reactions, and synthesis or degradation of fatty acids, amino acids, glucocorticoids, estrogens, androgens, and prostaglandins.
  • oxidoreductases oxidases, reductases, or dehydrogenases
  • they often have distinct cellular locations such as the cytosol, the plasma membrane, mitochondrial inner or outer membrane, and peroxisomes.
  • Short-chain alcohol dehydrogenases are a family of dehydrogenases that share only 15% to 30% sequence identity, with similarity predominantly in the coenzyme binding domain and the substrate binding domain.
  • SCADs are involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders.
  • retinol dehydrogenase is a SCAD-family member (Simon, A. et al. (1995) J. Biol. Chem.
  • retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et al. (1996) Genomics 36:424-430).
  • succinate:quinone reductases SQR
  • fumarate reductases QFR
  • succinate:quinone reductases EC 1.3.5.1
  • the complexes consist of two hydrophilic and one or two hydrophobic, membrane-integrated subunits.
  • the larger hydrophilic subunit A carries covalently bound flavin adenine dinucleotide; subunit B contains three iron-sulphur centers (Lancaster, CR. and A. Kroger (2000) Biochim. Biophys. Acta 1459:422-431).
  • the full-length cDNA sequence for the flavoprotein subunit of human heart succinate dehydrogenase (succinate: (acceptor) oxidoreductase; EC 1.3.99.1) is similar to the bovine succinate dehydrogenase in that it contains a cysteine triplet and in that the active site contains an additional cysteine that is not present in yeast or prokaryotic SQRs (Morris, A.A. et al. (1994) Biochim. Biophys. Acta 29:125-128).
  • Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabolism (Weiss, B. (1991) Neurotoxicology 12:379-386; Collins, S.M. et al. (1992) Ann. N.Y. Acad. Sci. 664:415-424; Brown, J.K. and H. Imam (1991) J. Inherit. Metab. Dis. 14:436-458). Many pathways of neurotransmitter metabolism require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as NADVNADH (Newsholme and Leech, supra, pp. 779-793).
  • a cofactor such as NADVNADH
  • Degradation of catecholamines requires alcohol dehydrogenase (in the brain) or aldehyde dehydrogenase (in peripheral tissue).
  • NAD + -dependent aldehyde dehydrogenase oxidizes 5- hydroxyindole-3 -acetate (the product of 5-hydroxytryptamine (serotonin) metabolism) in the brain, blood platelets, liver and pulmonary endothelium (Newsholme and Leech, supra, p. 786).
  • neurotransmitter degradation pathways that utilize NADVNADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme and Leech, supra, pp. 790, 792).
  • L-DOPA precursor of dopamine, a neuronal excitatory compound
  • glycine an inhibitory neurotransmitter in the brain and spinal cord
  • histamine liberated from mast cells during the inflammatory response
  • taurine an inhibitory neurotransmitter of the brain stem, spinal cord and retina
  • Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in diseases including Parkinson disease and inherited myoclonus (McCance, K.L. and S.E. Huether (1994) Pathophysiology,
  • Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid methionine. Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase, which includes three enzyme activities: tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase.
  • tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells.
  • 3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of NAD to NADH, in the mitochondria and peroxisomes of eukaryotic cells. In peroxisomes, 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabohsm produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands.
  • a ⁇ amyloid- ⁇
  • APP amyloid precursor protein
  • 3HACD has been shown to bind the A ⁇ peptide, and is overexpressed in neurons affected in Alzheimer's disease.
  • an antibody against 3HACD can block the toxic effects of A ⁇ in a cell culture model of Alzheimer's disease (Yan, S.
  • Steroids such as estrogen, testosterone, and corticosterone are generated from a common precursor, cholesterol, and interconverted. Enzymes acting upon cholesterol include dehydrogenases. Steroid dehydrogenases, such as the hydroxysteroid dehydrogenases, are involved in hypertension, fertility, and cancer (Duax, W.L. and D. Ghosh (1997) Steroids 62:95-100).
  • One such dehydrogenase is 3-oxo-5- ⁇ -steroid dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen-responsive tissues.
  • OASD 3-oxo-5- ⁇ -steroid dehydrogenase
  • OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen.
  • Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia.
  • a defect in OASD leads to defective formation of the external genitalia (Andersson, S. et al. (1991) Nature 354:159-161; Labrie, F. et al. (1992) Endocrinology 131:1571-1573; OMIM #264600).
  • 17 ⁇ -hydroxysteroid dehydrogenase plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone (DHTT).
  • 17 ⁇ HSD6 acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3 ⁇ -diol, to androsterone which is readily glucuronidated and removed.
  • 17 ⁇ HSD6 is active with both androgen and estrogen substrates in embryonic kidney 293 cells. Isozymes of 17 ⁇ HSD catalyze oxidation and/or reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M.G. and D.W. Russell (1997) J. Biol. Chem. 272:15959-15966).
  • 17 ⁇ HSDl preferentially reduces estradiol and is abundant in the ovary and placenta.
  • 17 ⁇ HSD2 catalyzes oxidation of androgens and is present in the endometrium and placenta.
  • 17 ⁇ HSD3 is exclusively a reductive enzyme in the testis (Geissler, W.M. et al. (1994) Nature Genet. 7:34-39).
  • An excess of androgens such as DHTT can contribute to diseases such as benign prostatic hyperplasia and prostate cancer.
  • the oxidoreductase isocitrate dehydrogenase catalyzes the conversion of isocitrate to a- ketoglutarate, a substrate of the citric acid cycle.
  • Isocitrate dehydrogenase can be either NAD or NADP dependent, and is found in the cytosol, mitochondria, and peroxisomes. Activity of isocitrate dehydrogenase is regulated developmentally, and by hormones, neurotransmitters, and growth factors.
  • HPR Hydroxypyruvate reductase
  • a peroxisomal 2-hydroxyacid dehydrogenase in the glycolate pathway catalyzes the conversion of hydroxypyruvate to glycerate with the oxidation of both NADH and NADPH.
  • the reverse dehydrogenase reaction reduces NAD + and NADP*.
  • HPR recycles nucleotides and bases back into pathways leading to the synthesis of ATP and GTP, which are used to produce DNA and RNA and to control various aspects of signal transduction and energy metabolism.
  • Purine nucleotide biosynthesis inhibitors are used as antiproliferative agents to treat cancer and viral diseases. HPR also regulates biochemical synthesis of serine and cellular serine levels available for protein synthesis.
  • the mitochondrial electron transport (or respiratory) chain is the series of oxidoreductase-type enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons from NADH to oxygen and the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP provides energy to drive energy-requiring reactions.
  • the key respiratory chain complexes are NADH:ubiquinone oxidoreductase (complex I), succinate :ubiquinone oxidoreductase (complex II), cytochrome c b oxidoreductase (complex HI), cytochrome c oxidase (complex IN), and ATP synthase (complex V) (Alberts, B.
  • dehydrogenase activities using NAD as a cofactor include 3-hydroxyisobutyrate dehydrogenase (3HBD), which catalyzes the NAD-dependent oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde within mitochondria.
  • 3-hydroxyisobutyrate levels are elevated in ketoacidosis, methylmalonic acidemia, and other disorders (Rougraff, P.M. et al. (1989) J. Biol. Chem. 264:5899-5903).
  • Another mitochondrial dehydrogenase important in amino acid metabolism is the enzyme isovaleryl-CoA-dehydrogenase (IVD).
  • IVD is involved in leucine metabolism and catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA.
  • Human IVD is a tetrameric flavoprotein synthesized in the cytosol with a mitochondrial import signal sequence. A mutation in the gene encoding IVD results in isovaleric acidemia (Vockley, J. et al. (1992) J. Biol. Chem. 267:2494-2501).
  • the family of glutathione peroxidases encompass tetrameric glutathione peroxidases (GPxl -3) and the monomeric phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4).
  • GPx4 is the only monomeric glutathione peroxidase found in mammals and the only mammalian glutathione peroxidase to show high affinity for and reactivity with phospholipid hydroperoxides, and to be membrane associated. A tandem mechanism for the antioxidant activities of GPx4 and vitamin E has been suggested. GPx4 has alternative transcription and translation start sites which determine its subcellular localization (Esworthy, R.S. et al. (1994) Gene 144:317-318; and Maiorino, M. et al. (1990) Meth. Enzymol. 186:448-450).
  • GST glutathione S-transferases
  • GSH glutathione
  • the absolute requirement for binding reduced GSH to a variety of chemicals necessitates a diversity in GST structures in various organisms and cell types.
  • GSTs are homodimeric or heterodimeric proteins localized in the cytosol.
  • the major isozymes share common structural and catalytic properties and include four major classes, Alpha, Mu, Pi, and Theta.
  • Each GST possesses a common binding site for GSH, and a variable hydrophobic binding site specific for its particular electrophilic substrates. Specific amino acid residues within GSTs have been identified as important for these binding sites and for catalytic activity. Residues Q67, T68, D101, E104, and R131 are important for the binding of GSH (Lee, H.-C. et al. (1995) J. Biol. Chem. 270:99-109). Residues R13, R20, and R69 are important for the catalytic activity of GST (Stenberg, G. et al. (1991) Biochem. J. 274:549-555). GSTs normally deactivate and detoxify potentially mutagenic and carcinogenic chemicals.
  • rat and human GSTs are reliable preneoplastic markers of carcinogenesis.
  • Dihalomethanes which produce liver tumors in mice, are believed to be activated by GST (Thier, R. et al. (1993) Proc. Natl. Acad. Sci. USA 90:8567-8580).
  • the mutagenicity of ethylene dibromide and ethylene dichloride is increased in bacterial cells expressing the human Alpha GST, Al-1, while the mutagenicity of aflatoxin Bl is substantially reduced by enhancing the expression of GST (Simula, T.P. et al. (1993) Carcinogenesis 14:1371-1376).
  • control of GST activity maybe useful in the control of mutagenesis and carcinogenesis.
  • MDR multi-drug resistance
  • Glutaredoxin is a glutathione (GSH)-dependent hydrogen donor for ribonucleotide diphosphate reductase and contains the active site consensus sequence -C-P-Y-C-. This sequence is conserved in glutaredoxins from such different organisms as Escherichia coli, vaccinia virus, yeast, plants, and mammalian cells.
  • Glutaredoxin has inherent GSH-disulfide oxidoreductase (thioltransferase) activity in a coupled system with GSH, NADPH, and GSH-reductase, catalyzing the reduction of low molecular weight disulfides as well as proteins.
  • Glutaredoxin has been proposed to exert a general thiol redox control of protein activity by acting both as an effective protein disulfide reductase, similar to thioredoxin, and as a specific GSH-mixed disulfide reductase (Padilla, CA. et al. (1996) FEBS Lett. 378:69-73).
  • glutaredoxin and other thioproteins provide effective antioxidant defense against oxygen radicals and hydrogen peroxide (Schallreuter, K.U. and J.M. Wood (1991) Melanoma Res. 1:159-167).
  • Glutaredoxin is the principal agent responsible for protein dethiolation in vivo and reduces dehydroascorbic acid in normal human neutrophils (Jung, CH and J.A. Thomas (1996) Arch. Biochem. Biophys. 335:61-72; Park, J.B. and M. Levine (1996) Biochem. J. 315:931-938).
  • the thioredoxin system serves as a hydrogen donor for ribonucleotide reductase and as a regulator of enzymes by redox control. It also modulates the activity of transcription factors such as NF- ⁇ B, AP-1, and steroid receptors.
  • transcription factors such as NF- ⁇ B, AP-1, and steroid receptors.
  • cytokines or secreted cytokine-like factors such as adult T-cell leukemia-derived factor, 3B6-interleukin-l, T-hybridoma-derived (MP-6) B cell stimulatory factor, and early pregnancy factor have been reported to be identical to thioredoxin (Holmgren, A. (1985) Annu. Rev. Biochem. 54:237-271; Abate, C et al.
  • the selenoprotein thioredoxin reductase is secreted by both normal and neoplastic cells and has been implicated as both a growth factor and as a polypeptide involved in apoptosis (Soderberg, A. et al. (2000) Cancer Res. 60:2281-2289).
  • An extracellular plasmin reductase secreted by hamster ovary cells (HT-1080) has been shown to participate in the generation of angiostatin from plasmin. In this case, the reduction of the plasmin disulfide bonds triggers the proteolytic cleavage of plasmin which yields the angiogenesis inhibitor, angiostatin (Stathakis, P. et al. (1997) J. Biol.
  • Another example of the importance of redox reactions in cell metabolism is the degradation of saturated and unsaturated fatty acids by mitochondrial and peroxisomal beta-oxidation enzymes which sequentially remove two-carbon units from Coenzyme A (CoA)-activated fatty acids.
  • the main beta- oxidation pathway degrades both saturated and unsarurated fatty acids while the auxiliary pathway performs additional steps required for the degradation of unsarurated fatty acids.
  • Mitochondria oxidize short-, medium-, and long-chain fatty acids to produce energy for cells.
  • Mitochondrial beta-oxidation is a major energy source for cardiac and skeletal muscle. In liver, it provides ketone bodies to the peripheral circulation when glucose levels are low as in starvation, endurance exercise, and diabetes (Eaton, S. et al. (1996) Biochem. J. 320:345-357).
  • Peroxisomes oxidize medium-, long-, and very-long-chain fatty acids, dicarboxylic fatty acids, branched fatty acids, prostaglandins, xenobiotics, and bile acid intermediates.
  • the chief roles of peroxisomal beta-oxidation are to shorten toxic lipophilic carboxylic acids to facihtate their excretion and to shorten very-long-chain fatty acids prior to mitochondrial beta-oxidation (Mannaerts, G.P. and P.P. Van Veldhoven (1993) Biochimie 75:147-158).
  • the auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzes the following reaction: trans-2, cis/trans-4-dienoyl-CoA + NADPH + H + — > trans-3-enoyl-CoA + NADP +
  • This reaction removes even-numbered double bonds from unsaturated fatty acids prior to their entry into the main beta-oxidation pathway (Koivuranta, K.T. et al. (1994) Biochem. J. 304:787-792).
  • the enzyme may also remove odd-numbered double bonds from unsaturated fatty acids (Smeland, T.E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6673-6677).
  • Rat 2,4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes (Dommes, V. et al. (1981) J. Biol. Chem. 256:8259-8262).
  • Two immunologically different forms of rat mitochondrial enzyme exist with molecular masses of 60 kDa and 120 kDa (Hakkola, E.H. and J.K. Hiltunen (1993) Eur. J. Biochem. 215:199-204).
  • the 120 kDa mitochondrial rat enzyme is synthesized as a 335 amino acid precursor with a 29 amino acid N-terminal leader peptide which is cleaved to form the mature enzyme (Hirose, A. et al. (1990) Biochim.
  • a human mitochondrial enzyme 83% similar to rat enzyme is synthesized as a 335 amino acid residue precursor with a 19 amino acid N-terminal leader peptide (Koivuranta et al., supra). These cloned human and rat mitochondrial enzymes function as homotetramers (Koivuranta et al., supra).
  • a Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is 295 amino acids long, contains a C-terminal peroxisomal targeting signal, and functions as a homodimer (Coe, J.G.S. et al. (1994) Mol. Gen. Genet.
  • This reaction hydrates the double bond between C-2 and C-3 of 2-trans-enoyl-CoA, which is generated from saturated and unsaturated fatty acids (Engel, C.K. et al. (1996) EMBO J. 15:5135- 5145).
  • This step is downstream from the step catalyzed by 2,4-dienoyl-reductase.
  • Different enoyl- CoA hydratases act on short-, medium-, and long-chain fatty acids (Eaton et al., supra). Mitochondrial and peroxisomal enoyl-CoA hydratases occur as both mono-functional enzymes and as part of multi-functional enzyme complexes.
  • Human liver mitochondrial short-chain enoyl-CoA hydratase is synthesized as a 290 amino acid precursor with a 29 amino acid N-terminal leader peptide (Kanazawa, M. et al. (1993) Enzyme Protein 47:9-13; and Janssen, U. et al. (1997) Genomics 40:470- 475).
  • Rat short-chain enoyl-CoA hydratase is 87% identical to the human sequence in the mature region of the protein and functions as a homohexamer (Kanazawa et al., supra; and Engel et al., supra).
  • a mitochondrial trifunctional protein exists that has long-chain enoyl-CoA hydratase, 3- hydroxyacyl-CoA dehydrogenase, and long-chain 3-oxothiolase activities (Eaton et al., supra).
  • enoyl-CoA hydratase activity is found in both a 327 amino acid residue mono- functional enzyme and as part of a multi-functional enzyme, also known as bifunctional enzyme, which possesses enoyl-CoA hydratase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoA hydrogenase activities (FitzPatrick, D.R. et al.
  • a 339 amino acid residue human protein with short-chain enoyl-CoA hydratase activity also acts as an AU-specific RNA binding protein (Nakagawa, J. et al. (1995) Proc. Natl. Acad. Sci. USA 92:2051-2055). All enoyl-CoA hydratases share homology near two active site glutamic acid residues, with 17 amino acid residues that are highly conserved (Wu, W.-J. et al. (1997) Biochemistry 36:2211-2220).
  • Mitochondrial beta-oxidation associated deficiencies include, e.g., carnitine palmitoyl transferase and carnitine deficiency, very-long-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, short-chain acyl-CoA dehydrogenase deficiency, electron transport flavoprotein and electron transport flavoprotein:ubiquinone oxidoreductase deficiency, trifunctional protein deficiency, and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (Eaton et al., supra).
  • Mitochondrial trifunctional protein including enoyl-CoA hydratase
  • enoyl-CoA hydratase deficient patients have reduced long-chain enoyl-CoA hydratase activities and suffer from non-ketotic hypoglycemia, sudden infant death syndrome, cardiomyopathy, hepatic dysfunction, and muscle weakness, and may die at an early age (Eaton et al., supra).
  • Reye's syndrome a disease characterized by hepatic dysfunction and encephalopathy that sometimes follows viral infection in children.
  • Reye's syndrome patients may have elevated serum levels of free fatty acids (Cotran, R.S. et al. (1994) Robbins Pathologic Basis of Disease, W.B. Saunders Co., Philadelphia PA, p.866).
  • Patients with mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and medium- chain 3-hydroxyacyl-CoA dehydrogenase deficiency also exhibit Reye-like illnesses (Eaton et al, supra; and Egidio, R.J. et al. (1989) Am. Fam. Physician 39:221-226).
  • Inherited conditions associated with peroxisomal beta-oxidation include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase deficiency, peroxisomal thiolase deficiency, and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum. Genet. 54:36-43; Hoefler et al., supra).
  • Peroxisomal beta-oxidation is impaired in cancerous tissue. Although neoplastic human breast epithelial cells have the same number of peroxisomes as do normal cells, fatty acyl-CoA oxidase activity is lower than in control tissue (el Bouhtoury, F. et al. (1992) J. Pathol. 166:27-35). Human colon carcinomas have fewer peroxisomes than normal colon tissue and have lower fatty-acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA hydratase) activities than normal tissue (Cable, S. et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 62:221-226).
  • 6-phosphogluconate dehydrogenase (6-PGDH) catalyses the NAD -dependent oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate with the production of NADPH.
  • 6-PGDH is the third enzyme of the pentose phosphate pathway (PPP) and is ubiquitous in nature. In some heterofermentatative species, NAD+ is used as a cofactor with the subsequent production of NADH.
  • 6-PGDH activity is regulated by the inhibitory effect of NADPH, and the activating effect of 6-phosphogluconate (Rippa, M. et al. (1998) Biochim. Biophys. Acta 1429:83-92). Deficiencies in 6-PGDH activity have been linked to chronic hemolytic anemia.
  • 6-PGDH e.g., enzymes found in trypanosomes
  • 6-PGDH enzymes found in trypanosomes
  • the Trypanosoma brucei enzyme is markedly more sensitive to inhibition by the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate, compared to the mammalian enzyme (Hanau, S. et al. (1996) Eur. J. Biochem. 240:592-599).
  • Ribonucleotide diphosphate reductase catalyzes the reduction of ribonucleotide diphosphates (i.e., ADP, GDP, CDP, and UDP) to their co ⁇ esponding deoxyribonucleotide diphosphates (i.e., dADP, dGDP, dCDP, and dUDP) which are used for the synthesis of DNA. Ribonucleotide diphosphate reductase thereby performs a crucial role in the de novo synthesis of deoxynucleotide precursors. Deoxynucleotides are also produced from deoxynucleosides by nucleoside kinases via the salvage pathway.
  • Mammalian ribonucleotide diphosphate reductase comprises two components, an effector- binding component (E) and a non-heme iron component (F).
  • Component E binds the nucleoside triphosphate effectors while component F contains the iron radical necessary for catalysis.
  • Molecular weight determinations of the E and F components, as well as the holoenzyme, vary according to the methods used in purification of the proteins and the particular laboratory. Component E is approximately 90-100 kDa, component F is approximately 100-120 kDa, and the holoenzyme is 200- 250 kDa.
  • Ribonucleotide diphosphate reductase activity is adversely effected by iron chelators, such as thiosemicarbazones, as well as EDTA.
  • Deoxyribonucleotide diphosphates also appear to be negative allosteric effectors of ribonucleotide diphosphate reductase.
  • Nucleotide triphosphates (both ribo- and deoxyribo-) appear to stimulate the activity of the enzyme.
  • 3-methyl-4-nitrophenol, a metabolite of widely used organophosphate pesticides, is a potent inhibitor of ribonucleotide diphosphate reductase in mammalian cells.
  • ribonucleotide diphosphate reductase activity in DNA virus e.g., herpes virus
  • DNA virus e.g., herpes virus
  • cancer cells are less sensitive to regulation by allosteric regulators and a co ⁇ elation exists between high ribonucleotide diphosphate reductase activity levels and high rates of cell proliferation (e.g., in hepatomas).
  • virus-encoded ribonucleotide diphosphate reductases, and those present in cancer cells are capable of maintaining an increased supply deoxyribonucleotide pool for the production of virus genomes or for the increased DNA synthesis which characterizes cancers cells.
  • Ribonucleotide diphosphate reductase is thus a target for therapeutic intervention (Nutter, L.M. and Y.-C Cheng (1984) Pharmac. Ther. 26:191-207; and Wright, J.A. (1983) Pharmac. Ther. 22:81-102).
  • Dihydrodiol dehydrogenases are monomeric, NAD(P) + -dependent, 34-37 kDa enzymes responsible for the detoxification of tr ⁇ ns-dihydrodiol and anti-diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) such as benzo[ ⁇ ]yrene, benz[ ⁇ ]anthracene, 7-methyl- benz[ ⁇ ]anthracene, 7,12-dirnethyl-benz[ ⁇ ]anthracene, chrysene, and 5-methyl-chrysene.
  • PAH polycyclic aromatic hydrocarbons
  • an environmental PAH toxin such as benzo[ ⁇ ]yrene is initially epoxidated by a microsomal cytochrome P450 to yield 7/?,8 ⁇ -arene-oxide and subsequently (-)-7i?,8i?-dihydrodiol ((-)- tran5-7,8-dihydroxy-7,8-dihydrobenzo[ ⁇ ]pyrene or (-)-tran ⁇ -B[ ⁇ ]P-diol)
  • This latter compound is further transformed to the ⁇ ntj-diol epoxide of benzo[ ⁇ ]pyrene (i.e., ( ⁇ )-anti-7 ?,8 ⁇ -dihydroxy-9 ⁇ ,10 ⁇ - epoxy-7,8,9,10-tetrahydrobenzo[ ⁇ ]pyrene), by the same enzyme or a different enzyme, depending on the species.
  • DD efficiently oxidizes the precursor of the anti-diol epoxide (i.e., trans-dihydrodiol) to transient catechols which auto-oxidize to quinones, also producing hydrogen peroxide and semiquinone radicals. This reaction prevents the formation of the highly carcinogenic ⁇ nti-diol.
  • Anti-diols are not themselves substrates for DD yet the addition of DD to a sample comprising an anti-di ⁇ i compound results in a significant decrease in the induced mutation rate observed in the Ames test. In this instance, DD is able to bind to and sequester the anti-diol, even though it is not oxidized.
  • DD plays an important role in the detoxification of metabolites of xenobiotic polycyclic compounds (Penning, T.M. (1993) Chemico-Biological Interactions 89:1-34).
  • 15-oxoprostaglandin 13-reductase (PGR) and 15-hydroxyprostaglandin dehydrogenase (15- PGDH) are enzymes present in the lung that are responsible for degrading circulating prostaglandins. Oxidative catabolism via passage through the pulmonary system is a common means of reducing the concentration of circulating prostaglandins.
  • 15-PGDH oxidizes the 15-hydroxyl group of a variety of prostaglandins to produce the co ⁇ esponding 15-oxo compounds.
  • the 15-oxo derivatives usually have reduced biological activity compared to the 15-hydroxyl molecule.
  • PGR further reduces the 13,14 double bond of the 15-oxo compound which typically leads to a further decrease in biological activity.
  • PGR is a monomer with a molecular weight of approximately 36 kDa.
  • the enzyme requires NADH or NADPH as a cofactor with a preference for NADH.
  • the 15-oxo derivatives of prostaglandins PGEi, PGE 2 , and PGE 2 ⁇ are all substrates for PGR; however, the non-derivatized prostaglandins (i.e., PGE j , PGE 2 , and PGE 2o ) are not substrates (Ensor, CM. et al. (1998) Biochem. J. 330:103-108).
  • 15-PGDH and PGR also catalyze the metabolism of lipoxin A 4 (LXA 4 ).
  • Lipoxins (LX) are autacoids, lipids produced at the sites of localized inflammation, which down-regulate polymorphonuclear leukocyte (PMN) function and promote resolution of localized trauma.
  • Lipoxin production is stimulated by the administration of aspirin in that cells displaying cyclooxygenase ⁇ (COX II) that has been acetylated by aspirin and cells that possess 5-lipoxygenase (5-LO) interact and produce lipoxin.
  • 15-PGDH generates 15-oxo-LXA 4 with PGR further converting the 15-oxo compound to 13,14-dihydro-15-oxo-LXA 4 (Clish, C.B. et al. (2000) J. Biol. Chem. 275:25372-25380).
  • This finding suggests a broad substrate specificity of the prostaglandin dehydrogenases and has implications for these enzymes in drug metabolism and as targets for therapeutic intervention to regulate inflammation.
  • the GMC (glucose-methanol-choline) oxidoreductase family of enzymes was defined based on sequence alignments of Drosophila melanogaster glucose dehydrogenase, Escherichia coli choline dehydrogenase, Aspergillus niger glucose oxidase, and Hansenula polymorpha methanol oxidase. Despite their different sources and substrate specificities, these four flavoproteins are homologous, being characterized by the presence of several distinctive sequence and structural features. Each molecule contains a canonical ADP-binding, beta-alpha-beta mononucleotide-binding motif close to the amino terminus.
  • This fold comprises a four-stranded parallel beta-sheet sandwiched between a three-stranded antiparallel beta-sheet and alpha-helices. Nucleotides bind in similar positions relative to this chain fold (Cavener, D.R. (1992) J. Mol. Biol. 223:811-814; Wierenga, R.K. et al. (1986) J. Mol. Biol. 187:101-107). Members of the GMC oxidoreductase family also share a consensus sequence near the central region of the polypeptide.
  • GMC oxidoreductase family include cholesterol oxidases from Brevibacterium sterolicum and Streptomyces; and an alcohol dehydrogenase from Pseudomonas oleovorans (Cavener, supra; Henikoff, S. and J.G. Henikoff (1994) Genomics 19:97-107; van Beilen, J.B. et al. (1992) Mol. Microbiol. 6:3121-3136).
  • IMP dehydrogenase and GMP reductase are two oxidoreductases which share many regions of sequence similarity.
  • IMP dehydrogenase (EC 1.1.1.205) catalyes the NAD-dependent reduction of IMP (inosine monophosphate) into XMP (xanthine monophosphate) as part of de novo GTP biosynthesis (Collart, F.R. and E. Huberman (1988) J. Biol. Chem. 263:15769-15772).
  • GMP reductase catalyzes the NADPH-dependent reductive deamination of GMP into IMP, helping to maintain the intracellular balance of adenine and guanine nucleotides (Andrews, S.C and J.R. Guest (1988) Biochem. J. 255:35-43).
  • Pyridine nucleotide-disulphide oxidoreductases are FAD flavoproteins involved in the transfer of reducing equivalents from FAD to a substrate. These flavoproteins contain a pair of redox-active cysteines contained within a consensus sequence which is characteristic of this protein family (Kurlyan, J. et al. (1991) Nature 352:172-174).
  • oxidoreductases include glutathione reductase (EC 1.6.4.2); thioredoxin reductase of higher eukaryotes (EC 1.6.4.5); trypanothione reductase (EC 1.6.4.8); lipoamide dehydrogenase (EC 1.8.1.4), the E3 component of alpha-ketoacid dehydrogenase complexes; and mercuric reductase (EC 1.16.1.1). Transferases
  • Transferases are enzymes that catalyze the transfer of molecular groups. The reaction may involve an oxidation, reduction, or cleavage of covalent bonds, and is often specific to a substrate or to particular sites on a type of substrate. Transferases participate in reactions essential to such functions as synthesis and degradation of cell components, and regulation of cell functions including cell signaling, cell proliferation, inflammation, apoptosis, secretion and excretion. Transferases are involved in key steps in disease processes involving these functions. Transferases are frequently classified according to the type of group transferred.
  • methyl transferases transfer one- carbon methyl groups
  • amino transferases transfer nitrogenous amino groups
  • similarly denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl, phosphorous-containing, sulfur-containing, or selenium-containing groups, as well as small enzymatic groups such as Coenzyme A.
  • Acyl transferases include peroxisomal carnitine octanoyl transferase, which is involved in the fatty acid beta-oxidation pathway, and mitochondrial carnitine palmitoyl transferases, involved in fatty acid metabolism and transport.
  • Choline O-acetyl transferase catalyzes the biosynthesis of the neurotransmitter acetylcholine.
  • N-acyltransferase enzymes catalyze the transfer of an amino acid conjugate to an activated carboxylic group. Endogenous compounds and xenobiotics are activated by acyl-CoA synthetases in the cytosol, microsomes, and mitochondria.
  • acyl-CoA intermediates are then conjugated with an amino acid (typically glycine, glutamine, or taurine, but also ornithine, arginine, histidine, serine, aspartic acid, and several dipeptides) by N-acyltransferases in the cytosol or mitochondria to form a metabolite with an amide bond.
  • an amino acid typically glycine, glutamine, or taurine, but also ornithine, arginine, histidine, serine, aspartic acid, and several dipeptides
  • N-acyltransferases amino acid N-acyltransferase (BAT) responsible for generating the bile acid conjugates which serve as detergents in the gastrointestinal tract (Falany, CN. et al. (1994) J. Biol. Chem. 269:19375-19379; Johnson, M.R. et al.
  • BAT is also useful as a predictive indicator for prognosis of hepatocellular carcinoma patients after partial hepatectomy (Furutani, M. et al. (1996) Hepatology 24:1441-1445).
  • Acetyltransferases Acetyltransferases have been extensively studied for their role in histone acetylation. Histone acetylation results in the relaxing of the chromatin structure in eukaryotic cells, allowing transcription factors to gain access to promoter elements of the DNA templates in the affected region of the genome (or the genome in general). In contrast, histone deacetylation results in a reduction in transcription by closing the chromatin structure and limiting access of transcription factors.
  • a common means of stimulating cell transcription is the use of chemical agents that inhibit the deacetylation of histones (e.g., sodium butyrate), resulting in a global (albeit artifactual) increase in gene expression.
  • histones e.g., sodium butyrate
  • the modulation of gene expression by acetylation also results from the acetylation of other proteins, including but not limited to, p53, GATA-1, MyoD, ACTR, TFHE, TFHF and the high mobility group proteins (HMG).
  • HMG high mobility group proteins
  • p53 acetylation results in increased DNA binding, leading to the stimulation of transcription of genes regulated by p53.
  • the prototypic histone acetylase (HAT) is Gcn5 from Saccharomyces cerevisiae.
  • Gcn5 is a member of a family of acetylases that includes Tetrahymena p55, human Gcn5, and human p300/CBP. Histone acetylation is reviewed in (Cheung, W.L. et al. (2000) Cu ⁇ . Opin. Cell Biol. 12:326-333 and Berger, S.L (1999) Curr. Opin. Cell Biol. 11:336-341). Some acetyltransferase enzymes possess the alpha/beta hydrolase fold (Center of Applied Molecular Engineering Inst.
  • N-acetyltransferases are cytosolic enzymes which utilize the cofactor acetyl-coenzyme A (acetyl-CoA) to transfer the acetyl group to aromatic amines and hydrazine containing compounds.
  • acetyl-CoA cofactor acetyl-coenzyme A
  • mice appear to have a third form of the enzyme, NAT3.
  • the human forms of N-acetyltransferase have independent regulation (NAT1 is widely-expressed, whereas NAT2 is in liver and gut only) and overlapping substrate preferences.
  • NAT1 does prefer some substrates (para-aminobenzoic acid, para-aminosalicylic acid, sulfamethoxazole, and sulfanilamide), while NAT2 prefers others (isoniazid, hydralazine, procainamide, dapsone, aminoglutethimide, and sulfamethazine).
  • tubedown-1 is homologous to the yeast NAT-1 N-acetyltransferases and encodes a protein associated with acetyltransferase activity. The expression patterns of tubedown-1 suggest that it may be involved in regulating vascular and hematopoietic development (Gendron, R.L. et al. (2000) Dev. Dyn. 218:300-315).
  • Amino transferases comprise a family of pyridoxal 5 -phosphate (PLP) -dependent enzymes that catalyze transformations of amino acids.
  • PPP pyridoxal 5 -phosphate
  • Amino transferases play key roles in protein synthesis and degradation, and they contribute to other processes as well.
  • GABA aminotransferase GABA-T
  • the activity of GABA-T is correlated to neuropsychiatric disorders such as alcoholism, epilepsy, and Alzheimer's disease (Sherif, F.M. and S.S. Ahmed (1995) Clin. Biochem. 28:145-154).
  • pyruvate aminotransferase branched-chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al. (1997) J. Biol. Chem. 272:21932-21937).
  • Kynurenine aminotransferase catalyzes the irreversible Iransamination of the L-tryptophan metabolite L-kynurenine to form kynurenic acid.
  • the enzyme may also catalyzes the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate.
  • Kynurenic acid is a putative modulator of glutamatergic neurotransmission, thus a deficiency in kynurenine aminotransferase may be associated with pleiotropic effects (Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
  • Glycosyl transferases include the mammalian UDP-glucouronosyl transferases, a family of membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid to lipophilic substrates in reactions that play important roles in detoxification and excretion of drugs, carcinogens, and other foreign substances.
  • Another mammalian glycosyl transferase mammalian UDP-galactose- ceramide galactosyl transferase, catalyzes the transfer of galactose to ceramide in the synthesis of galactocerebrosides in myelin membranes of the nervous system.
  • the UDP-glycosyl transferases share a conserved signature domain of about 50 amino acid residues (PROS ⁇ TE: PDOC00359, http://expasy.hcuge.ch/sprot/prosite.html).
  • Methyl transferases are involved in a variety of pharmacologically important processes. Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides and other pyridines, an important step in the cellular handling of drugs and other foreign compounds. Phenyle anolamine N-methyl transferase catalyzes the conversion of noradrenalin to adrenalin. 6-O-methylguanine-DNA methyl transferase reverses DNA methylation, an important step in carcinogenesis.
  • Uropo hyrin-i ⁇ C-methyl transferase which catalyzes the transfer of two methyl groups from S-adenosyl-L- methionine to uroporphyrinogen HI, is the first specific enzyme in the biosynthesis of cobalamin, a dietary enzyme whose uptake is deficient in pernicious anemia.
  • Protein-arginine methyl transferases catalyze the posttranslational methylation of arginine residues in proteins, resulting in the mono- and dimethylation of arginine on the guanidino group.
  • Substrates include histones, myelin basic protein, and heterogeneous nuclear ribonucleoproteins involved in mRNA processing, splicing, and transport.
  • Protein-arginine methyl transferase interacts with proteins upregulated by mitogens, with proteins involved in chronic lymphocytic leukemia, and with interferon, suggesting an important role for methylation in cytokine receptor signaling (Lin, W.-J. et al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich, C et al. (1997) EMBO J. 16:260-266; and Scott, H. S. et al. (1998) Genomics 48:330- 340).
  • Phospho transferases catalyze the transfer of high-energy phosphate groups and are important in energy-requiring and -releasing reactions.
  • the metabolic enzyme creatine kinase catalyzes the reversible phosphate transfer between creatine/creatine phosphate and ATP/ADP.
  • Glycocyamine kinase catalyzes phosphate transfer from ATP to guanidoacetate
  • arginine kinase catalyzes phosphate transfer from ATP to arginine.
  • a cysteme-containing active site is conserved in this family (PROSITE: PDOC00103).
  • Prenyl transferases are heterodimers, consisting of an alpha and a beta subunit, that catalyze the transfer of an isoprenyl group.
  • the Ras farnesyltransferase (FTase) enzyme transfers a farnesyl moiety from cytosolic farnesylpyrophosphate to a cysteine residue at the carboxyl te ⁇ riinus of the Ras oncogene protein. This modification is required to anchor Ras to the cell membrane so that it can perform its role in signal transduction.
  • FTase inhibitors block Ras function and demonstrate antitumor activity_(Buolamwini, J.K. (1999) Cu ⁇ . Opin. Chem. Biol. 3:500-509).
  • Saccharyl transferases are glycating enzymes involved in a variety of metabolic processes. Oligosaccharyl transferase-48, for example, is a receptor for advanced glycation endproducts, which accumulate in vascular complications of diabetes, macrovascular disease, renal insufficiency, and Alzheimer's disease (Thornalley, P. J. (1998) Cell Mol. Biol. (Noisy-Le-Grand) 44:1013-1023).
  • Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two carboxylic acids.
  • Succinyl CoA:3-oxoacid CoA transferase for example, transfers CoA from succinyl-CoA to a recipient such as acetoacetate.
  • Acetoacetate is essential to the metabolism of ketone bodies, which accumulate in tissues affected by metabolic disorders such as diabetes (PROSITE: PDOC00980).
  • Transglutaminase transferases are Ca 2+ dependent enzymes capable of forming isopeptide bonds by catalyzing the transfer of the ⁇ -carboxy group from protein-bound glutamine to the ⁇ -amino group of protein-bound lysine residues or other primary amines.
  • Tgases are the enzymes responsible for the cross-linking of cornified envelope (CE), the highly insoluble protein structure on the surface of corneocytes, into a chemically and mechanically resistant protein polymer. Seven known human Tgases have been identified.
  • transglutaminase gene products are specialized in the cross-linking of specific proteins or tissue structures, such as factor XHIa which stabilizes the fibrin clot inhemostasis, prostrate transglutaminase which functions in semen coagulation, and tissue transglutaminase which is involved in GTP-binding in receptor signaling.
  • Factor XHIa which stabilizes the fibrin clot inhemostasis
  • prostrate transglutaminase which functions in semen coagulation
  • tissue transglutaminase which is involved in GTP-binding in receptor signaling.
  • Four are expressed in terminally differentiating epithelia such as the epidermis.
  • Tgases are critical for the proper cross-linking of the CE as seen in the pathology of patients suffering from one form of the skin diseases refe ⁇ ed to as congenital ichthyosis which has been linked to mutations in the keratinocyte transglutaminase (TG K ) gene (Nemes, Z. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:8402-8407, Aeschlimann, D. et al. (1998) J. Biol. Chem. 273:3452-3460.)
  • Hydrolases are a class of enzymes that catalyze the cleavage of various covalent bonds in a substrate by the introduction of a molecule of water. The reaction involves a nucleophilic attack by the water molecule's oxygen atom on a target bond in the substrate. The water molecule is split across the target bond, breaking the bond and generating two product molecules. Hydrolases participate in reactions essential to such functions as synthesis and degradation of cell components, and for regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Hydrolases are involved in key steps in disease processes involving these functions.
  • Hydrolytic enzymes may be grouped by substrate specificity into classes including phosphatases, peptidases, lysophospholipases, phosphodiesterases, glycosidases, glyoxalases, aminohydrolases, carboxylesterases, sulfatases, phosphohydrolases, nucleotidases, lysozymes, and many others.
  • Phosphatases hydrolytically remove phosphate groups from proteins, an energy-providing step that regulates many cellular processes, including intracellular signaling pathways that in turn control cell growth and differentiation, cell-cell contact, the cell cycle, and oncogenesis.
  • l ⁇ Peptidases also called proteases, cleave peptide bonds that form the backbone of peptide or protein chains. Proteolytic processing is essential to cell growth, differentiation, remodeling, and homeostasis as well as inflammation and the immune response. Since typical protein half-lives range from hours to a few days, peptidases are continually cleaving precursor proteins to their active form, removing signal sequences from targeted proteins, and degrading aged or defective proteins.
  • Peptidases function in bacterial, parasitic, and viral invasion and replication within a host.
  • peptidases include trypsin and chymotrypsin (components of the complement cascade and the blood-clotting cascade) lysosomal cathepsins, calpains, pepsin, renin, and chymosin (Beynon, R. J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York, NY, pp. 1-5).
  • Lysophospholipases regulate intracellular lipids by catalyzing the hydrolysis of ester bonds to remove an acyl group, a key step in lipid degradation.
  • Small LPL isoforms approximately 15-30 kD, function as hydrolases; larger isoforms function both as hydrolases and transacylases.
  • Phosphodiesterases catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore crucial to a variety of cellular processes. Phosphodiesterases include DNA and RNA endo- and exo-nucleases, which are essential to cell growth and replication as well as protein synthesis. Endonuclease V (deoxyinosine 3'-endonuclease) is an example of a type II site-specific deoxyribonuclease, a putative DNA repair enzyme that cleaves DNAs containing hypoxanthine, uracil, or mismatched bases.
  • Endonuclease V deoxyinosine 3'-endonuclease
  • Escherichia coli endonuclease V has been shown to cleave DNA containing deoxyxanthosine at the second phosphodiester bond 3' to deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the nick site (He, B. et al. (2000) Mutat. Res. 459:109-114). It has been suggested that Escherichia coli endonuclease V plays a role in the removal of deaminated guanine, i.e., xanthine, from DNA, thus helping to protect the cell against the mutagenic effects of nitrosative deamination (Schouten, K.A. and B. Weiss (1999) Mutat.
  • POP1 ('processing of precursor RNAs') encodes a protein component of both RNase P and RNase MRP, another RNA processing protein. Mutations in yeast POP1 are lethal (Lygerou, Z. et al. (1994) Genes Dev. 8:1423-1433).
  • Another phosphodiesterase acid sphingomyehnase, hydrolyzes the membrane phospholipid sphingomyelin to ceramide and phosphorylcholine. Phosphorylcholine functions in synthesis of phosphatidylcholine, which is involved in intracellular signaling pathways. Ceramide is an essential precursor for the generation of gangliosides, membrane lipids found in high concentration in neural tissue. Defective acid sphingomyelinase phosphodiesterase leads to Niemann-Pick disease.
  • Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, which are compounds that contain one or more sugar.
  • Mammalian lactase-phlorizin hydrolase for example, is an intestinal enzyme that splits lactose.
  • Mammalian beta-galactosidase removes the terminal galactose from gangliosides, glycoproteins, and glycosaminoglycans, and deficiency of this enzyme is associated with a gangliosidosis known as Morquio disease type B (PROSITE PCDOC00910).
  • Vertebrate lysosomal alpha-glucosidase which hydrolyzes glycogen, maltose, and isomaltose
  • vertebrate intestinal sucrase-isomaltase which hydrolyzes sucrose, maltose, and isomaltose
  • the glyoxylase system is involved in gluconeogenesis, the production of glucose from storage compounds in the body.
  • glyoxylase I which catalyzes the formation of S-D- lactoylglutathione from methyglyoxal, a side product of triose-phosphate energy metabolism, and glyoxylase U, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and reduced glutathione.
  • Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes mellitus, the detoxification of bacterial toxins, and in the control of cell proliferation and microtubule assembly.
  • NG,NG-dimethylarginine dimethylaminohydrolase is an enzyme that hydrolyzes the endogenous nitric oxide synthase (NOS) inhibitors, NG-monomemyl-arginine and NG,NG-dimethyl-L- arginine, to L-citrulline.
  • NOS nitric oxide synthase
  • Inhibiting DDAH can cause increased intracellular concentration of NOS inhibitors to levels sufficient to inhibit NOS. Therefore, DDAH inhibition may provide a method of NOS inhibition, and changes in the activity of DDAH could play a role in pathophysiological alterations in nitric oxide generation (MacAllister, R.J. et al. (1996) Br. J. Pharmacol. 119:1533-1540).
  • DDAH oxidative stress- and nitric oxide-mediated events play a role in the pathogenesis of Alzheimer's disease (Smith, M.A. et al. (1998) Free Rad. Biol. Med. 25:898-902).
  • Acyl-CoA thioesterase is another member of the carboxylesterase family (Alexson, S.E. et al. (1993) Eur. J. Biochem. 214:719-727). Evidence suggests that acyl-CoA thioesterase has a regulatory role in steroidogenic tissues (Finkielstein, C. et al. (1998) Eur. J. Biochem. 256:60-66).
  • the alpha/beta hydrolase protein fold is common to several hydrolases of diverse phylogenetic origin and catalytic function. Enzymes with the alpha/beta hydrolase fold have a common core structure consisting of eight beta-sheets connected by alpha-helices. The most conserved structural feature of this fold is the loops of the nucleophile-histidine-acid catalytic triad. The histidine in the catalytic triad is completely conserved, while the nucleophile and acid loops accommodate more than one type of amino acid (Ollis, D.L. et al. (1992) Protein Eng. 5:197-211).
  • Sulfatases are members of a highly conserved gene family that share extensive sequence homology and a high degree of structural similarity. Sulfatases catalyze the cleavage of sulfate esters. To perform this function, sulfatases undergo a unique post-translational modification in the endoplasmic reticulum that involves the oxidation of a conserved cysteine residue. A human disorder called multiple sulfatase deficiency is due to a defect in this post-translational modification step, leading to inactive sulfatases (Recksiek, M. et al. (1998) J. Biol. Chem. 273:6096-6103).
  • Phosphohydrolases are enzymes that hydrolyze phosphate esters. Some phosphohydrolases contain a mutT domain signature sequence. MutT is a protein involved in the GO system responsible for removing an oxidatively damaged form of guanine from DNA. A region of about 40 amino acid residues, found in the N-terminus of mutT, is also found in other proteins, including some phosphohydrolases (PROSITE PDOC00695).
  • Serine hydrolases are a large functional class of hydrolytic enzymes that contain a serine residue in their active site. This class of enzymes contains proteinases, esterases, and lipases which hydrolyze a variety of substrates and, therefore, have different biological roles. Proteins in this superfamily can be further grouped into subfamilies based on substrate specificity or amino acid similarities (Puente, X.S. and C Lopez-Otin (1995) J. Biol. Chem. 270:12926-12932).
  • NTE Neuropathy target esterase
  • PV phenyl valerate
  • NTE contains at least two functional domains: an N-terminal putative regulatory domain and a C-terminal effector domain which contains the esterase activity and is, in part, conserved in proteins found in bacteria, yeast, nematodes and insects.
  • NTE's effector domain contains three predicted transmembrane segments, and the active-site serine residue lies at the center of one of these segments.
  • the isolated recombinant domain shows PV hydrolase activity only when incorporated into phospholipid Hposomes.
  • NTE's esterase activity is largely redundant in adult vertebrates, but organophosphates which react with NTE in vivo initiate unknown events which lead to a neuropathy with degeneration of long axons.
  • neuropathic organophosphates leave a negatively charged group covalently attached to the active-site serine residue, which causes a toxic gain of function in NTE (Glynn, P. (1999) Biochem. J. 344:625-631).
  • the Drosophila neurodegeneration gene swiss-cheese encodes a neuronal protein involved in glia-neuron interaction and is homologous to the above human NTE (Moser, M. et al. (2000) Mech. Dev. 90:279-282).
  • Chitinases are chitin-degrading enzymes present in a variety of organisms and participate in processes including cell wall remodeling, defense and catabolism. Chitinase activity has been found in human serum, leukocytes, granulocytes, and in association with fertilized oocytes in mammals (Escott, G.M. (1995) Infect. Immunol. 63:4770-4773; DeSouza, M.M. (1995) Endrocrinology 136:2485-2496). Glycolytic and proteolytic molecules in humans are associated with tissue damage in lung diseases and with increased tumorigenicity and metastatic potential of cancers (Mulligan, M.S. (1993) Proc. Natl. Acad. Sci.
  • Some of the mammalian members of the family such as a bovine whey chitotriosidase and human cartilage proteins which do not demonstrate specific chitinolytic activity, are expressed in association with tissue remodeling events (Rejman, JJ. (1988) Biochem. Biophys. Res. Commun. 150:329-334, Nyirkos, P. (1990) Biochem. J. 268:265-268). Elevated levels of human cartilage proteins have been reported in the synovial fluid and cartilage of patients with rheumatoid arthritis, a disease which produces a severe degradation of the cartilage and a proliferation of the synovial membrane in the affected joints (Hakala, B.E. (1993) J. Biol. Chem. 268:25803-25810).
  • S- adenosyl-L-homocysteine hydrolase also known as AdoHcyase or SAHH (PROSITE PDOC00603; EC 3.3.1.1)
  • AdoHcyase AdoHcyase
  • SAHH PROSITE PDOC00603; EC 3.3.1.1
  • SAHH is a cytosolic enzyme that has been found in all cells that have been tested, with the exception of Escherichia coli and certain related bacteria (Walker, R.D. et al. (1975) Can. J. Biochem. 53:312-319; Shimizu, S. et al. (1988) FEMS Microbiol. Lett. 51:177-180; Shimizu, S. et al. (1984) Eur. J. Biochem. 141:385-392). SAHH activity is dependent on NAD + as a cofactor.
  • Deficiency of SAHH is associated with hypermethioninemia (Online Mendelian Inheritance in Man (OMIM) #180960 Hype ⁇ nethioninemia), a pathologic condition characterized by neonatal cholestasis, failure to thrive, mental and motor retardation, facial dysmorphism with abnormal hair and teeth, and myocaridopathy (Labrune, P. et al. (1990) J. Pediat. 117:220-226).
  • OMIM Online Mendelian Inheritance in Man
  • hydrolases includes those enzymes which act on carbon-nitrogen (C-N) bonds other than peptide bonds. To this subclass belong those enzymes hydrolyzing amides, amidines, and other C-N bonds. This subclass is further subdivided on the basis of substrate specificity such as linear amides, cyclic amides, linear amidines, cyclic amidines, nitriles and other compounds.
  • a hydrolase belonging to the sub-subclass of enzymes acting on the cyclic amidines is adenosine deaminase (ADA). ADA catalyzes the breakdown of adenosine to inosine.
  • ADA adenosine deaminase
  • ADA is present in many mammalian tissues, including placenta, muscle, lung, stomach, digestive diverticulum, spleen, erythrocytes, thymus, seminal plasma, thyroid, T-cells, bone marrow stem cells, and liver.
  • a subclass of AD As, ADAR act on RNA and are classified as RNA editases.
  • An ADAR from Drosophila, dADAR, expressed in the developing nervous system may act on para voltage-gated Na+ channel transcripts in the central nervous system (Palladino, M.J. et al. (2000) RNA 6:1004-1018).
  • ADA deficiency causes profound lymphopenia with severe combined immunodeficiency (SQD).
  • Cells from patients with ADA deficiency contain low, sometimes undetectable, amounts of ADA catalytic activity and ADA protein.
  • ADA deficiency stems from genetic mutations in the ADA gene
  • Pancreatic ribonucleases are pyrimidine-specific endonucleases found in high quantity in the pancreas of certain mammalian taxa and of some reptiles (Beintema, J.J. et al (1988) Prog. Biophys. Mol. Biol. 51:165-192). Proteins in the mammalian pancreatic RNase superfamily are noncytosolic endonucleases that degrade RNA through a two-step transphosphorolytic-hydrolytic reaction (Beintema, J.J. et al. (1986) Mol. Biol. Evol. 3:262-275).
  • the enzymes are involved in endonucleolytic cleavage of 3 -phosphomononucleotides and 3 -phosphooligonucleotides ending in C-P or U-P with 2 ',3 -cyclic phosphate intermediates.
  • Ribonucleases can unwind the DNA helix by complexing with single-stranded DNA; the complex arises by an extended multi-site cation-anion interaction between lysine and arginine residues of the enzyme and phosphate groups of the nucleotides.
  • Some of the enzymes belonging to this family appear to play a purely digestive role, whereas others exhibit potent and unusual biological activities (D'Alessio, G. (1993) Trends Cell Biol. 3:106-109).
  • Proteins belonging to the pancreatic RNase family include: bovine seminal vesicle and brain ribonucleases; kidney non-secretory ribonucleases (Beintema, J.J. et al (1986) FEBS Lett. 194:338-343); Hver-type ribonucleases (Rosenberg, HF. et al. (1989) PNAS U.S.A. 86:4460-4464); angiogenin, which induces vascularisation of normal and maUgnant tissues; eosinophil cationic protein (Hofsteenge, J. et al.
  • pancreatic RNases contain 4 conserved disulfide bonds and 3 amino acid residues involved in the catalytic activity.
  • ADP-ribosylation is a reversible post-translational protein modification in which an ADP- ribose moiety is transfe ⁇ ed from ⁇ -NAD to a target amino acid such as arginine or cysteine.
  • ADP- ribosylarginine hydrolases regenerate arginine by removing ADP-ribose from the protein, completing the ADP-ribosylation cycle (Moss, J. et al. (1997) Adv. Exp. Med. Biol. 419:25-33).
  • ADP- ribosylation is a well-known reaction among bacterial toxins.
  • Cholera toxin for example, disrupts the adenylyl cyclase system by ADP-ribosylating the ⁇ -subunit of the stimulatory G-protein, causing an increase in intracellular cAMP (Moss, J. and M. Vaughan (Eds) (1990) ADP-ribosylating Toxins and G-Proteins: Insights into Signal Transduction. American Society for Microbiology, Washington, D.C). ADP-ribosylation may also have a regulatory function in eukaryotes, affecting such processes as cytoskeletal assembly (Zhou, H. et al. (1996) Arch. Biochem. Biophys. 334:214-222) and cell proliferation in cytotoxic T-cells (Wang, J. et al. (1996) J. Immunol. 156:2819-2827).
  • Nucleotidases catalyze the formation of free nucleosides from nucleotides.
  • the cytosolic nucleotidase cN-I (5' nucleotidase-I) cloned from pigeon heart catalyzes the formation of adenosine from AMP generated during ATP hydrolysis (Sala-Newby, G.B. et al. (1999) J. Biol. Chem. 274:17789-17793).
  • Increased adenosine concentration is thought to be a signal of metabolic stress, and adenosine receptors mediate effects including vasodilation, decreased stimulatory neuron firing and ischemic preconditioning in the heart (Schrader, J.
  • lysozyme c superfamily consists of conventional lysozymes c, calcium-binding lysozymes c, and ⁇ -lactalbumin (Prager, E.M. and P. Jolles (1996) EXS 75:9-31).
  • Lysozymes c are ubiquitous in a variety of tissues and secretions and can lyse the cell walls of certain bacteria (McKenzie, H.A. (1996) EXS 75:365-409).
  • Alpha-lactalbumin is a metallo-protein that binds calcium and participates in the synthesis of lactose (Iyer, L.K. and P.K. Qasba (1999) Protein Eng. 12:129-139).
  • Alpha-lactalbumin occurs in mammalian milk and colostrum (McKenzie, supra).
  • Lysozymes catalyze the hydrolysis of certain mucopolysaccharides of bacterial cell walls, specifically, the beta (l-4).glycosidic linkages between N-acetylmuramic acid and N- acetylglucosamine, and cause bacterial lysis. Lysozymes occur in diverse organisms including viruses, birds, and mammals. In humans, lysozymes are found in spleen, lung, kidney, white blood cells, plasma, saliva, milk, tears, and cartilage (OMfM #153450 Lysozyme; Weaver, L.H. et al. (1985) J. Mol. Biol. 184:739-741).
  • Lysozyme c functions in minants as a digestive enzyme, releasing proteins from ingested bacterial cells, and may perform the same function in human newborns (Braun, O.H. et al. (1995) Klin. Pediatr. 207:4-7).
  • lysozymes chicken-type and goose-type
  • Chicken-type and goose-type lysozymes have similar three-dimensional structures, but different amino acid sequences (Nakano, T. and T. Graf (1991) Biochim. Biophys. Acta 1090:273-276).
  • neutrophil granulocytes heterophils
  • chicken-type lysozyme is found in egg white.
  • chicken- type lysozyme mRNA is found in both adherent monocytes and macrophages and nonadherent promyelocytes and granulocytes as well as in cells of the bone marrow, spleen, bursa, and oviduct.
  • Goose-type lysozyme mRNA is found in non-adherent cells of the bone marrow and lung.
  • isozymes have been found in rabbits, including leukocytic, gastrointestinal, and possibly lymphoepithelial forms (OMIM #153450, supra; Nakano and Graf, supra; and GenBank GI 1310929).
  • a human lysozyme gene encoding a protein similar to chicken-type lysozyme has been cloned (Yoshimura, K. et al. (1988) Biochem. Biophys. Res. Commun. 150:794-801).
  • a consensus motif featuring regularly spaced cysteine residues has been derived from the lysozyme C enzymes of various species (PROSITE PS00128). Lysozyme C shares about 40% amino acid sequence identity with ⁇ -lactalbumin.
  • Lysozymes have several disease associations. Lysozymuria is observed in diabetic nephropathy (Shima, M. et al. (1986) Clin. Chem. 32:1818-1822), endemic nephropathy (Bruckner, I. et al. (1978) Med. Interne. 16:117-125), urinary tract infections (Heidegger, H. (1990) Minerva Ginecol. 42:243-250), and acute monocytic leukemia (Shaw, M.T. (1978) Am. J. Hematol. 4:97-103). Nakano and Graf (supra) suggested a role for lysozyme in host defense systems.
  • Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-S, C-(halide),
  • the group of C-C lyases includes carboxyl-lyases (decarboxylases), aldehyde-lyases (aldolases), oxo-acid-lyases, and other lyases.
  • the C-O lyase group includes hydro-lyases, lyases acting on polysaccharides, and other lyases.
  • the C-N lyase group includes ammonia-lyases, amidine- lyases, arnine-lyases (deaminases), and other lyases. Lyases are critical components of cellular biochemistry, with roles in metabolic energy production, including fatty acid metabolism and the tricarboxylic acid cycle, as well as other diverse enzymatic processes.
  • CA carbonic anhydrases
  • Hfi + CO 2 * » HCO 3 " + H + One important family of lyases are the carbonic anhydrases (CA), also called carbonate dehydratases, which catalyze the hydration of carbon dioxide in the reaction Hfi + CO 2 * » HCO 3 " + H + .
  • CA accelerates this reaction by a factor of over 10 6 by virtue of a zinc ion located in a deep cleft about 15 A below the protein's surface and co-ordinated to the imidazole groups of three His residues. Water bound to the zinc ion is rapidly converted to HCO 3 ⁇
  • CAI cytosolic isozymes
  • CAIII two membrane-bound forms
  • CAV mitochondrial form
  • CAVI secreted salivary form
  • PROSITE PDOC00146 Eukaryotic-type carbonic anhydrases signature isoenzymes CAI, CAJJ, and bovine CAUI have similar secondary structures and polypeptide-chain folds
  • CAI has 6 tryptophans
  • CAT! has 7
  • CAUI has 8 (Boren, K. et al. (1996) Protein Sci. 5:2479-2484).
  • CAJJ is the predominant CA isoenzyme in the brain of mammals.
  • CAs participate in a variety of physiological processes that involve pH regulation, CO 2 and
  • HCO 3 transport, ion transport, and water and electrolyte balance.
  • CAU contributes to H + secretion by gastric parietal cells, by renal tubular cells, and by osteoclasts that secrete H + to acidify the bone-resorbing compartment.
  • CAU promotes HCO 3 " secretion by pancreatic duct cells, cilary body epithelium, choroid plexus, salivary gland acinar cells, and distal colonal epithelium, thus playing a role in the production of pancreatic juice, aqueous humor, cerebrospinal fluid, and saliva, and contributing to electrolyte and water balance.
  • CAU also promotes CO 2 exchange in proximal tubules in the kidney, in erythrocytes, and in lung.
  • CATV has roles in several tissues: it facilitates HCO 3 " reabsorption in the kidney; promotes CO 2 flux in tissues including brain, skeletal muscle, and heart muscle; and promotes CO 2 exchange from the blood to the alveoli in the lung.
  • CAVI probably plays a role in pH regulation in saliva, along with CAU, and may have a protective effect in the esophagus and stomach.
  • Mitochondrial CAV appears to play important roles in gluconeogenesis and ureagenesis, based on the effects of CA inhibitors on these pathways.
  • CAJJ cerebrospinal fluid
  • OMTM #259730 Osteopetrosis with Renal Tubular Acidosis The concentration of CAJJ in the cerebrospinal fluid (CSF) appears to mark disease activity in patients with brain damage.
  • High CA concentrations have been observed in patients with brain infarction.
  • Patients with transient ischemic attack, multiple sclerosis, or epilepsy usually have CAJJ concentrations in the normal range, but higher CAU levels have been observed in the CSF of those with central nervous system infection, dementia, or trigeminal neuralgia (Parkkila, A.K. et al. (1997) Eur.
  • CA inhibitors such as acetazolamide are used in the treatment of glaucoma (Stewart, W.C (1999) Curr. Opin.
  • CA activity can be particularly useful as an indicator of long-term disease conditions, since the enzyme reacts relatively slowly to physiological changes.
  • CAI and zinc concentrations have been observed to decrease in hyperthyroid Graves' disease (Yoshida, K. (1996) Tohoku J. Exp. Med. 178:345-356) and glycosylated CAI is observed in diabetes mellitus (Kondo, T. et al. (1987) Clin. Chim. Acta 166:227-236).
  • a positive co ⁇ elation has been observed between CAI and CAJJ reactivity and endometriosis (Brinton, D.A. et al. (1996) Ann. Clin. Lab. Sci. 26:409-420; D'Cruz , O.J. et al. (1996) Fertil. Steril. 66:547-556).
  • ODC o ⁇ rithine decarboxylase
  • ODC is a pyridoxal-5'-phosphate (PLP)-dependent enzyme which is active as a homodimer. conserveed residues include those at the PLP binding site and a stretch of glycine residues thought to be part of a substrate binding region (PROSITE PDOC00685 Orn/DAP/Arg decarboxylase family 2 signatures). Mammalian ODCs also contain PEST regions, sequence fragments enriched in proline, glutamic acid, serine, and threonine residues that act as signals for intracellular degradation (Medina et al., supra). Many chemical carcinogens and tumor promoters increase ODC levels and activity.
  • PLP pyridoxal-5'-phosphate
  • ODC oncogenes
  • oncogenes may increase ODC levels by enhancing transcription of the ODC gene, and ODC itself may act as an oncogene when expressed at very high levels.
  • a high level of ODC is found in a number of precancerous conditions, and elevation of ODC levels has been used as part of a screen for tumor-promoting compounds (Pegg, A.E. et al. (1995) J. Cell. Biochem. Suppl. 22:132-138).
  • Inhibitors of ODC have been used to treat tumors in animal models and human clinical trials, and have been shown to reduce development of tumors of the bladder, brain, esophagus, gastrointestinal tract, lung, oral cavity, mammary gland, stomach, skin and trachea (Pegg et al., supra; McCann, P.P. and A.E. Pegg (1992) Pharmac. Ther. 54:195-215).
  • ODC also shows promise as a target for chemoprevention (Pegg et al., supra).
  • ODC inhibitors have also been used to treat infections by African trypanosomes, malaria, and Pneumocystis carinii, and are potentially useful for treatment of autoimmune diseases such as lupus and rheumatoid arthritis (McCann and Pegg, supra).
  • Another family of pyridoxal-dependent decarboxylases are the group II decarboxylases.
  • This family includes glutamate decarboxylase (GAD) which catalyzes the decarboxylation of glutamate into the neurotransmitter GABA; histidine decarboxylase (HDC), which catalyzes the decarboxylation of histidine to histamine; aromatic-L-amino-acid decarboxylase (DDC), also known as L-dopa decarboxylase or tryptophan decarboxylase, which catalyzes the decarboxylation of tryptophan to tryptamine and also acts on 5-hydroxy-tryptophan and dihydroxyphenylalanine (L-dopa); and cysteine sulfinic acid decarboxylase (CSD), the rate-limiting enzyme in the synthesis of taurine from cysteine (PROS ⁇ E PDOC00329 DDC/GAD/HDC/TyrDC pyridoxal-phosphate attachment site). Taurine is an abundant sulfonic amino acid in brain and is thought to act
  • Isomerases are a class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. This class includes racemases and epimerases, cis-trans- isomerases, intramolecular oxidoreductases, intramolecular transferases (mutases) and intramolecular lyases. Isomerases are critical components of cellular biochemistry with roles in metabolic energy production including glycolysis, as well as other diverse enzymatic processes (Stryer, supra, pp.483- 507).
  • Racemases are a subset of isomerases that catalyze inversion of a molecule's configuration around the asymmetric carbon atom in a substrate having a single center of asymmetry, thereby interconverting two racemers.
  • Epimerases are another subset of isomerases that catalyze inversion of configuration around an asymmetric carbon atom in a substrate with more than one center of symmetry, thereby interconverting two epimers.
  • Racemases and epimerases can act on amino acids and derivatives, hydroxy acids and derivatives, and carbohydrates and derivatives. The interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-4'-epimerase.
  • PPIases peptidyl prolyl cis-trans isomerases
  • CyP The cyclophilins (CyP) were originally identified as major receptors for the immunosuppressive drug cyclosporin A (CsA), an inhibitor of T-cell activation (Handschumacher, R.E. et al. (1984) Science 226:544-547; Harding, M.W. et al. (1986) J. Biol. Chem. 261:8547-8555).
  • CsA immunosuppressive drug
  • T-cell activation Handschumacher, R.E. et al. (1984) Science 226:544-547; Harding, M.W. et al. (1986) J. Biol. Chem. 261:8547-8555.
  • the peptidyl-prolyl isomerase activity of CyP may be part of the signaling pathway that leads to T-cell activation.
  • CyP's isomerase activity is essential for co ⁇ ect protein folding and/or protein trafficking, and may also be involved in assembly/disassembly of protein complexes and regulation of protein activity.
  • CyP NinaA is required for co ⁇ ect localization of rhodopsins
  • Cyp40 is part of the Hsp90/Hsp70 complex that binds steroid receptors.
  • the mammalian CyP (CypA) has been shown to bind the gag protein from human immunodeficiency virus 1 (HTV-1), an interaction that can be inhibited by cyclosporin.
  • CypA may play an essential function in HIV-1 replication.
  • Cyp40 has been shown to bind and inactivate the transcription factor c- Myb, an effect that is reversed by cyclosporin. This effect implicates CyP in the regulation of transcription, transformation, and differentiation (Bergsma, D.J. et al (1991) J. Biol. Chem. 266:23204- 23214; Hunter, T. (1998) Cell 92:141-143; and Leverson, J.D. and S.A. Ness (1998) Mol. Cell. 1:203- 211).
  • thioLdisulfide exchange that is necessary for co ⁇ ect protein assembly.
  • incubation of reduced, unfolded proteins in buffers with defined ratios of oxidized and reduced thiols can lead to native conformation, the rate of folding is slow and the attainment of native conformation decreases proportionately with the size and number of cysteines in the protein.
  • Certain cellular compartments such as the endoplasmic reticulum of eukaryotes and the periplasmic space of prokaryotes are maintained in a more oxidized state than the su ⁇ ounding cytosol.
  • Co ⁇ ect disulfide formation can occur in these compartments, but at a rate that is insufficient for normal cell processes and inadequate for synthesizing secreted proteins.
  • the protein disulfide isomerases, thioredoxins and glutaredoxins are able to catalyze the formation of disulfide bonds and regulate the redox environment in cells to enable the necessary thioLdisulfide exchanges (Loferer, H. (1995) J. Biol. Chem. 270:26178-26183).
  • Protein disulfide isomerases are found in the endoplasmic reticulum of eukaryotes and in the periplasmic space of prokaryotes. They function by exchanging their own disulfide for a thiol in a folding peptide chain. In contrast, the reduced thioredoxins and glutaredoxins are generally found in the cytoplasm and function by directly reducing disulfides in the substrate proteins.
  • Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the reversible transfer of electrons from a substrate that becomes oxidized to a substrate that becomes reduced. This class of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases. Proper maintenance of oxidoreductase levels is physiologically important. For example, genetically-linked deficiencies in lipoamide dehydrogenase can result in lactic acidosis (Robinson, B. H. et al. (1977) Pediat. Res. 11:1198-1202).
  • Transferases transfer a chemical group from one compound (the donor) to another compound (the acceptor).
  • the types of groups transferred by these enzymes include acyl groups, amino groups, phosphate groups (phosphotransferases or phosphomutases), and others.
  • the transferase carnitine palrmtoyltransferase is an important component of fatty acid metabolism. Genetically-linked deficiencies in this transferase can lead to myopathy (Scriver, C. et al. (1995) The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York NY, pp.1501-1533).
  • Topoisomerases are enzymes that affect the topological state of DNA. For example, defects in topoisomerases or their regulation can affect normal physiology. Reduced levels of topoisomerase II have been co ⁇ elated with some of the DNA processing defects associated with the disorder ataxia-telangiectasia (Singh, S.P. et al. (1988) Nucleic Acids Res. 16:3919-3929).
  • Ligases catalyze the formation of a bond between two substrate molecules. The process involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. Ligases are classified based on the nature of the type of bond they form, which can include carbon-oxygen, carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds.
  • Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA (tRNA) synthetases which are important RNA-associated enzymes with roles in translation. Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and co ⁇ ect attachment of an amino acid with its cognate tRNA.
  • the 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding "Rossman fold".
  • Class U enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet motif, as well as N- and C- terminal regulatory domains.
  • Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack, (1995) J. Mol. Evol. 40:519-530).
  • Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and co ⁇ elate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.
  • Ligases forming carbon-sulfur bonds mediate a large number of cellular biosynthetic intermediary metabolism processes involving intermolecular transfer of carbon atom-containing substrates (carbon substrates). Examples of such reactions include the tricarboxylic acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis of intermediary metabolites, and reactions involved in the amino acid degradation pathways. Some of these reactions require input of energy, usually in the form of conversion of ATP to either ADP or AMP and pyrophosphate.
  • a carbon substrate is derived from a small molecule containing at least two carbon atoms.
  • the carbon substrate is often covalently bound to a larger molecule which acts as a carbon substrate carrier molecule within the cell.
  • the carrier molecule is coenzyme A.
  • Coenzyme A is structurally related to derivatives of the nucleotide ADP and consists of 4'-phosphopantetheine linked via a phosphodiester bond to the alpha phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of 4 -phosphopantetheine acts as the site for carbon substrate bond formation.
  • the predominant carbon substrates which utihze CoA as a carrier molecule during biosynthesis and intermediary metabolism in the cell are acetyl, succinyl, and propionyl moieties, collectively refe ⁇ ed to as acyl groups.
  • Other carbon substrates include enoyl lipid, which acts as a fatty acid oxidation intermediate, and carnitine, which acts as an acetyl-CoA flux regulator/mitochondrial acyl group transfer protein.
  • Acyl-CoA and acetyl-CoA are synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase, respectively.
  • acyl-CoA synthetase activity i) acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxylic acids and is found in muscle mitochondria and the cytosol of other tissues; ii) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms (predominantly from dietary sources), and is present only in liver mitochondria; and iii) acyl CoA synthetase, which is specific for long chain fatty acids with between six and twenty carbon atoms, and is found in microsomes and the mitochondria.
  • acyl-CoA synthetase activity Proteins associated with acyl-CoA synthetase activity have been identified from many sources including bacteria, yeast, plants, mouse, and man. The activity of acyl-Co A synthetase may be modulated by phosphorylation of the enzyme by cAMP-dependent protein kinase.
  • Ligases forming carbon-nitrogen bonds include amide synthases such as glutamine synthetase
  • glutamine-ammonia ligase that catalyzes the amination of glutamic acid to glutamine by ammonia using the energy of ATP hydrolysis.
  • Glutamine is the primary source for the amino group in various amide transfer reactions involved in de novo pyrimidine nucleotide synthesis and in purine and pyrimidine ribonucleotide interconversions.
  • Overexpression of glutamine synthetase has been observed in primary liver cancer (Christa, L. et al. (1994) Gastroent. 106:1312-1320).
  • Acid-amino-acid ligases are represented by the ubiquitin conjugating enzymes which are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria.
  • UCS ubiquitin conjugation system
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression.
  • proteins targeted for degradation are conjugated to ubiquitin (Ub), a small heat stable protein.
  • Ub is first activated by a ubiquitin-activating enzyme (El), and then transfe ⁇ ed to one of several Ub- conjugating enzymes (E2).
  • E2 then links the Ub molecule through its C-terminal glycine to an internal lysine (acceptor lysine) of a target protein.
  • the ubiquitinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease.
  • the UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell 79:13-21).
  • Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and enzyme complexes that participate in the de novo pathways of purine and pyrimidine biosynthesis. Because these pathways are critical to the synthesis of nucleotides for replication of both RNA and DNA, many of these enzymes have been the targets of clinical agents for the treatment of cell proliferative disorders such as cancer and infectious diseases.
  • Purine biosynthesis occurs de novo from the amino acids glycine and glutamine, and other small molecules.
  • Three of the key reactions in this process are catalyzed by a trifunctional enzyme composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (ATRS), and glycinamide ribonucleotide transformylase (GART).
  • GAS glycinamide-ribonucleotide synthetase
  • GART glycinamide ribonucleotide transformylase
  • Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that converts inosinic acid to adenylosuccinate, a key step on the path to ATP synthesis.
  • This enzyme is also similar to another carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar reaction in the urea cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
  • Adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase may be considered as a functional unit, the purine nucleotide cycle.
  • This cycle converts AMP to inosine monophosphate (IMP) and reconverts IMP to AMP via adenylosuccinate, thereby producing NH 3 and forming fumarate from aspartate.
  • IMP inosine monophosphate
  • the purine nucleotide cycle functions, during intense exercise, in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of ATP formation and by providing Krebs cycle intermediates.
  • the purine nucleotide cycle accounts for the release of NH 3 under normal acid-base conditions.
  • the purine nucleotide cycle may contribute to ATP recovery.
  • Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features (Van den Berghe, G. et al. (1992) Prog Neurobiol.: 39:547-561).
  • a marked imbalance in the enzymic pattern of purine metabolism is linked with transformation and/or progression in cancer cells.
  • de novo synthesis of the pyrimidine nucleotides uridylate and cytidylate also arises from a common precursor, in this instance the nucleotide orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
  • PPRP phosphoribosyl pyrophosphate
  • ATCase aspartate transcarbamylase
  • carbamyl phosphate synthetase ⁇ d ⁇ rydroorotase
  • DHOase d ⁇ rydroorotase
  • Cytidine nucleotides are derived from uridine-5 -triphosphate (UTP) by the amidation of UTP using glutamine as the amino donor and the enzyme CTP synthetase. Regulatory mutations in the human CTP synthetase are believed to confer multi-drug resistance to agents widely used in cancer therapy (Yamauchi, M. et al. (1990) EMBO J. 9:2095-2099).
  • Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA carboxylase and pyruvate carboxylase.
  • Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA from CO 2 and H 2 O using the energy of ATP hydrolysis.
  • Acetyl-CoA carboxylase is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. Two isoforms of acetyl-CoA carboxylase, types I and types IJ, are expressed in human in a tissue-specific manner (Ha, J. et al. (1994) Eur. J. Biochem. 219:297- 306). Pyruvate carboxylase is a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.
  • Ligases forming phosphoric ester bonds include the DNA ligases involved in both DNA replication and repair.
  • DNA hgases seal phosphodiester bonds between two adjacent nucleotides in a DNA chain using the energy from ATP hydrolysis to first activate the free 5 -phosphate of one nucleotide and then react it with the 3 -OH group of the adjacent nucleotide.
  • This resealing reaction is used in DNA replication to join small DNA fragments called "Okazaki" fragments that are transiently formed in the process of replicating new DNA, and in DNA repair.
  • DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA, are co ⁇ ected before replication or transcription of the DNA can occur.
  • Bloom's syndrome is an inherited human disease in which individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts et al, supra, p. 247).
  • Pantothenate synthetase (D-pantoate; beta-alanine ligase (AlVff-forming); EC 6.3.2.1) is the last enzyme of the pathway of pantothenate (vitamin B(5)) synthesis. It catalyzes the condensation of pantoate with beta-alanine in an ATP-dependent reaction.
  • the enzyme is dimeric, with two well-defined domains per protomer: the N-terminal domain, a Rossmann fold, contains the active site cavity, with the C-terminal domain forming a hinged lid.
  • N-te ⁇ ninal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily (von Delft, F. et al. (2000) Structure (Camb) 9:439-450).
  • Farnesyl diphosphate synthase is an essential enzyme that is required both for cholesterol synthesis and protein prenylation.
  • the enzyme catalyzes the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentyl diphosphate.
  • FPPS is inhibited by nitrogen- containing biphosphonates, which can lead to the inhibition of osteoclast-mediated bone resorption by preventing protein prenylation (Dunford, J.E. et al. (2001) J. Pharmacol. Exp. Ther. 296:235-242).
  • 5-aminolevulinate synthase (ALAS; delta-aminolevuhnate synthase; EC 2.3.1.37) catalyzes the rate-limiting step in heme biosynthesis in both erythroid and non-erythroid tissues.
  • This enzyme is unique in the heme biosynthetic pathway in being encoded by two genes, the first encoding ALAS1, the non-erythroid specific enzyme which is ubiquitously expressed, and the second encoding ALAS2, which is expressed exclusively in erythroid cells.
  • the genes for ALAS1 and ALAS2 are located, respectively, on chromosome 3 and on the X chromosome. Defects in the gene encoding ALAS2 result in X-linked sideroblastic anemia. Elevated levels of ALAS are seen in acute hepatic porphyrias and can be lowered by zinc mesoporphyrin.
  • DMEs Drug Metabolizing Enzymes
  • the metabolism of a drug and its movement through the body are important in determining its effects, toxicity, and interactions with other drugs.
  • the three processes governing pharmacokinetics are the absorption of the drug, distribution to various tissues, and elimination of drug metabolites. These processes are intimately coupled to drug metabolism, since a variety of metabolic modifications alter most of the physicochemical and pharmacological properties of drugs, including solubikty, binding to receptors, and excretion rates.
  • the metabolic pathways which modify drugs also accept a variety of naturally occurring substrates such as steroids, fatty acids, prostaglandins, leukotrienes, and vitamins.
  • DMEs have broad substrate specificities, unlike antibodies, for example, which are diverse and highly specific. Since DMEs metabolize a wide variety of molecules, drug interactions may occur at the level of metabolism so that, for example, one compound may induce a DME that affects the metaboHsm of another compound.
  • Phase I reaction products are partially or fully inactive, and Phase U reaction products are the chief excreted species.
  • Phase I reaction products are sometimes more active than the original administered drugs; this metabolic activation principle is exploited by pro-drugs (e.g. L-dopa).
  • pro-drugs e.g. L-dopa
  • nontoxic compounds e.g. aflatoxin, benzo[ ⁇ ]pyrene
  • Phase I reactions are usually rate-limiting in drug metabolism. Prior exposure to the compound, or other compounds, can induce the expression of Phase I enzymes however, and thereby increase substrate flux through the metabolic pathways. (See Klaassen, CD.
  • Phase I enzymes include, but are not limited to, cytochrome P450 and flavin-containing monooxygenase.
  • Phase I-type catalytic cycles and reactions include, but are not limited to, NADPH cytochrome P450 reductase (CPR), the microsomal cytochrome b5/NADH cytochrome b5 reductase system, the fe ⁇ edoxin/ferredoxin reductase redox pair, aldo/keto reductases, and alcohol dehydrogenases.
  • CPR NADPH cytochrome P450 reductase
  • the major classes of Phase II enzymes include, but are not limited to, UDP glucuronyltransferase, sulfotransferase, glutathione S-transferase, N-acyltransferase, and N-acetyl transferase.
  • Cytochrome P450 and P450 catalytic cycle-associated enzymes include, but are not limited to, NADPH cytochrome P450 reductase (CPR), the microsomal cytochrome
  • Cytochromes P450 also known as P450 heme-thiolate proteins, usually act as terminal oxidases in multi-component electron transfer chains, called P450-containing monooxygenase systems.
  • Specific reactions catalyzed include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups. These reactions are involved in steroidogenesis of glucocorticoids, cortisols, estrogens, and androgens in animals; insecticide resistance in insects; herbicide resistance and flower coloring in plants; and environmental bioremediation by microorganisms. Cytochrome P450 actions on drugs, carcinogens, mutagens, and xenobiotics can result in detoxification or in conversion of the substance to a more toxic product.
  • Cytochromes P450 are abundant in the liver, but also occur in other tissues; the enzymes are located in microsomes. (See ExPASY ENZYME EC 1.14.14.1 ; Prosite PDOC00081 Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class P450 Group I signature; Graham- Lorence, S. and J.A. Peterson (1996) FASEB J. 10:206-214.)
  • cytochromes P450 have been identified in diverse organisms including bacteria, fungi, plants, and animals (Graham-Lorence and Peterson, supra). The B-class is found in prokaryotes and fungi, while the E-class is found in bacteria, plants, insects, vertebrates, and mammals. Five subclasses or groups are found within the larger family of E-class cytochromes P450 (PRINTS EP450I E-Class P450 Group I signature).
  • cytochromes P450 use a heme cofactor and share structural attributes. Most cytochromes P450 are 400 to 530 amino acids in length. The secondary structure of the enzyme is about 70% alpha-helical and about 22% beta-sheet. The region around the heme-binding site in the C- terminal part of the protein is conserved among cytochromes P450. A ten amino acid signature sequence in this heme-iron ligand region has been identified which includes a conserved cysteine involved in binding the heme iron in the fifth coordination site. In eukaryotic cytochromes P450, a membrane-spanning region is usually found in the first 15-20 amino acids of the protein, generally consisting of approximately 15 hydrophobic residues followed by a positively charged residue. (See Prosite PDOC00081, supra; Graham-Lorence and Peterson, supra.)
  • Cytochrome P450 enzymes are involved in cell proliferation and development.
  • the enzymes have roles in chemical mutagenesis and carcinogenesis by metabolizing chemicals to reactive intermediates that form adducts with DNA (Nebert, D.W. and F.J. Gonzalez (1987) Ann. Rev.
  • Cytochrome P450 expression in liver and other tissues is induced by xenobiotics such as polycyclic aromatic hydrocarbons, peroxisomal proliferators, phenobarbital, and the glucocorticoid dexamethasone (Dogra, S.C et al. (1998) Clin. Exp. Pharmacol. Physiol. 25:1-9).
  • a cytochrome P450 protein may participate in eye development as mutations in the P450 gene CYPIB 1 cause primary congenital glaucoma (OMTM #601771 Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYPIB 1).
  • Cytochromes P450 are associated with inflammation and infection. Hepatic cytochrome P450 activities are profoundly affected by various infections and inflammatory stimuli, some of which are suppressed and some induced (Morgan, E.T. (1997) Drug Metab. Rev. 29:1129-1188). Effects observed in vivo can be mimicked by proinflammatory cytokines and interferons. Autoantibodies to two cytochrome P450 proteins were found in patients with autoimmune polyenodocrinopathy- candidiasis-ectodermal dystrophy (APECED), a polyglandular autoimmune syndrome (OMTM #240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy).
  • APECED autoimmune polyenodocrinopathy- candidiasis-ectodermal dystrophy
  • OMTM #240300 Autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy autoimmune polyenodocr
  • cytochromes P450 have been linked to metabolic disorders, including congenital adrenal hyperplasia, the most common adrenal disorder of infancy and childhood; pseudovitamin D- deficiency rickets; cerebrotendinous xanthomatosis, a lipid storage disease characterized by progressive neurologic dysfunction, premature atherosclerosis, and cataracts; and an inherited resistance to the anticoagulant drugs coumarin and warfarin (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, Inc. New York, NY, pp. 1968-1970; Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka, S. et al. (1998) N.
  • the cytochrome P450 catalytic cycle is completed through reduction of cytochrome P450 by NADPH cytochrome P450 reductase (CPR).
  • CPR NADPH cytochrome P450 reductase
  • Another microsomal electron transport system consisting of cytochrome b5 and NADPH cytochrome b5 reductase has been widely viewed as a minor contributor of electrons to the cytochrome P450 catalytic cycle.
  • CYP51 Candida albicans cytochrome P450
  • CYP51 Candida albicans cytochrome P450
  • Cytochrome b5 reductase is also responsible for the reduction of oxidized hemoglobin (methemoglobin, or ferrihemoglobin, which is unable to cany oxygen) to the active hemoglobin (fe ⁇ ohemoglobin) in red blood cells.
  • Methemoglobinemia results when there is a high level of oxidant drugs or an abnormal hemoglobin (hemoglobin M) which is not efficiently reduced.
  • Methemoglobinemia can also result from a hereditary deficiency in red cell cytochrome b5 reductase (Reviewed in Mansour, A. and A.A. Lurie (1993) Am. J. Hematol. 42:7-12).
  • Vitamin D exists as two biologically equivalent prohormones, ergocalciferol (vitamin D 2 ), produced in plant tissues, and cholecalciferol (vitamin D 3 ), produced in animal tissues.
  • ergocalciferol vitamin D 2
  • cholecalciferol vitamin D 3
  • the latter form, cholecalciferol is formed upon the exposure of 7-dehydrocholesterol to near ultraviolet light (i.e., 290-310 nm), normally resulting from even minimal periods of skin exposure to sunlight (reviewed in Miller, W.L and A.A. Portale (2000) Trends Endocrinol. Metab. 11:315-319).
  • Both prohormone forms are further metabolized in the liver to 25-hydroxyvitamin D (25(OH)D) by the enzyme 25-hydroxylase.
  • 25(OH)D is the most abundant precursor form of vitamin D which must be further metabolized in the kidney to the active form, l ⁇ ,25-dmydroxyvitamin D (l ⁇ ,25(OH) 2 D), by the enzyme 25-hydroxyvitamin D l ⁇ -hydroxylase (l ⁇ -hydroxylase). Regulation of l ⁇ ,25(OH) 2 D production is primarily at this final step in the synthetic pathway.
  • l ⁇ -hydroxylase depends upon several physiological factors including the circulating level of the enzyme product (l ⁇ ,25(OH) 2 D) and the levels of parathyroid hormone (PTH), calcitonin, insulin, calcium, phosphorus, growth hormone, and prolactin. Furthermore, extrarenal l ⁇ -hydroxylase activity has been reported, suggesting that tissue-specific, local regulation of l ⁇ ,25(OH)2D production may also be biologically important.
  • 24-hydroxylase can also use 25(OH)D as a substrate (Shinki, T. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12920-12925; Miller and Portale, supra; and references within).
  • Vitamin D 25-hydroxylase, l ⁇ -hydroxylase, and 24-hydroxylase are all NADPH-dependent, type I (mitochondrial) cytochrome P450 enzymes that show a high degree of homology with other members of the family. Vitamin D 25-hydroxylase also shows a broad substrate specificity and may also perform 26-hydroxylation of bile acid intermediates and 25, 26, and 27 -hydroxylation of cholesterol (Dilworth, F.J. et al. (1995) J. Biol. Chem. 270:16766-16774; Miller and Portale, supra; and references within).
  • vitamin D The active form of vitamin D (l ⁇ ,25(OH) 2 D) is involved in calcium and phosphate homeostasis and promotes the differentiation of myeloid and skin cells.
  • Vitamin D deficiency resulting from deficiencies in the enzymes involved in vitamin D metabolism causes hypocalcemia, hypophosphatemia, and vitamin D-dependent (sensitive) rickets, a disease characterized by loss of bone density and distinctive clinical features, including bandy or bow leggedness accompanied by a waddling gait.
  • vitamin D 25-hydroxylase a lipid-storage disease characterized by the deposition of cholesterol and cholestanol in the Achilles' tendons, brain, lungs, and many other tissues. The disease presents with progressive neurologic dysfunction, including postpubescent cerebellar ataxia, atherosclerosis, and cataracts. Vitamin D 25-hydroxylase deficiency does not result in rickets, suggesting the existence of alternative pathways for the synthesis of 25(OH)D (Griffin, J.E. and J.E. Zerwekh (1983) J. Oin. Invest. 72:1190-1199; Gamblin, G.T. et al. (1985) J. Clin. Invest. 75:954-960; and Miller and Portale, supra).
  • Fe ⁇ edoxin and fe ⁇ edoxin reductase are electron transport accessory proteins which support at least one human cytochrome P450 species, cytochrome P450c27 encoded by the CYP27 gene (Dilworth, F.J. et al. (1996) Biochem. J. 320:267-71).
  • a Streptomyces griseus cytochrome P450, CYP104D1 was heterologously expressed in Escherichia coli and found to be reduced by the endogenous fe ⁇ edoxin and fe ⁇ edoxin reductase enzymes (Taylor, M. et al. (1999) Biochem. Biophys. Res. Commun.
  • Flavin-containing monooxygenases oxidize the nucleophilic nitrogen, sulfur, and phosphorus heteroatom of an exceptional range of substrates.
  • FMOs are microsomal and use NADPH and O 2 ; there is also a great deal of substrate overlap with cytochromes P450.
  • the tissue distribution of FMOs includes liver, kidney, and lung.
  • Isoforms of FMO in mammals include FMOl, FMO2, FMO3, FMO4, and FMO5, which are expressed in a tissue-specific manner.
  • the isoforms differ in their substrate specificities and properties such as inhibition by various compounds and stereospecificity of reaction.
  • FMOs have a 13 amino acid signature sequence, the components of which span the N-terminal two-thirds of the sequences and include the FAD binding region and the FATGY motif found in many N-hydroxylating enzymes (Stehr, M. et al. (1998) Trends Biochem. Sci. 23:56-57; PRINTS FMOXYGENASE Flavin- containing monooxygenase signature).
  • Specific reactions include oxidation of nucleophilic tertiary amines to N-oxides, secondary amines to hydroxylamines and nitrones, primary amines to hydroxylamines and oximes, and sulfur-containing compounds and phosphines to S- and P-oxides. Hydrazines, iodides, selenides, and boron-containing compounds are also substrates. FMOs are more heat labile and less detergent-sensitive than cytochromes P450 in vitro though FMO isoforms vary in thermal stability and detergent sensitivity.
  • FMOs play important roles in the metabolism of several drugs and xenobiotics.
  • FMO FMO3 in liver
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S metabolizing
  • S non-oxidized
  • cimetidine an H 2 -antagonist widely used for the treatment of gastric ulcers.
  • Liver-expressed forms of FMO are not under the same regulatory control as cytochrome P450.
  • phenobarbital treatment leads to the induction of cytochrome P450, but the repression of FMO1.
  • Lysyl oxidase (lysine 6-oxidase, LO) is a copper-dependent amine oxidase involved in the formation of connective tissue matrices by crosslinking collagen and elastin.
  • LO is secreted as an N- glycosylated precursor protein of approximately 50 kDa and cleaved to the mature form of the enzyme by a metalloprotease, although the precursor form is also active.
  • the copper atom in LO is involved in the transport of electrons to and from oxygen to facilitate the oxidative deamination of lysine residues in these extracellular matrix proteins. While the coordination of copper is essential to LO activity, insufficient dietary intake of copper does not influence the expression of the apoenzyme.
  • LO activity is increased in response to ozone, cadmium, and elevated levels of hormones released in response to local tissue trauma, such as transforming growth factor-beta, platelet-derived growth factor, angiotensin II, and fibroblast growth factor. Abnormalities in LO activity have been linked to Menkes syndrome and occipital horn syndrome.
  • DHFR Dihydrofolate reductases
  • the enzymes can be inhibited by a number of dihydrofolate analogs, including trimethroprim and methotrexate. Since an abundance of dTMP is required for DNA synthesis, rapidly dividing cells require the activity of DHFR.
  • the replication of DNA viruses i.e., herpesvirus
  • drugs that target DHFR have been used for cancer chemotherapy and to inhibit DNA virus replication.
  • thymidylate synthetases are also target enzymes.
  • Drugs that inhibit DHFR are preferentially cytotoxic for rapidly dividing cells (or DNA virus-infected cells) but have no specificity, resulting in the ⁇ discriminate destruction of dividing cells.
  • cancer cells may become resistant to drugs such as methotrexate as a result of acquired transport defects or the duplication of one or more DHFR genes (Stryer, L. (1988) Biochemistry. W.H Freeman and Co., Inc. New York. pp. 511-519).
  • drugs such as methotrexate as a result of acquired transport defects or the duplication of one or more DHFR genes (Stryer, L. (1988) Biochemistry. W.H Freeman and Co., Inc. New York. pp. 511-519).
  • Aldo/keto reductases are monomeric NADPH-dependent oxidoreductases with broad substrate specificities (Bohren, K.M. et al. (1989) J. Biol. Chem. 264:9547-9551). These enzymes catalyze the reduction of carbonyl-containing compounds, including carbonyl-containing sugars and aromatic compounds, to the co ⁇ esponding alcohols. Therefore, a variety of carbonyl-containing drugs and xenobiotics are likely metabolized by enzymes of this class.
  • aldose reductase One known reaction catalyzed by a family member, aldose reductase, is the reduction of glucose to sorbitol, which is then further metabolized to fructose by sorbitol dehydrogenase. Under normal conditions, the reduction of glucose to sorbitol is a minor pathway. In hyperglycemic states, however, the accumulation of sorbitol is impUcated in the development of diabetic complications (OMTM #103880 Aldo-keto reductase family 1, member Bl). Members of this enzyme family are also highly expressed in some liver cancers (Cao, D. et al. (1998) J. Biol. Chem. 273:11429-11435). Alcohol dehydrogenases
  • Alcohol dehydrogenases oxidize simple alcohols to the co ⁇ esponding aldehydes.
  • ADH is a cytosolic enzyme, prefers the cofactor NAD + , and also binds zinc ion.
  • Liver contains the highest levels of ADH, with lower levels in kidney, lung, and the gastric mucosa.
  • Known ADH isoforms are dimeric proteins composed of 40 kDa subunits. There are five known gene loci which encode these subunits (a, b, g, p, c), and some of the loci have characterized allelic variants (b j , b 2 , b 3 , g g 2 ).
  • the subunits can form homodimers and heterodimers; the subunit composition determines the specific properties of the active enzyme.
  • the holoenzymes have therefore been categorized as Class I (subunit compositions aa, ab, ag, bg, gg), Class U (pp), and Class HI (cc).
  • Class I ADH isozymes oxidize ethanol and other small aliphatic alcohols, and are inhibited by pyrazote.
  • Class II isozymes prefer longer chain aliphatic and aromatic alcohols, are unable to oxidize methanol, and are not inhibited by pyrazote.
  • Class JJI isozymes prefer even longer chain ahphatic alcohols (five carbons and longer) and aromatic alcohols, and are not inhibited by pyrazote.
  • the short-chain alcohol dehydrogenases include a number of related enzymes with a variety of substrate specificities. Included in this group are the mammahan enzymes D-beta-hydroxybutyrate dehydrogenase, (R)-3-hydroxybutyrate dehydrogenase, 15-hydroxyprostaglandin dehydrogenase, NADPH-dependent carbonyl reductase, corticosteroid 11-beta-dehydrogenase, and estradiol 17-beta- dehydrogenase, as well as the bacterial enzymes acetoacetyl-CoA reductase, glucose 1- dehydrogenase, 3-beta-hydroxysteroid dehydrogenase, 20-beta-hydroxysteroid dehydrogenase, ribitol dehydrogenase, 3-oxoacyl reductase, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, sorbito
  • Sulfate conjugation occurs on many of the same substrates which undergo O-glucuronidation to produce a highly water-soluble sulfuric acid ester.
  • Sulfotransferases catalyze this reaction by transferring SO 3 " from the cofactor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the substrate.
  • ST substrates are predominantly phenols and ahphatic alcohols, but also include aromatic amines and aliphatic amines, which are conjugated to produce the co ⁇ esponding sulfamates. The products of these reactions are excreted mainly in urine.
  • STs are found in a wide range of tissues, including liver, kidney, intestinal tract, lung, platelets, and brain.
  • the enzymes are generally cytosolic, and multiple forms are often co-expressed. For example, there are more than a dozen forms of ST in rat liver cytosol.
  • These biochemically characterized STs fall into five classes based on their substrate preference: arylsulfotransferase, alcohol sulfotransferase, estrogen sulfotransferase, tyrosine ester sulfotransferase, and bile salt sulfotransferase.
  • ST enzyme activity varies greatly with sex and age in rats. The combined effects of developmental cues and sex-related hormones are thought to lead to these differences in ST expression profiles, as well as the profiles of other DMEs such as cytochromes P450. Notably, the high expression of STs in cats partially compensates for their low level of UDP glucuronyltransferase activity.
  • thermostable enzyme catalyzes the sulfation of phenols such as para-nitrophenol, minoxidil, and acetaminophen; the thermolabile enzyme prefers monoamine substrates such as dopamine, epinephrine, and levadopa.
  • STs include an estrogen sulfotransferase and an N-acetylglucosamine-6-O- sulfotransferase.
  • This last enzyme is illustrative of the other major role of STs in cellular biochemistry, the modification of carbohydrate structures that may be important in cellular differentiation and maturation of proteoglycans.
  • an inherited defect in a sulfotransferase has been impUcated in macular corneal dystrophy, a disorder characterized by a failure to synthesize mature keratan sulfate proteoglycans (Nakazawa, K. et al. (1984) J. Biol. Chem. 259:13751-13757; OMTM #217800 Macular dystrophy, corneal).
  • Galactosyltransferases are a subset of glycosyltransferases that transfer galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) oUgosaccharide chains that are part of glycoproteins or glycoUpids that are free in solution (Kolbinger, F. et al. (1998) J. Biol. Chem. 273:433-440; Amado, M. et al. (1999) Biochim. Biophys. Acta 1473:35-53). Galactosyltransferases have been detected on the cell surface and as soluble extracellular proteins, in addition to being present in the Golgi.
  • ⁇ l,3- galactosyltransferases form Type I carbohydrate chains with Gal ( ⁇ l-3)GlcNAc linkages.
  • Known human and mouse ⁇ 1,3 -galactosyltransferases appear to have a short cytosoUc domain, a single transmembrane domain, and a catalytic domain with eight conserved regions. (Kolbinger et al., supra; and Hennet, T. et al. (1998) J. Biol. Chem. 273:58-65).
  • region 1 is located at amino acid residues 78-83, region 2 is located at amino acid residues 93-102, region 3 is located at amino acid residues 116-119, region 4 is located at amino acid residues 147-158, region 5 is located at amino acid residues 172-183, region 6 is located at amino acid residues 203-206, region 7 is located at amino acid residues 236-246, and region 8 is located at amino acid residues 264-275.
  • a variant of a sequence found within mouse UDP-galactose: ⁇ -N-acetylglucosamine ⁇ l,3-galactosyltransferase-I region 8 is also found in bacterial galactosyltransferases, suggesting that this sequence defines a galactosyltransferase sequence motif (Hennet et al., supra). Recent work suggests that brainiac protein is a ⁇ 1,3 -galactosyltransferase (Yuan, Y. et al. (1997) CeU 88:9-11; and Hennet et al., supra).
  • UDP-Gal:GlcNAc-l,4-galactosyltransferase (-1,4-GalT) (Sato, T. et al., (1997) EMBO J. 16:1850-1857) catalyzes the formation of Type JJ carbohydrate chains with Gal ( ⁇ l-4)GlcNAc linkages.
  • a soluble form of the enzyme is formed by cleavage of the membrane-bound form.
  • Amino acids conserved among ⁇ l,4- galactosyltransferases include two cysteines linked through a disulfide-bond and a putative UDP- galactose-binding site in the catalytic domain (Yadav, S. and K. Brew (1990) J. Biol. Chem. 265:14163-14169; Yadav, S.P. and K. Brew (1991) J. Biol. Chem. 266:698-703; and Shaper, N.L. et al. (1997) J. Biol. Chem. 272:31389-31399).
  • ⁇ l,4-galactosyltransferases have several speciaUzed roles in addition to synthesizing carbohydrate chains on glycoproteins or glycohpids.
  • mammals a ⁇ l,4-galactosyltransferase, as part of a heterodimer with ⁇ -lactalbumin, functions in lactating mammary gland lactose production.
  • a ⁇ l,4-galactosyltransferase on the surface of sperm functions as a receptor that specificaUy recognizes the egg.
  • CeU surface ⁇ l,4-galactosyltransferases also function in ceU adhesion, cell/basal lamina interaction, and normal and metastatic ceU migration. (Shur, B. (1993) Cu ⁇ . Opin. CeU Biol. 5:854-863; and Shaper, J. (1995) Adv. Exp. Med. Biol. 376:95-104).
  • Gamma-glutamyl transpeptidases are ubiquitously expressed enzymes that initiate extracehular glutathione (GSH) breakdown by cleaving gamma-glutamyl amide bonds. The breakdown of GSH provides ceUs with a regional cysteine pool for biosynthetic pathways. Gamma-glutamyl transpeptidases also contribute to cellular antioxidant defenses and expression is induced by oxidative stress. The cell surface-locaUzed glycoproteins.are expressed at high levels in cancer ceUs.
  • Aminotransferases comprise a family of pyridoxal 5 -phosphate (PLP) -dependent enzymes that catalyze transformations of amino acids. Aspartate aminotransferase (AspAT) is the most extensively studied PLP-containing enzyme. It catalyzes the reversible transamination of dicarboxyUc L-amino acids, aspartate and glutamate, and the co ⁇ esponding 2-oxo acids, oxalacetate and 2-oxoglutarate.
  • PLP pyridoxal 5 -phosphate
  • pyruvate aminotransferase branched-chain amino acid aminotransferase, tyrosine aminotransferase, aromatic aminotransferase, alanine:glyoxylate aminotransferase (AGT), and kynurenine aminotransferase (Vacca, R.A. et al. (1997) J. Biol. Chem. 272:21932-21937).
  • Primary hyperoxaluria type-1 is an autosomal recessive disorder resulting in a deficiency in the Uver-specific peroxisomal enzyme, alanine:glyoxylate aminotransferase- 1.
  • the phenotype of the disorder is a deficiency in glyoxylate metaboUsm.
  • glyoxylate is oxidized to oxalate rather than being transaminated to glycine.
  • the result is the deposition of insoluble calcium oxalate in the kidneys and urinary tract, ultimately causing renal failure (Lumb, M.J. et al. (1999) J. Biol. Chem. 274:20587-20596).
  • Kynurenine aminotransferase catalyzes the i ⁇ eversible transamination of the L-tryptophan metaboUte L-kynurenine to form kynurenic acid.
  • the enzyme may also catalyze the reversible transamination reaction between L-2-aminoadipate and 2-oxoglutarate to produce 2-oxoadipate and L-glutamate.
  • Kynurenic acid is a putative modulator of glutamatergic neurotransmission; thus a deficiency in kynurenine aminotransferase may be associated with pleotrophic effects (BuchU, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
  • Catechol-0-methyltransferase catalyzes the transfer of the methyl group of S- adenosyl-L-methionine (AdoMet; SAM) donor to one of the hydroxyl groups of the catechol substrate (e.g., L-dopa, dopamine, or DBA). Methylation of the 3 -hydroxyl group is favored over methylation of the 4 -hydroxyl group and the membrane bound isoform of COMT is more regiospecific than the soluble form.
  • SAM S- adenosyl-L-methionine
  • Translation of the soluble form of the enzyme results from utiUzation of an internal start codon in a full-length mRNA (1.5 kb) or from the translation of a shorter mRNA (1.3 kb), transcribed from an internal promoter.
  • the proposed S N 2-like methylation reaction requires Mg ++ and is inhibited by Ca ++ .
  • the binding of the donor and substrate to COMT occurs sequentially.
  • AdoMet first binds COMT in a Mg ++ -independent manner, foUowed by the binding of Mg ++ and the binding of the catechol substrate.
  • inhibitors have been developed for in vitro use (e.g., gaUates, tropolone, U-0521, and 3 ',4 -dihydroxy-2-methyl-propiophetropolone) and for clinical use (e.g., nitrocatechol-based compounds and tolcapone). Administration of these inhibitors results in the increased half-Ufe of L-dopa and the consequent formation of dopamine.
  • Inhibition of COMT is also likely to increase the half-Hfe of various other catechol-structure compounds, including but not Umited to epinephrine/norepinephrine, isoprenaline, rimiterol, dobutarnine, fenoldopam, apomorphine, and ⁇ - methyldopa.
  • a deficiency in norepinephrine has been Unked to clinical depression, hence the use of COMT inhibitors could be usefuU in the treatment of depression.
  • COMT inhibitors are generaUy weU tolerated with rninimal side effects and are ultimately metaboUzed in the Uver with only minor accumulation of metaboUtes in the body (Mannist ⁇ , P.T. and S. Kaakkola (1999) Pharmacol. Rev. 51:593-628).
  • Copper-zinc superoxide dismutases are compact homodimeric metalloenzymes involved in ceUular defenses against oxidative damage.
  • the enzymes contain one atom of zinc and one atom of copper per subunit and catalyze the dismutation of superoxide anions into O 2 and The rate of dismutation is diffusion-limited and consequently enhanced by the presence of favorable electrostatic interactions between the substrate and enzyme active site. Examples of this class of enzyme have been identified in the cytoplasm of aU the eukaryotic ceUs as weU as in the periplasm of several bacterial species.
  • Copper-zinc superoxide dismutases are robust enzymes that are highly resistant to proteolytic digestion and denaturing by urea and SDS.
  • the presence of the metal ions and intrasubunit disulfide bonds is beUeved to be responsible for enzyme stabiUty.
  • the enzymes undergo reversible denaturation at temperatures as high as 70 °C (Battistoni, A. et al. (1998) J. Biol. Chem. 273:5655-5661).
  • M. tuberculosis expresses almost two orders of magnitude more superoxide dismutase than the nonpathogenic mycobacterium M. smegmatis, and secretes a much higher proportion of the expressed enzyme. The result is the secretion of -350-fold more enzyme by M. tuberculosis than M. smegmatis, providing substantial resistance to oxidative stress (Harth, G. and M.A. Horwitz (1999) J. Biol. Chem. 274:4281-4292).
  • Phosphotriesterases are enzymes that hydrolyze toxic organophosphorus compounds and have been isolated from a variety of tissues. Phosphotriesterases play a central role in the detoxification of insecticides by mammals. Birds and insects lack PTE, and as a result have reduced tolerance for organophosphorus compounds (Vilanova, E. and M.A. Sogorb (1999) Crit. Rev. Toxicol. 29:21-57). Phosphotriesterase activity varies among individuals and is lower in infants than adults. PTE knockout mice are markedly more sensitive to the organophosphate-based toxins diazoxon and chlorpyrifos oxon (Furlong, C.E., et al.
  • Glycerophosphoryl diester phosphodiesterase (also known as glycerophosphodiester phosphodiesterase) is a phosphodiesterase which hydrolyzes deacetylated phosphoUpid glycerophosphodiesters to produce sn-glycerol-3 -phosphate and an alcohol.
  • GlycerophosphochoUne, glycerophosphoethanolamine, glycerophosphoglycerol, and glycerophosphoinositol are examples of substrates for glycerophosphoryl diester phosphodiesterases.
  • a glycerophosphoryl diester phosphodiesterase from E. coli has broad specificity for glycerophosphodiester substrates (Larson, TJ. et al. (1983) J. Biol. Chem. 248:5428-5432).
  • CycUc nucleotide phosphodiesterases are crucial enzymes in the regulation of the cycUc nucleotides cAMP and cGMP.
  • cAMP and cGMP function as intracellular second messengers to transduce a variety of extraceUular signals including hormones, Ught, and neurotransmitters.
  • PDEs degrade cycUc nucleotides to their co ⁇ esponding monophosphates, thereby regulating the intracellular concentrations of cycUc nucleotides and their effects on signal transduction. Due to their roles as regulators of signal transduction, PDEs have been extensively studied as chemotherapeutic targets (Perry, M.J. and G.A. Higgs (1998) Cu ⁇ . Opin. Chem. Biol. 2:472-481; Torphy, J.T. (1998) Am. J. Resp. Crit. Care Med. 157:351-370).
  • Famines of mammaUan PDEs have been classified based on their substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory agents (Beavo, J.A. (1995) Physiol. Rev. 75:725-748; Conti, M. et al. (1995) Endocrine Rev. 16:370-389).
  • Several of these famiUes contain distinct genes, many of which are expressed in different tissues as spUce variants.
  • Within PDE famiUes there are multiple isozymes and multiple spUce variants of these isozymes (Conti, M. and S.- L.C Jin (1999) Prog. Nucleic Acid Res. Mol. Biol. 63:1-38).
  • Type 1 PDEs are Ca 2+ /calmoduUn-dependent and appear to be encoded by at least three different genes, each having at least two different spUce variants (Kakkar, R. et al. (1999) CeU Mol. Life Sci. 55:1164-1186).
  • PDEls have been found in the lung, heart, and brain.
  • Some PDEl isozymes are regulated in vitro by phosphorylation/dephosphorylation. Phosphorylation of these PDE1 isozymes decreases the affinity of the enzyme for calmodulin, decreases PDE activity, and increases steady state levels of cAMP (Kakkar et al., supra).
  • PDEls may provide useful therapeutic targets for disorders of the central nervous system and the cardiovascular and immune systems, due to the involvement of PDEls in both cycUc nucleotide and calcium signaling (Perry and Higgs, supra).
  • PDE2s are cGMP-stimulated PDEs that have been found in the cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle (Sadhu, K. et al. (1999) J. Histochem. Cytochem. 47:895-906).
  • PDE2s are thought to mediate the effects of cAMP on catecholamine secretion, participate in the regulation of aldosterone (Beavo, supra), and play a role in olfactory signal transduction (Juilfs, D.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:3388-3395).
  • PDE3s have high affinity for both cGMP and cAMP, and so these cycUc nucleotides act as competitive substrates for PDE3s.
  • PDE3s play roles in stimulating myocardial contractiUty, inhibiting platelet aggregation, relaxing vascular and airway smooth muscle, inhibiting proUferation of T- lymphocytes and cultured vascular smooth muscle cells, and regulating catecholamine-induced release of free fatty acids from adipose tissue.
  • the PDE3 family of phosphodiesterases are sensitive to specific inhibitors such as cilostamide, enoximone, and Uxazinone. Isozymes of PDE3 can be regulated by cAMP-dependent protein kinase, or by insulin-dependent kinases (Degerman, E. et al. (1997) J. Biol. Chem. 272:6823-6826).
  • PDE4s are specific for cAMP; are locaUzed to airway smooth muscle, the vascular endotheUum, and aU inflammatory ceUs; and can be activated by cAMP-dependent phosphorylation. Since elevation of cAMP levels can lead to suppression of inflammatory ceU activation and to relaxation of bronchial smooth muscle, PDE4s have been studied extensively as possible targets for novel anti-inflammatory agents, with special emphasis placed on the discovery of asthma treatments. PDE4 inhibitors are cu ⁇ ently undergoing clinical trials as treatments for asthma, chronic obstructive pulmonary disease, and atopic eczema.
  • AU four known isozymes of PDE4 are susceptible to the inhibitor roUpram, a compound which has been shown to improve behavioral memory in mice (Barad, M. et al. (1998) Proc. Natl. Acad. Sci. USA 95:15020-15025).
  • PDE4 inhibitors have also been studied as possible therapeutic agents against acute lung injury, endotoxemia, rheumatoid arthritis, multiple sclerosis, and various neurological and gastrointestinal indications (Doherty, A.M. (1999) Cu ⁇ . Opin. Chem. Biol. 3:466-473).
  • PDE5 is highly selective for cGMP as a substrate (Turko, IN. et al. (1998) Biochemistry 37:4200-4205), and has two aUosteric cGMP-specific binding sites (McAUister-Lucas, L.M. et al. (1995) J. Biol. Chem. 270:30671-30679). Binding of cGMP to these allosteric binding sites seems to be important for phosphorylation of PDE5 by cGMP-dependent protein kinase rather than for direct regulation of catalytic activity. High levels of PDE5 are found in vascular smooth muscle, platelets, lung, and kidney. The inhibitor zaprinast is effective against PDE5 and PDEls.
  • PDE6s the photoreceptor cycUc nucleotide phosphodiesterases
  • PDE6s hydrolyze cGMP to regulate cGMP-gated cation channels in photoreceptor membranes.
  • PDE6s also have two Ingh-affinity cGMP-binding sites which are thought to play a regulatory role in PDE6 function (Artemyev, N.O. et al. (1998) Methods 14:93-104). Defects in PDE6s have been associated with retinal disease. Retinal degeneration in the rd mouse (Yan, W.
  • the PDE7 family of PDEs consists of only one known member having multiple spUce variants (Bloom, TJ. and J.A. Beavo (1996) Proc. Natl. Acad. Sci. USA 93:14188-14192).
  • PDE7s are cAMP specific, but Uttle else is known about their physiological function.
  • mRNAs encoding PDE7s are found in skeletal muscle, heart, brain, lung, kidney, and pancreas, expression of PDE7 proteins is restricted to specific tissue types (Han, P. et al. (1997) J. Biol. Chem. 272:16152-16157; Perry and Higgs, supra).
  • PDE7s are very closely related to the PDE4 family; however, PDE7s are not inhibited by roUpram, a specific inhibitor of PDE4s (Beavo, supra).
  • PDE8s are cAMP specific, and are closely related to the PDE4 family. PDE8s are expressed in thyroid gland, testis, eye, Uver, skeletal muscle, heart, kidney, ovary, and brain. The cAMP-hydrolyzing activity of PDE8s is not inhibited by the PDE inhibitors rohpram, vinpocetine, milrinone, IBMX (3 -isobutyl- 1-methylxanthine), or zaprinast, but PDE8s are inhibited by dipyridamole (Fisher, D.A. et al. (1998) Biochem. Biophys. Res. Commun. 246:570-577; Hayashi, M. et al. (1998) Biochem.
  • PDE9s are cGMP specific and most closely resemble the PDE8 family of PDEs. PDE9s are expressed in kidney, Uver, lung, brain, spleen, and small intestine.
  • PDE9s are not inhibited by sildenafil (VIAGRA; Pfizer, Inc., New York NY), roUpram, vinpocetine, dipyridamole, or IBMX (3 -isobutyl- 1- methylxanthine), but they are sensitive to the PDE5 inhibitor zaprinast (Fisher, D.A. et al. (1998) J. Biol. Chem. 273:15559-15564; SoderUng, S.H. et al. (1998) J. Biol. Chem. 273:15553-15558). PDElOs are dual-substrate PDEs, hydrolyzing both cAMP and cGMP.
  • PDElOs are expressed in brain, thyroid, and testis.
  • PDEs are composed of a catalytic domain of about 270-300 amino acids, an N-terrninal regulatory domain responsible for binding cofactors, and, in some cases, a hydrophiUc C-terminal domain of unknown function (Conti and Jin, supra).
  • N-terminal regulatory domains include non- catalytic cGMP-binding domains in PDE2s, PDE5s, and PDE6s; calmodulin-binding domains in PDEls; and domains containing phosphorylation sites in PDE3s and PDE4s.
  • the N-terminal cGMP-binding domain spans about 380 amino acid residues and comprises tandem repeats of a conserved sequence motif (McAlHster-Lucas, L.M. et al. (1993) J. Biol. Chem. 268:22863-22873).
  • the NKXnD motif has been shown by mutagenesis to be important for cGMP binding (Turko, IN. et al. (1996) J. Biol. Chem. 271:22240-22244).
  • PDE famiUes display approximately 30% amino acid identity within the catalytic domain; however, isozymes within the same family typicahy display about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore, within a family there is extensive similarity (>60%) outside the catalytic domain; while across famiUes, there is Uttle or no sequence similarity outside this domain.
  • PDE3 inhibitors are being developed as antithrombotic agents, antihypertensive agents, and as cardiotonic agents useful in the treatment of congestive heart failure.
  • RoUpram a PDE4 inhibitor, has been used in the treatment of depression, and other PDE4 inhibitors have an anti-inflammatory effect. RoUpram may inhibit HTV-1 repUcation (Angel, J.B. et al. (1995) ADDS 9:1137-1144).
  • roUpram suppresses the production of cytokines such as T ⁇ F-a and b and interferon g, and thus is effective against encephalomyeUtis.
  • RoUpram may also be effective in treating tardive dyskinesia and multiple sclerosis (Sommer, ⁇ . et al. (1995) Nat. Med. 1:244-248; Sasaki, H. et al. (1995) Eur. J. Pharmacol. 282:71-76).
  • TheophylUne is a nonspecific PDE inhibitor used in treatment of bronchial asthma and other respiratory diseases.
  • TheophylUne is beUeved to act on airway smooth muscle function and in an anti-inflammatory or immunomodulatory capacity (Banner, K.H. and C.P. Page (1995) Eur. Respir. J. 8:996-1000).
  • PentoxifylUne is another nonspecific PDE inhibitor used in the treatment of intermittent claudication and diabetes-induced peripheral vascular disease. PentoxifylUne is also known to block TNF-a production and may inhibit HJV-1 repUcation (Angel et al., supra).
  • PDEs have been reported to affect ceUular proUferation of a variety of ceU types (Conti et al. (1995) Endocrine Rev. 16:370-389) and have been impUcated in various cancers. Growth of prostate carcinoma cell lines DU145 and LNCaP was inhibited by deUvery of cAMP derivatives and PDE inhibitors (Bang, YJ. et al. (1994) Proc. Natl. Acad. Sci. USA 91:5330-5334). These ceUs also showed a permanent conversion in phenotype from epitheUal to neuronal morphology. It has also been suggested that PDE inhibitors can regulate mesangial ceU proUferation (Matousovic, K.
  • UDP glucuronyltransferase family catalyze the transfer of a glucuronic acid group from the cofactor uridine diphosphate-glucuronic acid (UDP-glucuronic acid) to a substrate.
  • the transfer is generally to a nuckophiUc heteroatom (O, N, or S).
  • Substrates include xenobiotics which have been functionaUzed by Phase I reactions, as weU as endogenous compounds such as biUrubin, steroid hormones, and thyroid hormones. Products of glucuronidation are excreted in urine if the molecular weight of the substrate is less than about 250 g/mol, whereas larger glucuronidated substrates are excreted in bile.
  • UGTs are located in the microsomes of Uver, kidney, intestine, skin, brain, spleen, and nasal mucosa, where they are on the same side of the endoplasmic reticulum membrane as cytochrome P450 enzymes and flavin-containing monooxygenases.
  • UGTs have a C-terminal membrane-spanning domain which anchors them in the endoplasmic reticulum membrane, and a conserved signature domain of about 50 amino acid residues in their C terrninal section (PROSITE PDOC00359 UDP- glycosyltransferase signature).
  • UGTs involved in drug metaboUsm are encoded by two gene famiUes, UGT1 and UGT2.
  • Members of the UGT1 family result from alternative spUcing of a single gene locus, which has a variable substrate binding domain and constant region involved in cofactor binding and membrane insertion.
  • Members of the UGT2 family are encoded by separate gene loci, and are divided into two famiUes, UGT2A and UGT2B.
  • the 2 A subfamily is expressed in olfactory epitheUum
  • the 2B subfamily is expressed in Uver microsomes.
  • UGT hyperbiUrubinemia
  • Crigler-Najjar syndrome characterized by intense hyperbiUrubinemia from birth
  • OMLM #191740 UGT1 a milder form of hyperbiUrubinemia termed Gilbert's disease
  • Thioesterases Two soluble thioesterases involved in fatty acid biosynthesis have been isolated from mammahan tissues, one which is active only toward long-chain fatty-acyl thioesters and one which is active toward thioesters with a wide range of fatty-acyl chain-lengths.
  • thioesterases catalyze the chain-te ⁇ ninating step in the de novo biosynthesis of fatty acids.
  • Chain termination involves the hydrolysis of the thioester bond which Unks the fatty acyl chain to the 4'-phosphopantetheine prosthetic group of the acyl carrier protein (ACP) subunit of the fatty acid synthase (Smith, S. (1981a) Methods Enzymol. 71:181-188; Smith, S. (1981b) Methods Enzymol. 71:188-200).
  • ACP acyl carrier protein
  • E. coli contains two soluble thioesterases, thioesterase I which is active only toward long- chain acyl thioesters, and thioesterase II (TEH) which has a broad chain-length specificity (Naggert, J. et al. (1991) J. Biol. Chem. 266:11044-11050).
  • E. coli TEII does not exhibit sequence similarity with either of the two types of mammaUan thioesterases which function as cham-te ⁇ ninating enzymes in de novo fatty acid biosynthesis.
  • mammaUan thioesterases E. coli contains two soluble thioesterases, thioesterase I which is active only toward long- chain acyl thioesters, and thioesterase II (TEH) which has a broad chain-length specificity (Naggert, J. et al. (1991) J. Biol. Chem. 266:11044-11050
  • coli TEH lacks the characteristic serine active site gly-X-ser-X-gly sequence motif and is not inactivated by the serine modifying agent diisopropyl fluorophosphate.
  • modification of histidine 58 by iodoacetamide and diethylpyrocarbonate aboUshed TEII activity.
  • Overexpression of TEII did not alter fatty acid content in E. coli, which suggests that it does not function as a chain-terminating enzyme in fatty acid biosynthesis (Naggert et al., supra). For that reason, Naggert et al. (supra) proposed that the physiological substrates for E. coli TELL may be coenzyme A (CoA)-fatty acid esters instead of ACP- phosphopanthetheine-fatty acid esters.
  • CoA coenzyme A
  • MammaUan carboxylesterases are a multigene family expressed in a variety of tissues and ceU types. AcetylchoUnesterase, butyrylcholinesterase, and carboxylesterase are grouped into the serine superfamily of esterases (B-esterases). Other carboxylesterases include thyroglobuUn, thrombin, Factor LX, ghotactin, and plasminogen. Carboxylesterases catalyze the hydrolysis of ester- and amide- groups from molecules and are involved in detoxification of drugs, environmental toxins, and carcinogens.
  • Substrates for carboxylesterases include short- and long-chain acyl-glycerols, acylcarnitine, carbonates, dipivefrin hydrochloride, cocaine, saUcylates, capsaicin, palmitoyl-coenzyme A, imidapril, haloperidol, pyrroUzidine alkaloids, steroids, p-nitrophenyl acetate, malathion, butaniUcaine, and isocarboxazide.
  • Carboxylesterases are also important for the conversion of prodrugs to free acids, which may be the active form of the drug (e.g., lovastatin, used to lower blood cholesterol) (reviewed in Satoh, T.
  • NeuroUgins are a class of molecules that (i) have N-terminal signal sequences, (ii) resemble ceU- surface receptors, (iii) contain carboxylesterase domains, (iv) are highly expressed in the brain, and (v) bind to neurexins in a calcium-dependent manner. Despite the homology to carboxylesterases, neuroUgins lack the active site serine residue, implying a role in substrate binding rather than catalysis (Ichtchenko, K. et al. (1996) J. Biol. Chem. 271:2676-2682). Squalene epoxidase
  • Squalene epoxidase (squalene monooxygenase, SE) is a microsomal membrane-bound, FAD- dependent oxidoreductase that catalyzes the first oxygenation step in the sterol biosynthetic pathway of eukaryotic cells.
  • Cholesterol is an essential structural component of cytoplasmic membranes acquired via the LDL receptor-mediated pathway or the biosynthetic pathway.
  • SE converts squalene to 2,3(5)-oxidosquatene, which is then converted to lanosterol and then cholesterol.
  • HMG-CoA reductase is responsible for the first committed step in cholesterol biosynthesis, conversion of 3-hydroxyl-3-methyl-glutaryl CoA (HMG-CoA) to mevalonate.
  • HMG-CoA is the target of a number of pharmaceutical compounds designed to lower plasma cholesterol levels, but inhibition of MHG-CoA also results in the reduced synthesis of non-sterol intermediates required for other biochemical pathways.
  • SE catalyzes a rate-limiting reaction that occurs later in the sterol synthesis pathway with cholesterol as the only end product
  • SE is a better ideal target for the design of anti-hyperUpidemic drugs (Nakamura, Y. et al. (1996) 271:8053-8056).
  • Epoxide hydrolases catalyze the addition of water to epoxide-containing compounds, thereby hydrolyzing epoxides to their co ⁇ esponding 1,2-diols. They are related to bacterial haloalkane dehalogenases and show sequence similarity to other members of the ⁇ / ⁇ hydrolase fold family of enzymes. This family of enzymes is important for the detoxification of xenobiotic epoxide compounds which are often highly electrophiUc and destructive when introduced. Examples of epoxide hydrolase reactions include the hydrolysis of some teukotoxin to leukotoxin diol, and isoleukotoxin to isoleukotoxin diol.
  • Epoxide hydrolases possess a catalytic triad composed of Asp, Asp, and His (Arand, M. et al. (1996) J. Biol. Chem. 271:4223-4229; Rink, R. et al. (1997) J. Biol. Chem. 272:14650-14657; Argiriadi, M.A. et al. (2000) J. Biol. Chem. 275:15265-15270).
  • Enzymes involved in the degradation of tyrosine to succinate and pyruvate include 4-hydroxyphenylpyruvate oxidase, 4-hydroxyphenylacetate 3-hydroxylase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase, 5-carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase, trans, cis- 5-carboxymethyl-2-hydroxymuconate isomerase, homoprotocatechuate isomerase/decarboxylase, c 5'-2-oxohept-3-ene-l,7-dioate hydratase, 2,4-dihydroxyhept-traM5-2-ene-l,7-dioate aldolase, and succinic semialdehyde dehydrogenase.
  • Enzymes involved in the degradation of tyrosine to fumarate and acetoacetate include 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1,2-dioxygenase, maleylacetoacetate isomerase, fumarylacetoacetase and 4-hydroxyphenylacetate.
  • Additional enzymes associated with tyrosine metaboUsm in different organisms include 4-chlorophenylacetate-3,4-dioxygenase, aromatic aminotransferase, 5-oxopent-3-ene-l,2,5-tricarboxylate decarboxylase, 2-oxo-hept-3-ene-l,7-dioate hydratase, and 5-carboxymethyl-2-hydroxymuconate isomerase (Elhs, L.B.M. et al. (1999) Nucleic Acids Res. 27:373-376; Wackett, L.P. and EUis, L.B.M. (1996) J. Microbiol. Meth. 25:91-93; and Schmidt, M. (1996) Amer. Soc. Microbiol. News 62:102).
  • hereditary tyrosinemia 1 is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase, the last enzyme in the pathway in organisms that metaboUze tyrosine to fumarate and acetoacetate.
  • HTl is characterized by progressive Uver damage beginning at infancy, and increased risk for Uver cancer (Endo, F. et al. (1997) J. Biol. Chem. 272:24426-24432).
  • Microa ⁇ ays are analytical tools used in bioanalysis.
  • a microa ⁇ ay has a pluraUty of molecules spatially distributed over, and stably associated with, the surface of a soUd support.
  • Microa ⁇ ays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of apphcations, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • One area in particular in which microa ⁇ ays find use is in gene expression analysis.
  • a ⁇ ay technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • a ⁇ ays When the expression of a single gene is examined, a ⁇ ays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, a ⁇ ays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • FamiUal adenomatous polyposis is caused by mutations in the adenomatous polyposis coU gene (APC), resulting in truncated or inactive forms of the protein.
  • APC adenomatous polyposis coU gene
  • This tumor suppressor gene has been mapped to chromosome 5q.
  • Hereditary nonpolyposis colorectal cancer is caused by mutations in mis-match repair genes.
  • hereditary colon cancer syndromes occur in a smaU percentage of the population and most colorectal cancers are considered sporadic, knowledge from studies of the hereditary syndromes can be generaUy apphed. For instance, somatic mutations in APC occur in at least 80% of sporadic colon tumors.
  • APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently. Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in aU of these genes lead to gene expression changes in colon cancer.
  • Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S. Lung cancers are divided into four histopathologicaUy distinct groups. Three groups (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are classified as non-smaU ceU lung cancers (NSCLCs). The fourth group of cancers is refe ⁇ ed to as smaU ceU lung cancer (SCLC). Deletions on chromosome 3 are common in this disease and are thought to indicate the presence of a tumor suppressor gene in this region. Activating mutations in K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.
  • Breast cancer is the most frequently diagnosed type of cancer in American women and the second most frequent cause of cancer death.
  • the Ufetime risk of an American woman developing breast cancer is 1 in 8, and one-third of women diagnosed with breast cancer die of the disease.
  • a number of risk factors have been identified, including hormonal and genetic factors.
  • One genetic defect associated with breast cancer results in a loss of heterozygosity (LOH) at multiple loci such as p53, Rb, BRCA1, and BRCA2.
  • LHO heterozygosity
  • Another genetic defect is gene ampUfication involving genes such as c-myc and c-erbB2 (Her2-neu gene).
  • Steroid and growth factor pathways are also altered in breast cancer, notably the estrogen, progesterone, and epidermal growth factor (EGF) pathways.
  • Breast cancer evolves through a multi-step process whereby premaUgnant mammary epitheUal ceUs undergo a relatively defined sequence of events leading to tumor formation.
  • An early event in tumor development is ductal hyperplasia.
  • CeUs undergoing rapid neoplastic growth graduaUy progress to invasive carcinoma and become metastatic to the lung, bone, and potentially other organs.
  • Variables that may influence the process of tumor progression and mahgnant transformation include genetic factors, environmental factors, growth factors, and hormones.
  • Ovarian cancer is the leading cause of death from a gynecologic cancer.
  • the majority of ovarian cancers are derived from epitheUal cells, and 70% of patients with epitheUal ovarian cancers present with late-stage disease. As a result, the long-term survival rates for this disease is very low. Identification of early-stage markers for ovarian cancer would significantly increase the survival rate.
  • Genetic variations involved in ovarian cancer development include mutation of p53 and microsateUite instabiUty. Gene expression patterns likely vary when normal ovary is compared to ovarian tumors.
  • Tangier disease is a genetic disorder characterized by near absence of circulating high density Upoprotein (HDL) and the accumulation of cholesterol esters in many tissues, including tonsils, lymph nodes, Uver, spleen, thymus, and intestine.
  • Low levels of HDL represent a clear predictor of premature coronary artery disease and homozygous TD co ⁇ elates with a four- to six-fold increase in cardiovascular disease compared to controls.
  • HDL plays a cardio-protective role in reverse cholesterol transport, the flux of cholesterol from peripheral ceUs such as tissue macrophages through plasma Upoproteins to the Uver.
  • Parkinson's disease is a neurodegenerative disorder characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway, and the presence of Lewy bodies. Genetic Unkages for the Parkin gene to chromosome 6q25.2-27, for PARK3 to chromosome 2p (West, A. B. (2001) Eur. J. Hum. Genet. 9:659-666), and for PARK6 to chromosome Ip35-p36 have been identified (Valente, E. M. et al. (2002) Ann. Neurol. 51:14-18). Clinical disorders classified as parkinsonism include PD, dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), and essential tremor.
  • DLB dementia with Lewy bodies
  • PSP progressive supranuclear palsy
  • Several neurodegenerative diseases share pathogenic mechanisms involving tau or synuclein aggregation. These disorders include Alzheimer's disease, and Pick's disease as weU as PD and progressive supranuclear palsy (Hardy, J. (2001) J. Alzheimers Dis. 3:109-116).
  • Several genetically distinct forms of PD can be caused by mutations in single genes. Genes for monogenicaUy inherited forms of Parkinson's disease have been mapped and/or cloned. In some famiUes with autosomal dominant inheritance and typical Lewy-body pathology, mutations have been identified in the gene for alpha-synuclein. Aggregation of this protein in Lewy-bodies may be a crucial step in the molecular pathogenesis of famiUal and sporadic PD.
  • Parkin-mutations appear to be a common cause of PD in patients with very early onset. Mutations in the parkin gene of early-onset PD are autosomal recessive mutations in which nigral degeneration is not accompanied by Lewy-body formation. Parkin has been impUcated in the ceUular protein degradation pathways, as it has been shown that it functions as a ubiquitin Ugase. A mutation in the gene for ubiquitin C-terminal hydrolase LI in this pathway has been identified in another small family with PD. Other loci have been mapped to chromosome 2p and 4p, respectively, in famiUes with dominantly inherited PD. These early-onset forms differ from the common sporadic form of PD.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metaboUsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU proUferative disorders, including cancer.
  • ⁇ NZM' purified polypeptides, enzymes, referred to collectively as ⁇ NZM' and individuaUy as 'ENZM-1,' ⁇ NZM-2,' ⁇ NZM-3,' ⁇ NZM-4,' ⁇ NZM- 5,' ⁇ NZM-6,' ⁇ NZM-7,' ⁇ NZM-8,' ⁇ NZM-9,' ⁇ NZM-10,' ⁇ NZM-11,' ⁇ NZM-12,' ⁇ NZM- 13,' ⁇ NZM-14,' ⁇ NZM-15,' 'ENZM-16,' ⁇ NZM-17,' ⁇ NZM-18,' ⁇ NZM-19,' ⁇ NZM-20,' ⁇ NZM-21,' ⁇ NZM-22,' ⁇ NZM-23,' ⁇ NZM-24,' ⁇ NZM-25,' ⁇ NZM-26,' ⁇ NZM-27,
  • Embodiments also provide methods for utiUzing the purified enzymes and/or their encoding polynucleotides for facihtating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utiUzing the purified enzymes and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JO NO:l- 57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an a ino acid sequence selected from the group consisting of SEQ LD NO: 1-57, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO:l-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ LD NO:l-57.
  • StiU another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NO:l-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ LD NO: 1-57. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ LD NO:58-l 14.
  • StiU another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably Unked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an a ino acid sequence selected from the group consisting of SEQ LD NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57.
  • Another embodiment provides a ceU transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57.
  • the method comprises a) culturing a ceU under conditions suitable for expression of the polypeptide, wherein said ceU is transformed with a recombinant polynucleotide comprising a promoter sequence operably Unked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57.
  • StiU yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-l 14, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-l 14, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specificaUy hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
  • the method can include detecting the amount of the hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • StiU yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, b) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a
  • the method comprises a) ampUfying said target polynucleotide or fragment thereof using polymerase chain reaction ampUfication, and b) detecting the presence or absence of said ampUfied target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the ampUfied target polynucleotide or fragment thereof.
  • compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOJ-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, and a pharmaceuticaUy acceptable excipient.
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ LD NOJ-57.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional ENZM, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, b) a polypeptide comprising a naturaUy occurring arnino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NOJ-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-57.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional ENZM, comprising administering to a patient in need of such treatment the composition.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ D NOJ-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NOJ-57.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional ENZM, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ JD NO:l-57, b) a polypeptide comprising a naturaUy occu ⁇ ing amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ LD NOJ-57, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specificaUy binds to the polypeptide.
  • Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ LD NO:l-57, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an arnino acid sequence selected from the group consisting of SEQ LD NO: 1-57, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ LD NO: 1-57.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:58-114, u) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, Hi) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ LD NO:58-114, Hi) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of H), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention.
  • the probabiUty scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 Hsts the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA Ubraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA Ubraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with appUcable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • a reference to “a host cell” includes a pluraUty of such host ceUs, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skiUed in the art, and so forth.
  • ENZM refers to the amino acid sequences of substantiaUy purified ENZM obtained from any species, particularly a mammaUan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or rriimics the biological activity of
  • ENZM may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ENZM either by directly interacting with ENZM or by acting on components of the biological pathway in which ENZM participates.
  • AUeUc variant is an alternative form of the gene encoding ENZM.
  • AUeUc variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered.
  • a gene may have none, one, or many aUeUc variants of its naturaUy occurring form.
  • Common mutational changes which give rise to alleUc variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding ENZM include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as ENZM or a polypeptide with at least one functional characteristic of ENZM. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oHgonucleotide probe of the polynucleotide encoding ENZM, and improper or unexpected hybridization to alleUc variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding ENZM.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionahy equivalent ENZM.
  • DeUberate amino acid substitutions may be made on the basis of one or more similarities in polarity, charge, solubiUty, hydrophobicity, hydrophiUcity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of ENZM is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having similar hydrophiUcity values may include: asparagine and glutamine; and serine and threonine.
  • Arnino acids with uncharged side chains having similar hydrophiUcity values may include: leucine, isoleucine, and vaUne; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oUgopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules.
  • amino acid sequence is recited to refer to a sequence of a naturaUy occurring protein molecule
  • amino acid sequence and Uke terms are not meant to Umit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • “AmpUfication” relates to the production of additional copies of a nucleic acid. AmpUfication may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid ampUfication technologies weU known in the art.
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of ENZM.
  • Antagonists may include proteins such as antibodies, anticaUns, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of ENZM either by directly interacting with ENZM or by acting on components of the biological pathway in which ENZM participates.
  • antibody refers to intact immunoglobuUn molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind ENZM polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oUgopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaUy coupled to peptides include bovine serum albumin, thyroglobuUn, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • an antigenic determinant may compete with the intact antigen (i.e., the immunogen used to eUcit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oUgonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial Ubraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-Uke molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2 -OH group of a ribonucleotide may be replaced by 2 -F or 2 -NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer Ufetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate Ugands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • introduction refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (BUnd, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-Uke molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense"
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oUgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oUgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oUgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription.
  • the complementary antisense molecule base-pahs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occu ⁇ ing molecule.
  • immunologicalaUy active or “immunogenic” refers to the capabiUty of the natural, recombinant, or synthetic ENZM, or of any oUgopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding ENZM or fragments of ENZM may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabiUzing agent such as a carbohydrate.
  • the probe In hybridizations, the probe maybe deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncaUed bases, extended using the XL-PCR kit (AppUed Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVLEW fragment assembly system (Accelrys, BurUngton MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original arnino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha heUcal conformation,
  • deletion refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemicaUy modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or arnino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of ENZM or a polynucleotide encoding ENZM which can be identical in sequence to, but shorter in length than, the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentiaUy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:58-114 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ED NO:58-l 14, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ED NO:58-l 14 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and ampUfication technologies and in analogous methods that distinguish SEQ ED NO:58-l 14 from related polynucleotides.
  • a fragment of SEQ ED NOJ-57 is encoded by a fragment of SEQ ED NO:58-l 14.
  • a fragment of SEQ ED NO: 1-57 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ LD NOJ-57.
  • a fragment of SEQ ED NOJ-57 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ED NOJ-57.
  • the precise length of a fragment of SEQ ED NOJ-57 and the region of SEQ ED NO:l-57 to which the fragment co ⁇ esponds can be dete ⁇ nined based on the intended purpose for the fragment usmg one or more analytical methods described herein or otherwise known in the art.
  • a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “fuU length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • Homology refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aUgned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize aUgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be deterrnined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151- 153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191).
  • the "weighted" residue weight table is selected as the default.
  • BLAST Basic Local AHgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local AHgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to ahgn a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences that is used for direct pairwise comparison of two nucleotide sequences.
  • BLAST 2 Sequences can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/bl2.htrnl.
  • the "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below).
  • BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ LD number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar arnino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that aU encode substantially the same protein.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences ahgned using a standardized algorithm.
  • Methods of polypeptide sequence aUgnment are weU-known. Some aUgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.
  • percent similarity and % similarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aUgned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.
  • Gap x drop-off 50 Expect: 10 Word Size: 3
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ED number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • "Human artificial chromosomes" are linear rnicrochromosomes which may contain
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and stiU retains its original binding abiUty.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive anneaUng conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for anneaUng of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity.
  • Permissive anneaUng conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 ⁇ g/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • An equation for calculating T m and conditions for nucleic acid hybridization are weU known and can be found in Sambrook, J. and D.W. RusseU (2001: Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68 °C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar rote for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or Rot analysis) or formed between one nucleic acid present in solution and another nucleic acid immobiUzed on a soUd support (e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • a soUd support e.g., paper, membranes, filters, chips, pins or glass sUdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed.
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more arnino acid residues or nucleotides, respectively.
  • Immunune response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect ceUular and systemic defense systems.
  • an “immunogenic fragment” is a polypeptide or oUgopeptide fragment of ENZM which is capable of eUciting an immune response when introduced into a Uving organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oUgopeptide fragment of ENZM which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microa ⁇ ay refers to an a ⁇ angement of a pluraUty of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and "a ⁇ ay element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microa ⁇ ay.
  • modulate refers to a change in the activity of ENZM. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of ENZM.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oUgonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • “Operably Unked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably Unked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably Unked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oUgonucleotide of at least about 5 nucleotides in length Unked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubiUty to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their Ufespan in the ceU.
  • Post-translational modification of an ENZM may involve Upidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications wiU vary by ceU type depending on the enzymatic miUeu of ENZM.
  • Probe refers to nucleic acids encoding ENZM, their complements, or fragments thereof, which are used to detect identical, aUeUc or related nucleic acids. Probes are isolated oUgonucleotides or polynucleotides attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, Ugands, cher luminescent agents, and enzymes.
  • "Primers" are short nucleic acids, usuaUy DNA oUgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for ampUfication (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR). Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence.
  • probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, maybe used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • OUgonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oUgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabiUties.
  • the PrimOU primer selection program (available to the pubUc from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the pubUc from the Whitehead Institute/MET Center for Genome Research, Cambridge MA) aUows the user to input a "mispriming Ubrary," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oUgonucleotides for microa ⁇ ays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence aUgnments, thereby aUo ing selection of primers that hybridize to either the most conserved or least conserved regions of aUgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oUgonucleotides and polynucleotide fragments.
  • oUgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partially complementary polynucleotides in a sample of nucleic acids. Methods of oUgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturaUy occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accompUshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and RusseU (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably Unked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stabiUty.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionucUdes; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that all occu ⁇ ences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing ENZM, nucleic acids encoding ENZM, or fragments thereof may comprise a bodily fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition.
  • the interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
  • substitution refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, sUdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capiUaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, Upofection, and particle bombardment.
  • transformed cells includes stably transformed ceUs in which the inserted DNA is capable of repUcation either as an autonomously repUcating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques weU known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the ceU, by way of deUberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertiUzation, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook and RusseU (supra).
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "aUeUc” (as defined above), “spUce,” “species,” or “polymorphic” variant.
  • a spUce variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate spUcing during mRNA processing.
  • the co ⁇ esponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another.
  • the resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence similarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence similarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human enzymes (ENZM), the polynucleotides encoding ENZM, and the use of these compositions for the diagnosis, treatment, or prevention of autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metaboUsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU proUferative disorders, including cancer.
  • Table 1 summarizes the nomenclature for the fuU length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its co ⁇ esponding polypeptide are co ⁇ elated to a single Incyte project identification number (Encyte Project ED).
  • Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Encyte Polypeptide LD) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ED NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide LD) as shown.
  • Column 6 shows the Incyte ED numbers of physical, fuU length clones co ⁇ esponding to the polypeptide and polynucleotide sequences of the invention.
  • the fuU length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ED NO:) and the co ⁇ esponding Incyte polypeptide sequence number (Incyte Polypeptide ED) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ED NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers
  • PROTEOME ED NO: of the nearest PROTEOME database homologs.
  • Column 4 shows the probabiUty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where appUcable, all of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and
  • SEQ ED NO: polypeptide sequence identification number
  • Incyte Polypeptide ID Incyte polypeptide sequence number
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Accelrys, BurUngton MA).
  • Column 6 shows amino acid residues comprising signature sequences, domains, and motifs.
  • Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were appUed.
  • SEQ ED NO:6 is 411 amino acids in length and is 100% identical, from residue Ml to residue W372, to human cytochrome P450 (GenBank ED gl 1275334) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabiUty score is 1.9e-205, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance.
  • SEQ ED NO:6 also has homology to proteins that are locaUzed to the endoplasmic reticulum, have oxidative functions, and are cytochrome P450 proteins, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ED NO:6 also contains a cytochrome P450 domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein famines/domains. (See Table 3.)
  • HMM hidden Markov model
  • SEQ ED NO:52 is 100% identical, from residue G161 to residue V423, and from residue Ml to residue S162, to a human cytochrome (GenBank ID gl81300) as determined by the Basic Local AUgnment Search Tool (BLAST). (See Table 2.) The BLAST probabiUty score is 3.6e-227, which indicates the probabiUty of obtaining the observed polypeptide sequence aUgnment by chance. SEQ ID NO:52 also has homology to proteins that are locaUzed within Uver microsomes, have oxidative function, and are cytochrome P450 enzymes, as determined by BLAST analysis using the PROTEOME database.
  • BLAST Basic Local AUgnment Search Tool
  • SEQ LD NO:52 also contains a cytochrome P450 domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein famiUes/domains. (See Table 3.) Data from BLIMPS, MOTEFS, and PROFELESCAN analyses provide further co ⁇ oborative evidence that SEQ ED NO:52 is a cytochrome P450 enzyme.
  • SEQ ED NO:l-5, SEQ ED NO:7-51, and SEQ ED NO:53-57 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ TD NOJ-57 are described in Table 7.
  • the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 Usts the polynucleotide sequence identification number (Polynucleotide SEQ ED NO:), the co ⁇ esponding Incyte polynucleotide consensus sequence number (Incyte ED) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or ampUfication technologies that identify SEQ ED NO:58-114 or that distinguish between SEQ ED NO:58-114 and related polynucleotides.
  • polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA Ubraries or from pooled cDNA
  • polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the fuU length polynucleotides.
  • polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The S anger Centre, Cambridge, UK) database (i.e., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm. For example, a polynucleotide sequence identified as
  • FL_XXXXXXX_N j _N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was appUed, and YYYYY is the number of the prediction generated by the algorithm, and N 1 23 , if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the "exon-stretching" algorithm was appUed, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • RefSeq identifier (denoted by " ⁇ M,” “NP,” or “NT”) maybe used in place of the GenBank identifier (i.e., gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • Table Ust examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example TV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA Ubraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA Ubrary is the Encyte cDNA Ubrary which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA Ubraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ED NO:) and the co ⁇ esponding Incyte project identification number (PED) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for the EST in which the SNP was detected (EST ED), and column 4 shows the identification number for the SNP (SNP TD).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position of the SNP within the full- length polynucleotide sequence (CB1 SNP).
  • Column 7 shows the aUele found in the EST sequence.
  • Columns 8 and 9 show the two aUeles found at the SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the aUele found in the EST.
  • Columns 11-14 show the frequency of aUele 1 in four different human populations.
  • ENZM variants can have at least about 80%, at least about 90%, or at least about 95% amino acid sequence identity to the ENZM amino acid sequence, and can contain at least one functional or structural characteristic of ENZM.
  • Various embodiments also encompass polynucleotides which encode ENZM.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ED NO:58-114, which encodes ENZM.
  • the polynucleotide sequences of SEQ ED NO:58-114 as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occu ⁇ ences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding ENZM.
  • such a variant polynucleotide wiU have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a polynucleotide encoding ENZM.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:58-114 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ED NO:58-l 14. Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of ENZM.
  • a polynucleotide variant of the invention is a spUce variant of a polynucleotide encoding ENZM.
  • a spUce variant may have portions which have significant sequence identity to a polynucleotide encoding ENZM, but wiU generaUy have a greater or lesser number of polynucleotides due to additions or deletions of blocks of sequence arising from alternate spUcing during mRNA processing.
  • a spUce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding ENZM over its entire length; however, portions of the spUce variant wiU have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding ENZM.
  • a polynucleotide comprising a sequence of SEQ ED NO:59, a polynucleotide comprising a sequence of SEQ ED NO:67, and a polynucleotide comprising a sequence of SEQ ED NO:68 are spUce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ED NO:62 and a polynucleotide comprising a sequence of SEQ TD NO:73 are spUce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ED NO:71, a polynucleotide comprising a sequence of SEQ ED NO:72, and a polynucleotide comprising a sequence of SEQ ED NO:76 are spUce variants of each other;
  • polynucleotides which encode ENZM and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring ENZM under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding ENZM or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utiUzed by the host.
  • RNA transcripts having more desirable properties such as a greater half-Ufe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode ENZM and ENZM derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many available expression vectors and ceU systems using reagents well known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding ENZM or any fragment thereof.
  • Embodiments of the invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ED NO:58-l 14 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L.
  • Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (AppUed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE ampUfication system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 Uquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (AppUed Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (AppUed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are weU known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding ENZM may be extended utiUzing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to ampUfy unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods AppUc. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to ampUfy unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and su ⁇ ounding sequences (TrigUa, T.
  • a third method involves PCR ampUfication of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods AppUc. 1:111-119).
  • multiple restriction enzyme digestions and Ugations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers may be designed using commerciaUy available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C
  • Ubraries When screening for full length cDNAs, it is preferable to use Ubraries that have been size-selected to include larger cDNAs. In addition, random-primed Ubraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oUgo d(T) Ubrary does not yield a fuU-length cDNA. Genomic Ubraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capiUary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/Ught intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppUed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controUed.
  • CapiUary electrophoresis is especiaUy preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode ENZM maybe cloned in recombinant DNA molecules that direct expression of ENZM, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express ENZM.
  • the polynucleotides of the invention can be engineered using methods generaUy known in the art in order to alter ENZM-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oUgonucleotides may be used to engineer the nucleotide sequences.
  • oUgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce spUce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDLNG (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C. et al. (1999) Nat. BiotechnoL 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of ENZM, such as its biological or enzymatic activity or its abiUty to bind to other molecules or compounds.
  • MOLECULARBREEDLNG Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458; Chang, C-C et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F.C.
  • DNA shuffling is a process by which a Ubrary of gene variants is produced using PCR-mediated recombination of gene fragments. The Ubrary is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These prefe ⁇ ed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
  • fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturaUy occurring genes in a directed and controUable manner.
  • polynucleotides encoding ENZM may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • ENZM itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or soUd-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp. 55-60; Roberge, J.Y. et al. (1995) Science 269:202-204). Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (AppUed Biosystems). Additionally, the amino acid sequence of ENZM, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance Uquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • the polynucleotides encoding ENZM or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding ENZM. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding ENZM. Such signals include the ATG initiation codon and adjacent sequences, e.g.
  • Methods which are well known to those skiUed in the art may be used to construct expression vectors containing polynucleotides encoding ENZM and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15). A variety of expression vector/host systems may be utiUzed to contain and express polynucleotides encoding ENZM.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauUflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal ceU systems (Sambrook and RusseU, supra; Ausubel et al., supra; Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids maybe used for deUvery of polynucleotides to the targeted organ, tissue, or ceU population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; BuUer, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242).
  • the invention is not limited by the host ceU employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding ENZM.
  • routine cloning, subcloning, and propagation of polynucleotides encoding ENZM can be achieved using a multifunctional E. coli vector such as PBLUESCRLPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRLPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Invitrogen.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of ENZM may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of ENZM.
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation (Ausubel et al., supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, CA. et al. (1994) Bio/Technology 12:181-184).
  • Plant systems may also be used for expression of ENZM. Transcription of polynucleotides encoding ENZM may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:1631). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters maybe used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; BrogUe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant ceUs by direct DNA transformation or pathogen-mediated transfection (The McGraw HiU Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utiUzed.
  • polynucleotides encoding ENZM may be Ugated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses ENZM in host cells (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammaUan host ceUs.
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • HACs Human artificial chromosomes
  • HACs may also be employed to deUver larger fragments of DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and deUvered via conventional delivery methods (Uposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355).
  • ENZM in ceU lines For long term production of recombinant proteins in mammaUan systems, stable expression of ENZM in ceU lines is prefe ⁇ ed.
  • polynucleotides encoding ENZM can be transformed into ceU lines using expression vectors which may contain viral origins of repUcation and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. FoUowing the introduction of the vector, ceUs maybe aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not Umited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk and apr ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetaboUte, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD Additional selectable genes have been described, e.g., trpB and hisD, which alter ceUular requirements for metaboUtes (Hartman, S.C.
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), ⁇ - glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding ENZM is inserted within a marker gene sequence
  • transformed ceUs containing polynucleotides encoding ENZM can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding ENZM under the control of a single promoter. Expression of the marker gene in response to induction or selection usuaUy indicates expression of the tandem gene as well.
  • host cells that contain the polynucleotide encoding ENZM and that express ENZM may be identified by a variety of procedures known to those of skill in the art.
  • Immunological methods for detecting and measuring the expression of ENZM using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated ceU sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding ENZM include oUgolabeUng, nick translation, end-labeling, or PCR ampUfication using a labeled nucleotide.
  • polynucleotides encoding ENZM, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 an appropriate RNA polymerase
  • Suitable reporter molecules or labels which may be used for ease of detection include radionucUdes, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with polynucleotides encoding ENZM may be cultured under conditions suitable for the expression and recovery of the protein from ceU culture.
  • the protein produced by a transformed ceU may be secreted or retained intraceUularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode ENZM may be designed to contain signal sequences which direct secretion of ENZM through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its abiUty to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not Umited to, acetylation, carboxylation, glycosylation, phosphorylation, Upidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Cohection (ATCC, Manassas VA) and may be chosen to ensure the co ⁇ ect modification and processing of the foreign protein.
  • ATCC American Type Culture Cohection
  • Manassas VA American Type Culture Cohection
  • natural, modified, or recombinant polynucleotides encoding ENZM may be Ugated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric ENZM protein containing a heterologous moiety that can be recognized by a commerciaUy available antibody may faciUtate the screening of peptide Ubraries for inhibitors of ENZM activity.
  • Heterologous protein and peptide moieties may also faciUtate purification of fusion proteins using commerciaUy available affinity matrices.
  • Such moieties include, but are not Umited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiUzed glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy available monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the ENZM encoding sequence and the heterologous protein sequence, so that ENZM may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16). A variety of commerciaUy available kits may also be used to faciUtate expression and purification of fusion proteins. In another embodiment, synthesis of radiolabeled ENZM may be achieved in vitro using the
  • TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S-methionine.
  • ENZM, fragments of ENZM, or variants of ENZM may be used to screen for compounds that specifically bind to ENZM.
  • One or more test compounds may be screened for specific binding to ENZM. In various embodiments, 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to ENZM. Examples of test compounds can include antibodies, anticalins, oUgonucleotides, proteins (e.g., Ugands or receptors), or smaU molecules.
  • variants of ENZM can be used to screen for binding of test compounds, such as antibodies, to ENZM, a variant of ENZM, or a combination of ENZM and/or one or more variants ENZM.
  • a variant of ENZM can be used to screen for compounds that bind to a variant of ENZM, but not to ENZM having the exact sequence of a sequence of SEQ ED NOJ-57.
  • ENZM variants used to perform such screening can have a range of about 50% to about 99% sequence identity to ENZM, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to ENZM can be closely related to the natural Ugand of ENZM, e.g., a Ugand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner (CoUgan, J.E. et al. (1991) Current Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural Ugand of a receptor ENZM (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22:132- 140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to ENZM can be closely related to the natural receptor to which ENZM binds, at least a fragment of the receptor, or a fragment of the receptor including all or a portion of the Ugand binding site or binding pocket.
  • the compound may be a receptor for ENZM which is capable of propagating a signal, or a decoy receptor for ENZM which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Cu ⁇ . Opin. Cell Biol. 11:255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328-336).
  • the compound can be rationaUy designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer Unked to the Fc portion of human IgGj (Taylor, P.C. et al. (2001) Cu ⁇ . Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having similar or, alternatively, different specificities can be screened for specific binding to ENZM, fragments of ENZM, or variants of ENZM.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of ENZM.
  • an antibody can be selected such that its binding specificity aUows for preferential identification of specific fragments or variants of ENZM.
  • an antibody can be selected such that its binding specificity aUows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of ENZM.
  • anticaUns can be screened for specific binding to ENZM, fragments of ENZM, or variants of ENZM.
  • AnticaUns are Ugand-binding proteins that have been constructed based on a UpocaUn scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Ske ⁇ a, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of Hpocalins can include a beta-ba ⁇ el having eight antiparallel beta-strands, which supports four loops at its open end.
  • loops form the natural Ugand-binding site of the Hpocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specificaUy bind to, stimulate, or inhibit ENZM involves producing appropriate ceUs which express ENZM, either as a secreted protein or on the ceU membrane.
  • Prefe ⁇ ed ceUs can include cells from mammals, yeast, Drosophila, or E. coli.
  • CeUs expressing ENZM or cell membrane fractions which contain ENZM are then contacted with a test compound and binding, stimulation, or inhibition of activity of either ENZM or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with ENZM, either in solution or affixed to a soUd support, and detecting the binding of ENZM to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor. AdditionaUy, the assay may be carried out using ceU-free preparations, chemical Ubraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a soUd support.
  • An assay can be used to assess the abiUty of a compound to bind to its natural Ugand and/or to inhibit the binding of its natural Ugand to its natural receptors.
  • assays include radio- labeUng assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No. 6,372,724.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its abiUty to bind to its natural Ugands (Matthews, D.J. and J.A. WeUs. (1994) Chem. Biol. 1:25-30).
  • one or more arnino acid substitutions can be introduced into a polypeptide compound (such as a Hgand) to improve or alter its abiUty to bind to its natural receptors (Cunningham, B.C. and J.A. WeUs (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
  • a polypeptide compound such as a Hgand
  • ENZM, fragments of ENZM, or variants of ENZM maybe used to screen for compounds that modulate the activity of ENZM.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for ENZM activity, wherein ENZM is combined with at least one test compound, and the activity of ENZM in the presence of a test compound is compared with the activity of ENZM in the absence of the test compound. A change in the activity of ENZM in the presence of the test compound is indicative of a compound that modulates the activity of ENZM.
  • test compound is combined with an in vitro or ceU-free system comprising ENZM under conditions suitable for ENZM activity, and the assay is performed.
  • a test compound which modulates the activity of ENZM may do so indirectly and need not come in direct contact with the test compound. At least one and up to a pluraUty of test compounds may be screened.
  • polynucleotides encoding ENZM or their mammaHan homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the co ⁇ esponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) CUn. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgicaUy transfe ⁇ ed to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding ENZM may also be manipulated in vitro in ES ceUs derived from human blastocysts.
  • Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types. These ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147
  • Polynucleotides encoding ENZM can also be used to create "knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding ENZM is injected into animal ES ceUs, and the injected sequence integrates into the animal cell genome.
  • Transformed ceUs are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress ENZM e.g., by secreting ENZM in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).
  • ENZM appears to play a role in autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metaboUsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU proUferative disorders, including cancer.
  • disorders associated with increased ENZM expression or activity it is desirable to decrease the expression or activity of ENZM.
  • disorders associated with decreased ENZM expression or activity it is desirable to increase the expression or activity of ENZM.
  • ENZM or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ENZM.
  • disorders include, but are not Umited to, an autoinimune/inflammatory disorder such as acquired immunodeficiency syndrome (AEDS), Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes meUitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaHs, erythema nod
  • AEDS acquired
  • a vector capable of expressing ENZM or a fragment or derivative thereof may be adrninistered to a subject to treat or prevent a disorder associated with decreased expression or activity of ENZM including, but not Umited to, those described above.
  • a composition comprising a substantiaUy purified ENZM in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ENZM including, but not limited to, those provided above.
  • an agonist which modulates the activity of ENZM may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of ENZM including, but not Umited to, those Usted above.
  • an antagonist of ENZM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ENZM.
  • disorders include, but are not limited to, those autoimmune/inflammatory disorders, infectious disorders, immune deficiencies, disorders of metaboUsm, reproductive disorders, neurological disorders, cardiovascular disorders, eye disorders, and ceU proUferative disorders, including cancer described above.
  • an antibody which specificaUy binds ENZM may be used directly as an antagonist or indirectly as a targeting or deUvery mechanism for bringing a pharmaceutical agent to ceUs or tissues which express ENZM.
  • a vector expressing the complement of the polynucleotide encoding ENZM may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of ENZM including, but not Umited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments maybe administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skiU in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of ENZM may be produced using methods which are generaUy known in the art.
  • purified ENZM may be used to produce antibodies or to screen Ubraries of pharmaceutical agents to identify those which specificaUy bind ENZM.
  • Antibodies to ENZM may also be generated using methods that are weU known in the art.
  • Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • neutraUzing antibodies i.e., those which inhibit dimer formation
  • Single chain antibodies may be potent enzyme inhibitors and may have appUcation in the design of peptide mimetics, and in the development of immuno-adsorbents and biosensors (Muyldermans, S. (2001) J. Biotechnol. 74:277-302).
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with ENZM or with any fragment or oUgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not Umited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
  • BCG BaciUi Calmette-Guerin
  • Corynebacterium parvum are especiaUy preferable.
  • the oUgopeptides, peptides, or fragments used to induce antibodies to ENZM have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wiU consist of at least about 10 amino acids. It is also preferable that these oUgopeptides, peptides, or fragments are substantiaUy identical to a portion of the amino acid sequence of the natural protein. Short stretches of ENZM amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to ENZM may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not Umited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole, S.P. et al. (1984) Mol. CeU Biol. 62:109-120).
  • chimeric antibodies such as the spUcing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison, S.L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).
  • techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce ENZM-specific single chain antibodies.
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobuUn Ubraries (Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn Ubraries or panels of highly specific binding reagents as disclosed in the Uterature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for ENZM may also be generated.
  • fragments include, but are not Umited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression Ubraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al. (1989) Science 246:1275-1281).
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • K a is defined as the molar concentration of ENZM-antibody complex divided by the molar concentrations of free antigen and free antibody under equiUbrium conditions.
  • K a is defined as the molar concentration of ENZM-antibody complex divided by the molar concentrations of free antigen and free antibody under equiUbrium conditions.
  • the K a determined for a preparation of monoclonal antibodies, which are monospecific for a particular ENZM epitope, represents a true measure of affinity
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are prefe ⁇ ed for use in immunoassays in which the ENZM- antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are prefe ⁇ ed for use in immunopurification and similar procedures which ultimately require dissociation of ENZM, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, ERL Press, Washington DC; LiddeU, J.E.
  • polyclonal antibody preparations may be further evaluated to determine the quahty and suitabiUty of such preparations for certain downstream appUcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of ENZM-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quaUty and usage in various appUcations, are generaUy available (Catty, supra; CoUgan et al., supra).
  • polynucleotides encoding ENZM may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oUgonucleotides) to the coding or regulatory regions of the gene encoding ENZM.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified oUgonucleotides
  • antisense oUgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding ENZM (Agrawal, S., ed. (1996) Antisense Therapeutics. Humana Press, Totawa NJ).
  • Antisense sequences can be deUvered intraceUularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the ceUular sequence encoding the target protein (Slater, J.E. et al. (1998) J. AUergy Clin. Immunol. 102:469-475; Scanlon, K.J. et al. (1995) 9:1288-1296).
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors (MiUer, A.D.
  • polynucleotides encoding ENZM may be used for somatic or germUne gene therapy.
  • Gene therapy may be performed to (i) co ⁇ ect a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCED)-Xl disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C et al.
  • SCED severe combined immunodeficiency
  • ADA adenosine deaminase
  • H express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated ceU proUferation), or (Hi) express a protein which affords protection against intraceUular parasites (e.g., against human retroviruses, such as human Hnmunodeficiency virus (HTV) (Baltimore, D. (1988)
  • diseases or disorders caused by deficiencies in ENZM are treated by constructing mammaUan expression vectors encoding ENZM and introducing these vectors by mechanical means into ENZM-deficient ceUs.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into mdividual ceUs, (H) baUistic gold particle deUvery, (Hi) Hposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Cu ⁇ . Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of ENZM m include, but are not Umited to, the PCDNA 3.1, EPETAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRLPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • ENZM maybe expressed using (i) a constirutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (H) an inducible promoter (e.g., the tetracycUne-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Cu ⁇ . Opin. Biotechnol.
  • a constirutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -act
  • CommerciaUy available Uposome transformation kits e.g., the PERFECT LLPED TRANSFEC ⁇ ON KET, available from Invitrogen
  • aUow one with ordinary skiU Hi the art to dehver polynucleotides to target ceUs in culture and requHe minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammaUan transfection protocols.
  • diseases or disorders caused by genetic defects with respect to ENZM expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding ENZM under the control of an mdependent promoter or the retrovirus long terminal repeat (LTR) promoter, (H) appropriate RNA packaging signals, and (Hi) a Rev-responsive element (RRE) along with additional retrovirus cis-actmg RNA sequences and coding sequences requked for efficient vector propagation.
  • Retro virus vectors e.g., PFB and PFBNEO
  • Retro virus vectors are commerciaUy available (Stratagene) and are based on pubUshed data (Riviere, I. et al. (1995) Proc.
  • the vector is propagated Hi an appropriate vector producing ceU line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Vkol. 61:1647-1650; Bender, M.A. et al. (1987) J. Vkol. 61:1639-1646; Adam, M.A. and A.D. MiUer (1988) J. VHol. 62:3802-3806; DuU, T. et al. (1998) J. VHol.
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging cell Unes and is hereby mcorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T-cells), and the return of transduced ceUs to a patient are procedures weU known to persons skiUed H the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J.
  • an adenovkus-based gene therapy deUvery system is used to deUver polynucleotides encodmg ENZM to ceUs which have one or more genetic abnormaUties with respect to the expression of ENZM.
  • the construction and packaging of adenovkus-based vectors are weU known to those with ordinary skiU Hi the art.
  • RepUcation defective adenovirus vectors have proven to be versatile for importing genes encoding Hnmunoregulatory proteins into intact islets Hi the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenovkal vectors are described in U.S. Patent No.
  • Adenovirus vectors for gene therapy hereby incorporated by reference.
  • adenovkal vectors see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
  • a herpes-based, gene therapy deUvery system is used to deUver polynucleotides encodmg ENZM to target ceUs which have one or more genetic abnormaUties with respect to the expression of ENZM.
  • herpes simplex virus (HSV)-based vectors may be especially valuable for introducing ENZM to ceUs of the central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are weU known to those with ordmary skiU Hi the art.
  • a repUcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to deUver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395).
  • the construction of a HSV-1 virus vector has also been disclosed in detail Hi U.S. Patent No.
  • an alphavirus (positive, single-stranded RNA virus) vector is used to deUver polynucleotides encodmg ENZM to target ceUs.
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • SFV Semliki Forest Virus
  • alphavirus RNA repUcation a subgenomic RNA is generated that normaUy encodes the vkal capsid proteins.
  • This subgenomic RNA repUcates to higher levels than the fuU length genomic RNA, resulting Hi the overproduction of capsid protems relative to the vkal proteins with enzymatic activity (e.g., protease and polymerase).
  • inserting the coding sequence for ENZM mto the alphavirus genome Hi place of the capsid-coding region results Hi the production of a large number of ENZM-coding RNAs and the synthesis of high levels of ENZM Hi vector transduced ceUs.
  • alphavirus infection is typically associated with ceU lysis within a few days
  • the abiUty to estabUsh a persistent infection Hi hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SEN) indicates that the lytic repUcation of alphavkuses can be altered to suit the needs of the gene therapy appUcation (Dryga, S.A. et al. (1997) Vkology 228:74-83).
  • the specific transduction of a subset of ceUs Hi a population may requHe the sorting of ceUs prior to transduction.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the ill transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules
  • Ribozymes may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding ENZM.
  • RNA sequences within any potential RNA target are initiaUy identified by scanning the target molecule for ribozyme cleavage sites, including the foUowing sequences: GUA, GUU, and GUC
  • short RNA sequences of between 15 and 20 ribonucleotides, co ⁇ esponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oUgonucleotide inoperable.
  • the suitabiUty of candidate targets may also be evaluated by testing accessibiUty to hybridization with complementary oUgonucleotides using ribonuclease protection assays.
  • RNA molecules maybe generated by in vitro and in vivo transcription of DNA molecules encoding ENZM. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be mtroduced into ceU Unes, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stabiUty and half-Hfe. Possible modifications mclude, but are not Umited to, the addition of flanking sequences at the 5 ' and/or 3 ' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent Hi the production of PNAs and can be extended Hi aU of these molecules by the mclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • RNAi RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is a post- transcriptional mode of gene silencing H which double-stranded RNA (dsRNA) introduced into a targeted ceU specifically suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantiaUy reduces the expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accompUshed by use of DNA or DNA fragments as weU.
  • RNAi methods are described by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T. (2000; Nature 404:804-808).
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue usmg gene deUvery and/or vkal vector deUvery methods described herein or known Hi the art.
  • RNAi can be induced Hi mammaUan cells by the use of smaU interfering RNA also known as siRNA.
  • siRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides H length) that result in vivo from cleavage of mtroduced dsRNA by the action of an endogenous ribonuclease.
  • SiRNA appear to be the mediators of the RNAi effect n mammals.
  • the most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3' overhangs.
  • the use of siRNA for inducing RNAi Hi mammaUan ceUs is described by Elbashk, S.M. et al. (2001; Nature 411:494-498).
  • SiRNA can either be generated indkectly by introduction of dsRNA into the targeted cell, or dkectly by mammaUan transfection methods and agents described herein or known Hi the art (such as Uposome-mediated transfection, vkal vector methods, or other polynucleotide deUvery/introductory methods).
  • Suitable SiRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occu ⁇ ence of each nucleotide and the 3' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being prefe ⁇ ed.
  • target polynucleotide e.g., mRNA
  • Regions to be avoided for target siRNA sites mclude the 5 ' and 3 ' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these may be richer in regulatory protein binding sites. UTR-binding protems and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known Hi the art. Target sequences with significant homology to other codmg sequences can be eliminated from consideration.
  • the selected SiRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy available methods and kits such as the SILENCER siRNA construction kit (Ambion, Austin TX).
  • long-term gene silencing and/or RNAi effects can be induced in selected tissue using expression vectors that contmuously express siRNA.
  • This can be accompUshed usmg expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known Hi the art (see, e.g., BrummeUcamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958).
  • shRNAs can be deUvered to target ceUs using expression vectors known in the art.
  • siRNA An example of a suitable expression vector for deUvery of siRNA is the PSELENCER1.0-U6 (ckcular) plasmid (Ambion). Once deUvered to the target tissue, shRNAs are processed in vivo mto siRNA-like molecules capable of carrying out gene- specific silencing.
  • the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
  • Expression levels of the mRNA of a targeted gene can be determined by northern analysis methods usmg, for example, the NORTHERNMAX-GLY kit (Ambion); by microa ⁇ ay methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known Hi the art or described herem.
  • Expression levels of the protein encoded by the targeted gene can be determined by Western analysis usmg standard techniques known H the art.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encodmg ENZM.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not Umited to, oUgonucleotides, antisense oUgonucleotides, triple heUx-forming oUgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either Hhibitors or promoters of polynucleotide expression.
  • a compound which specificaUy inhibits expression of the polynucleotide encodmg ENZM may be therapeuticaUy useful
  • Hi the treatment of disorders associated with decreased ENZM expression or activity, a compound which specificaUy promotes expression of the polynucleotide encodmg ENZM may be therapeutically useful.
  • one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known Hi the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commerciaUy-available or proprietary Ubrary of naturaUy-occu ⁇ ing or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a Ubrary of chemical compounds created combinatorially or randomly.
  • a sample comprising a polynucleotide encodmg ENZM is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabiUzed ceU, or an in vitro ceU-free or reconstituted biochemical system.
  • Hi the expression of a polynucleotide encodmg ENZM are assayed by any method commonly known Hi the art.
  • the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encodmg ENZM.
  • the amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective n altering the expression of the polynucleotide.
  • a screen for a compound effective Hi altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU Une such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
  • a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human ceU Une such as HeLa cell (Clarke, M.L. et al. (2000) Biochem
  • a particular embodiment of the present mvention involves screening a combinatorial Ubrary of oUgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oUgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oUgonucleotides
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. DeUvery by transfection, by Uposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (Goldman, C.K. et al. (1997) Nat. Biotechnol. 15:462- 466).
  • any of the therapeutic methods described above may be appUed to any subject Hi need of such therapy, mcluding, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the adrninistration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, celluloses, gums, and protems.
  • Various formulations are commonly known and are thoroughly discussed H the latest edition of Remington's Pharmaceutical Sciences (Maack PubUsbing, Easton PA).
  • Such compositions may consist of ENZM, antibodies to ENZM, and mimetics, agonists, antagonists, or inhibitors of ENZM.
  • compositions described herein such as pharmaceutical compositions
  • Compositions for pulmonary administration may be prepared in Uquid or dry powder form.
  • compositions are generaUy aerosoUzed immediately prior to inhalation by the patient.
  • aerosol deUvery of fast- acting formulations is weU-known in the art.
  • macromolecutes e.g. larger peptides and proteins
  • Pulmonary deUvery aUows adrninistration without needle injection, and obviates the need for potentiaUy toxic penetration enhancers.
  • compositions suitable for use Hi the mvention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is weU within the capabiUty of those skilled in the art.
  • SpeciaUzed forms of compositions may be prepared for dkect intraceUular deUvery of macromolecutes comprising ENZM or fragments thereof.
  • Uposome preparations containing a ceU-Hnpermeable macromolecule may promote ceU fusion and intraceUular deUvery of the macromolecule.
  • ENZM or a fragment thereof may be joined to a short cationic N- terminal portion from the HEV Tat-1 protein. Fusion protems thus generated have been found to transduce into the ceUs of aU tissues, including the brain, Hi a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeutically effective dose can be estimated initiaUy either Hi ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of adrninistration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example ENZM or fragments thereof, antibodies of ENZM, and agonists, antagonists or inhibitors of ENZM, which ameUorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained Hi such compositions is preferably within a range of ckculating concentrations that includes the ED 50 with Uttle or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desked effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of adrninistration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-Ufe and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of adrninistration.
  • Guidance as to particular dosages and methods of deUvery is provided Hi the Uterature and generaUy available to practitioners Hi the art. Those skiUed Hi the art will employ different formulations for nucleotides than for proteins or thek inhibitors. Similarly, deUvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind ENZM may be used for the diagnosis of disorders characterized by expression of ENZM, or H assays to monitor patients being treated with ENZM or agonists, antagonists, or inhibitors of ENZM.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for ENZM mclude methods which utiUze the antibody and a label to detect ENZM H human body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known H the art and may be used.
  • ENZM ELISAs, RIAs, and FACS
  • ELISAs ELISAs
  • RIAs RIAs
  • FACS fluorescence-activated cell sorting
  • Normal or standard values for ENZM expression are estabUshed by combining body fluids or ceU extracts taken from normal mammaUan subjects, for example, human subjects, with antibodies to ENZM under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of ENZM expressed Hi subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values estabUshes the parameters for diagnosing disease.
  • polynucleotides encodmg ENZM may be used for diagnostic purposes.
  • the polynucleotides which may be used mclude oUgonucleotides, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression Hi biopsied tissues Hi which expression of ENZM may be co ⁇ elated with disease.
  • the diagnostic assay may be used to dete ⁇ nine absence, presence, and excess expression of ENZM, and to monitor regulation of ENZM levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, mcluding genomic sequences, encoding ENZM or closely related molecules may be used to identify nucleic acid sequences which encode ENZM.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or ampUfication wiU determine whether the probe identifies only naturaUy occurring sequences encodmg ENZM, alleUc variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the ENZM encoding sequences.
  • the hybridization probes of the subject mvention may be DNA or RNA and may be derived from the sequence of SEQ TD NO:58-l 14 or from genomic sequences including promoters, enhancers, and introns of the ENZM gene.
  • Means for producing specific hybridization probes for polynucleotides encoding ENZM m include the cloning of polynucleotides encodmg ENZM or ENZM derivatives into vectors for the production of mRNA probes.
  • Such vectors are known H the art, are commerciaUy available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • reporter groups for example, by radionucUdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding ENZM may be used for the diagnosis of disorders associated with expression of ENZM.
  • disorders include, but are not Umited to, an autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome (ALDS), Addison's disease, adult respkatory distress syndrome, aUergies, ankylosing spondyUtis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes meUitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaUs, erythema nodosum, atrophic gastritis,
  • Polynucleotides encodmg ENZM may be used Hi Southern or northern analysis, dot blot, or other membrane-based technologies; Hi PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and Hi microa ⁇ ays utiUzing fluids or tissues from patients to detect altered ENZM expression. Such quaUtative or quantitative methods are weU known H the art.
  • polynucleotides encodmg ENZM may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encodmg ENZM may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding ENZM in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in cUnical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is estabUshed. This may be accompUshed by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encodmg ENZM, under conditions suitable for hybridization or ampUfication.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained Hi this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to estabUsh the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed Hi the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • Hi biopsied tissue from an mdividual may indicate a predisposition for the development, of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earUer, thereby preventing the development or further progression of the cancer.
  • oUgonucleotides designed from the sequences encoding ENZM may involve the use of PCR. These oUgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
  • OUgomers wiU preferably contain a fragment of a polynucleotide encodmg ENZM, or a fragment of a polynucleotide complementary to the polynucleotide encodmg ENZM, and wiU be employed under optimized conditions for identification of a specific gene or condition. OUgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oUgonucleotide primers derived from polynucleotides encoding ENZM may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease Hi humans. Methods of SNP detection mclude, but are not Umited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oUgonucleotide primers derived from polynucleotides encoding ENZM are used to ampUfy DNA usmg the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the DNA maybe derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the Hke.
  • SNPs Hi the DNA cause differences in the secondary and tertiary structures of PCR products H smgle-stranded form, and these differences are detectable using gel electrophoresis H non-denaturing gels.
  • the oUgonucleotide primers are fluorescently labeled, which allows detection of the ampUmers Hi high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy, sequence database analysis methods, termed in siUco SNP (isSNP) are capable of identifying polymorphisms by comparing the sequence of mdividual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA). SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitus. SNPs are also useful for examining differences H disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease.
  • variants in the mannose-binding lectin, MBL2 have been shown to be co ⁇ elated with deleterious pulmonary outcomes Hi cystic fibrosis.
  • SNPs also have utiUty Hi pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as Ufe-threatening toxicity.
  • a variation in N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, while a variation H the core promoter of the ALOX5 gene results Hi diminished clinical response to treatment with an anti-asthma drug that targets the 5-Upoxygenase pathway.
  • oUgonucleotides or longer fragments derived from any of the polynucleotides described herem may be used as elements on a microa ⁇ ay.
  • the microarray can be used Hi transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microa ⁇ ay may also be used to identify genetic variants, mutations, and polymorphisms. This information maybe used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents Hi the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient H order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • ENZM, fragments of ENZM, or antibodies specific for ENZM may be used as elements on a microa ⁇ ay.
  • the microa ⁇ ay may be used to monitor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present mvention to generate a transcript image of a tissue or cell type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No. 5,840,484; hereby expressly incorporated by reference herem).
  • a transcript image may be generated by hybridizing the polynucleotides of the present mvention or their complements to the totaUty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in mgh-throughput format, wherein the polynucleotides of the present mvention or thek complements comprise a subset of a pluraUty of elements on a microa ⁇ ay.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images maybe generated using transcripts isolated from tissues, ceU Unes, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as H the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
  • Transcript images which profile the expression of the polynucleotides of the present mvention may also be used in conjunction with in vitro model systems and precUnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naturaUy-occurring environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysk, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113 :467-471). If a test compound has a signature similar to that of a compound with known toxicity, it is Ukely to share those toxic properties.
  • the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present mvention, so that transcript levels correspondmg to the polynucleotides of the present mvention may be quantified.
  • the transcript levels Hi the treated biological sample are compared with levels Hi an untreated biological sample. Differences Hi the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression H a particular tissue or ceU type.
  • proteome expression patterns, or profiles are analyzed by quantifying the number of expressed protems and their relative abundance under given conditions and at a given time.
  • a profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, H which protems from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuaUzed Hi the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stams.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protem spots from different samples are compared to identify any changes Hi protein spot density related to the treatment.
  • the proteins H the spots are partiaUy sequenced usmg, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protem in a spot may be determmed by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for ENZM to quantify the levels of ENZM expression.
  • the antibodies are used as elements on a microa ⁇ ay, and protem expression levels are quantified by exposing the microa ⁇ ay to the sample and detecting the levels of protem bound to each a ⁇ ay element (Lueking, A. et al. (1999) Anal. Biochem. 270:103- 111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
  • Detection maybe performed by a variety of methods known in the art, for example, by reacting the protems in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed Hi parallel with toxicant signatures at the transcript level.
  • There is a poor co ⁇ elation between transcript and protem abundances for some proteins in some tissues (Anderson, N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful Hi the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts H body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reUable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound.
  • Proteins that are expressed Hi the treated biological sample are separated so that the amount of each protem can be quantified.
  • the amount of each protem is compared to the amount of the co ⁇ esponding protein in an untreated biological sample. A difference in the amount of protem between the two samples is indicative of a toxic response to the test compound Hi the treated sample.
  • Individual protems are identified by sequencing the amino acid residues of the individual protems and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present mvention. The amount of protein recognized by the antibodies is quantified.
  • the amount of protein in the treated biological sample is compared with the amount Hi an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microa ⁇ ays may be prepared, used, and analyzed usmg methods known Hi the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT appUcation WO95/25116; Shalon, D. et al. (1995) PCT appUcation WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S. Patent No. 5,605,662).
  • Various types of microa ⁇ ays are well known and thoroughly described Hi Schena, M., ed. (1999; DNA Microa ⁇ ays: A Practical Approach, Oxford University Press,
  • nucleic acid sequences encoding ENZM may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence. Either codmg or noncodmg sequences may be used, and Hi some instances, noncoding sequences may be preferable over codmg sequences. For example, conservation of a codmg sequence among members of a multi-gene family may potentiaUy cause undesked cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA Ubraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA Ubraries
  • nucleic acid sequences may be used to develop genetic Unkage maps, for example, which co ⁇ elate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization may be co ⁇ elated with other physical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the OnUne MendeUan Inheritance Hi Man (OMEM) World Wide Web site. Co ⁇ elation between the location of the gene encoding ENZM on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • FISH Fluorescent in situ hybridization
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
  • placement of a gene on the chromosome of another mammaUan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional clonmg or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences H the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • ENZM in another embodiment of the mvention, ENZM, its catalytic or immunogenic fragments, or oUgopeptides thereof can be used for screening Ubraries of compounds H any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a soUd support, borne on a ceU surface, or located intraceUularly.
  • the formation of bmdmg complexes between ENZM and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT appUcation WO84/03564).
  • This method large numbers of different smaU test compounds are synthesized on a soUd substrate. The test compounds are reacted with ENZM, or fragments thereof, and washed.
  • Bound ENZM is then detected by methods weU known Hi the art.
  • Purified ENZM can also be coated dkectly onto plates for use in the aforementioned drug screening techniques.
  • non-neutraUzing antibodies can be used to capture the peptide and immobiUze it on a soUd support.
  • nucleotide sequences which encode ENZM may be used Hi any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are cu ⁇ ently known, includmg, but not Umited to, such properties as the triplet genetic code and specific base pak interactions.
  • nucleotide sequences that are cu ⁇ ently known, includmg, but not Umited to, such properties as the triplet genetic code and specific base pak interactions.
  • poly(A)+ RNA was isolated using oUgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was provided with RNA and constructed the co ⁇ esponding cDNA Ubraries. Otherwise, cDNA was synthesized and cDNA Ubraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription was initiated usmg oUgo d(T) or random primers. Synthetic oUgonucleotide adapters were Ugated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes.
  • cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs were Ugated mto compatible restriction enzyme sites of the polyUnker of a suitable plasmid, e.g., PBLUESCREPT plasmid
  • Plasmids obtained as described in Example I were recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN.
  • Plasmids were resuspended Hi 0.1 ml of distiUed water and stored, with or without lyophiUzation, at 4°C
  • plasmid DNA was ampUfied from host ceU lysates using direct link PCR Hi a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps were carried out Hi a single reaction mixture.
  • Incyte cDNA recovered Hi plasmids as described H Example II were sequenced as foUows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (AppUed Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the
  • cDNA sequencing reactions were prepared using reagents provided by Amersham Biosciences or suppUed Hi ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (AppUed Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (AppUed Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art.
  • Reading frames within the cDNA sequences were identified usmg standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences were selected for extension usmg the techniques disclosed Hi Example VLU.
  • the polynucleotide sequences derived from Incyte cDNAs were vaUdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, usmg algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof were then queried against a selection of pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto CA); hidden Markov model (HMM)-based protem family databases such as PFAM, LNCY, and TIGRFAM (Haft, D.H.
  • HMM hidden Markov model
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabiUstic approach which analyzes consensus primary structures of gene famiUes; see, for example, Eddy, S.R. (1996) Cu ⁇ . Opin. Struct. Biol. 6:361-365.
  • the queries were performed usmg programs based on BLAST, FASTA, BLEMPS, and HMMER.
  • the Incyte cDNA sequences were assembled to produce full length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted codmg sequences were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the fuU length polynucleotide sequences were translated to derive the co ⁇ esponding fuU length polypeptide sequences.
  • a polypeptide may begin at any of the methionme residues of the fuU length translated polypeptide.
  • FuU length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protem databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family databases such as PFAM, LNCY, and TIGRFAM; and HMM-based protem domain databases such as SMART.
  • GenBank protem databases Genpept
  • SwissProt the PROTEOME databases
  • BLOCKS BLOCKS
  • PRINTS DOMO
  • PRODOM Prosite
  • Prosite Prosite
  • HMM-based protein family databases such as PFAM, LNCY, and TIGRFAM
  • HMM-based protem domain databases such as SMART.
  • FuU length polynucleotide sequences are also analyzed usmg MACDNASIS PRO software (Mirai
  • Polynucleotide and polypeptide sequence aUgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence aUgnment program (DNASTAR), which also calculates the percent identity between aUgned sequences.
  • Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides appUcable descriptions, references, and threshold parameters.
  • the fkst column of Table 7 shows the tools, programs, and algorithms used
  • the second column provides brief descriptions thereof
  • the third column presents appropriate references, aU of which are incorporated by reference herem Hi thek entirety
  • the fourth column presents, where appUcable, the scores, probabiUty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probabiUty value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C and S. Karlin (1998) Cu ⁇ . Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionme to a stop codon.
  • the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • Genscan The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode enzymes, the encoded polypeptides were analyzed by querying against PFAM models for enzymes. Potential enzymes were also identified by homology to Incyte cDNA sequences that had been annotated as enzymes. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri pubUc databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct e ⁇ ors in the sequence predicted by Genscan, such as extra or omitted exons.
  • Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described Hi Example EV. Partial cDNAs assembled as described in Example ELI were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed usmg an algorithm based on graph theory and dynamic prograiriming to integrate cDNA and genomic information, generating possible spUce variants that were subsequently confirmed, edited, or extended to create a fuU length sequence. Sequence mtervals in which the entke length of the interval was present on more than one sequence Hi the cluster were identified, and mtervals thus identified were considered to be equivalent by transitivity.
  • Enco ⁇ ect exons predicted by Genscan were co ⁇ ected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary. "Stretched" Sequences
  • Partial DNA sequences were extended to fuU length with an algorithm based on BLAST analysis.
  • First, partial cDNAs assembled as described in Example EQ were queried against pubUc databases such as the GenBank primate, rodent, mammaUan, vertebrate, and eukaryote databases using the BLAST program.
  • the nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example EV.
  • a chimeric protein was generated by usmg the resultant high-scoring segment paks (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur Hi the chimeric protein with respect to the original GenBank protem homolog.
  • GenBank protem homolog the chimeric protein, or both were used as probes to search for homologous genomic sequences from the pubUc human genome databases. Partial DNA sequences were therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene. VI. Chromosomal Mapping of ENZM Encoding Polynucleotides
  • sequences which were used to assemble SEQ ED NO:58-114 were compared with sequences from the Encyte LEFESEQ database and pubUc domain databases usmg BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ED NO:58-l 14 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from pubUc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to deterrnine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence Hi a cluster resulted Hi the assignment of aU sequences of that cluster, includmg its particular SEQ ED NO:, to that map location.
  • pubUc resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon were used to deterrnine
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p- arm.
  • centiMorgan cM
  • centiMorgan is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA Hi humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included Hi each of the clusters.
  • Lewy body Parkinson disease has been thought to be a specific autosomal dominant disorder (Wakabayashi, K. et al. (1998) Acta Neuropath. 96:207-210).
  • Juvenile parkinsonism may be a specific autosomal recessive disorder (Matsumine, H. et al. (1997) Am. J. Hum. Genet. 60: 588-596).
  • Lod score is a statistical method used to test the Unkage of two or more loci within famiUes having a genetic disease.
  • the lod score is the logarithm to base 10 of the odds in favor of Unkage.
  • Linkage is defined as the tendency of two genes located on the same chromosome to be inherited together through meiosis (Genetics in Medicine, Fifth Edition, (1991) Thompson, M.W. et al., W.B. Saunders Co. Philadelphia).
  • a lod score of +3 or greater indicates a probabiUty of 1 Hi 1000 that a particular marker was found solely by chance Hi affected individuals, which is strong evidence that two genetic loci are Unked.
  • PARK3 maps to 2pl3 (Gasser, T. et al. (1998) Nature Genet. 18:262-265).
  • a marker at chromosomal position D2S441 was found to have a lod score of 3.2 Hi the region of PARK3. This marker supported the disease association of PARK3 Hi the chromosomal interval from D2S134 to D2S286 (Gasser et al., supra).
  • markers were obtained with lod scores greater than 3 includmg D1S199, D1S2732, D1S2828, D1S478, D1S2702, D1S2734, D1S2674 (Valente, E.M. et al, supra). These markers were used to determine the PD-relevant range of chromosome loci and identify sequences that map to chromosome 1 between D1S199 and D1S2885.
  • RFLP Restriction fragment length polymorphism
  • a preliminary step used an algorithm, similar to MEGABLAST, to define the mRNA sequence /masked genomic DNA contig pairings.
  • the cDNA/genomic pairings identified by the first algorithm were confirmed, and the ENZM polynucleotides mapped to DNA contigs, usmg SEM4 (Florea, L. et al. (1998) Genome Res. 8:967-974, version May 2000) which had been optimized for high throughput processing and strand assignment confidence.
  • the STM4 output of the mRNA sequence/genomic contig paks was further processed to determine the co ⁇ ect location of the ENZM polynucleotides on the genomic contig, as weU as thek strand identity.
  • SEQ LD NO:82 was mapped to Contig GBI:NT_005420_002.8 from Genbank release February 2002, covering a 9.65 Mb region of the genome that also contains PD-associated genetic markers D2S134 and D2S286.
  • SEQ ED NO:82 is in proximity with genetic markers shown to consistently associate with PD.
  • SEQ ED NO:82 can be used for one or more of the foUowing: i) Unkage analysis of persons and/or famiUes to the PD disease region at 2pl3, H) diagnostic assays for PD, and Hi) developing therapeutics and/or other treatments for PD. VII. Analysis of Polynucleotide Expression
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7; Ausubel et al., supra, ch. 4).
  • the product score takes into account both the degree of similarity between two sequences and the length of the sequence match.
  • the product score is a normaUzed value between 0 and 100, and is calculated as foUows: the BLAST score is multipUed by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches Hi a high-scoring segment pak (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pak with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quaUty in a BLAST aUgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotides encoding ENZM are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example HI). Each cDNA sequence is derived from a cDNA Ubrary constructed from a human tissue.
  • Each human tissue is classified mto one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitaUa, female; genitaUa, male; germ ceUs; hemic and immune system; Uver; musculoskeletal system; nervous system; pancreas; respkatory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of Ubraries Hi each category is counted and divided by the total number of Ubraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, cell Une, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of Ubraries in each category is counted and divided by the total number of Ubraries across aU categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding ENZM.
  • cDNA sequences and cDNA Ubrary/tissue information are found Hi the LEFESEQ GOLD database (Incyte Genomics, Palo Alto CA). VIII.
  • FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using oUgonucleotide primers designed from this fragment. One primer was synthesized to initiate 5 'extension of the known fragment, and the other primer was synthesized to initiate 3 ' extension of the known fragment.
  • the initial primers were designed using OLEGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides Hi length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C Any stretch of nucleotides which would result Hi hairpin structures and primer-primer dimerizations was avoided.
  • the concentration of DNA Hi each weU was dete ⁇ nined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 ⁇ l of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent.
  • the plate was scanned Hi a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l aUquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose gel to determine which reactions were successful in extending the sequence.
  • the extended nucleotides were desalted and concentrated, transfe ⁇ ed to 384-weU plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to reUgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to reUgation into pUC 18 vector
  • the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega).
  • Extended clones were reUgated using T4 Ugase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fiU-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37 °C Hi 384-weU plates Hi LB/2x carb Uquid media.
  • fuU length polynucleotides are verified using the above procedure or are used to obtain 5 'regulatory sequences usmg the above procedure along with oUgonucleotides designed for such extension, and an appropriate genomic Ubrary.
  • SNPs single nucleotide polymorphisms
  • Preliminary filters removed the majority of basecaU e ⁇ ors by requiring a niinimum Phred quaUty score of 15, and removed sequence aUgnment e ⁇ ors and e ⁇ ors resulting from improper trirnming of vector sequences, chimeras, and spUce variants.
  • An automated procedure of advanced chromosome analysis analysed the original chromatogram files in the vicinity of the putative SNP.
  • Clone e ⁇ or filters used statistically generated algorithms to identify e ⁇ ors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering e ⁇ or filters used statistically generated algorithms to identify e ⁇ ors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences. A final set of filters removed dupUcates and SNPs found in immunoglobulins or T-ceU receptors.
  • Certain SNPs were selected for further characterization by mass spectrometry usmg the high throughput MASSARRAY system (Sequenom, Inc.) to analyze aUele frequencies at the SNP sites Hi four different human populations.
  • the Caucasian population comprised 92 individuals (46 male, 46 female), includmg 83 from Utah, four French, three deciualan, and two Amish mdividuals.
  • the African population comprised 194 mdividuals (97 male, 97 female), aU African Americans.
  • the Hispanic population comprised 324 individuals (162 male, 162 female), aU Mexican Hispanic.
  • the Asian population comprised 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian.
  • AUele frequencies were first analyzed Hi the Caucasian population; Hi some cases those SNPs which showed no aUeUc variance Hi this population were not further tested Hi the other three populations.
  • Hybridization probes derived from SEQ ED NO:58-114 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeUng of oUgonucleotides, consisting of about 20 base paks, is specificaUy described, essentially the same procedure is used with larger nucleotide fragments.
  • OUgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled oUgonucleotides are substantiaUy purified using a SEPHADEX G-25 superfine size exclusion dextranbead column (Amersham Biosciences).
  • blots are sequentiaUy washed at room temperature under conditions of up to, for example, 0.1 x saUne sodium citrate and 0.5% sodium dodecyl sulfate.
  • Hybridization patterns are visuaUzed usmg autoradiography or an alternative imaging means and compared.
  • the Unkage or synthesis of a ⁇ ay elements upon a microa ⁇ ay can be achieved utiUzing photoUthography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate Hi each of the aforementioned technologies should be uniform and soUd with a non-porous surface (Schena, M., ed. (1999) DNA Microa ⁇ ays: A Practical Approach, Oxford University Press, London). Suggested substrates include siUcon, siUca, glass sUdes, glass chips, and siUcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to a ⁇ ange and Unk elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical a ⁇ ay may be produced usmg available methods and machines well known to those of ordmary skiU Hi the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oUgomers thereof may comprise the elements of the microarray. Fragments or oUgomers suitable for hybridization can be selected usmg software weU known Hi the art such as LASERGENE software (DNASTAR).
  • the array elements are hybridized with polynucleotides Hi a biological sample.
  • the polynucleotides Hi the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each a ⁇ ay element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microa ⁇ ay may be assessed.
  • microa ⁇ ay preparation and usage is described in detail below.
  • RNA is isolated from tissue samples using the guamdinium thiocyanate method and poly(A) + RNA is purified usmg the oUgo-(dT) ceUulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oUgo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMBRIGHT kits (Incyte Genomics).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeUng) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
  • Samples are purified using two successive CHROMA SPLN 30 gel filtration spin columns (Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion usmg a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • a SpeedVAC SpeedVAC
  • Sequences of the present invention are used to generate a ⁇ ay elements.
  • Each a ⁇ ay element is ampUfied from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR ampUfication uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are ampUfied Hi thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • AmpUfied a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified a ⁇ ay elements are immobiUzed on polymer-coated glass sUdes.
  • Glass microscope sUdes (Corning) are cleaned by ultrasound H 0.1% SDS and acetone, with extensive distiUed water washes between and after treatments.
  • Glass sUdes are etched Hi 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively H distiUed water, and coated with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis MO) in 95% ethanol.
  • Coated sUdes are cured Hi a 110°C oven.
  • a ⁇ ay elements are appUed to the coated glass substrate using a procedure described Hi U.S.
  • Patent No. 5,807,522 incorporated herem by reference.
  • 1 ⁇ l of the a ⁇ ay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capiUary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of a ⁇ ay element sample per sUde.
  • Microa ⁇ ays are UV-crossUnked using a STRATALLNKER UV-crossUnker (Stratagene).
  • Microarrays are washed at room temperature once in 0.2% SDS and three times Hi distilled water.
  • Non-specific bmdmg sites are blocked by incubation of microa ⁇ ays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C foUowed by washes Hi 0.2%
  • PBS phosphate buffered saline
  • Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
  • Cy5 labeled cDNA synthesis products Hi 5X SSC, 0.2% SDS hybridization buffer.
  • the sample mixture is heated to 65° C for 5 minutes and is aUquoted onto the microa ⁇ ay surface and covered with an 1.8 cm 2 coversUp.
  • the arrays are transfe ⁇ ed to a waterproof chamber having a cavity just sUghtly larger than a microscope sUde.
  • the chamber is kept at 100% humidity internally by the addition of 140 ⁇ l of 5X SSC H a corner of the chamber.
  • the chamber containing the a ⁇ ays is incubated for about 6.5 hours at 60°C
  • the a ⁇ ays are washed for 10 min at 45°C in a fkst wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45° C H a second wash buffer (0.1X SSC), and dried.
  • Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an
  • Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral Unes at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser Ught is focused on the a ⁇ ay using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the sUde containing the a ⁇ ay is placed on a computer-controUed X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm a ⁇ ay used H the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiUne laser excites the two fluorophores sequentiaUy. Emitted Ught is spUt, based on wavelength, into two photomultipUer tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores. Appropriate filters positioned between the array and the photomultipUer tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each a ⁇ ay is typicaUy scanned twice, one scan per fluorophore usmg the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typicaUy caUbrated usmg the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the a ⁇ ay contains a complementary DNA sequence, aUowing the intensity of the signal at that location to be co ⁇ elated with a weight ratio of hybridizing species of 1:100,000.
  • the caUbration is done by labeUng samples of the caUbrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output of the photomultipUer tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) instaUed Hi an EBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a Unear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore 's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value co ⁇ esponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte Genomics).
  • Array elements that exhibit at least about a two-fold change Hi expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed.
  • Expression for example, SEQ ED NO:64 showed differential expression H tumorous or diseased tissue versus non-tumorous or healthy tissues, as determined by microa ⁇ ay analysis.
  • Lung cancer is the leading cause of cancer death for men and the second leading cause of cancer death for women in the U.S.
  • Lung cancers are divided mto four histopathologically distinct groups. Three groups (squamous ceU carcinoma, adenocarcinoma, and large ceU carcinoma) are classified as non-smaU ceU lung cancers (NSCLCs). The fourth group of cancers is refe ⁇ ed to as small cell lung cancer (SCLC).
  • Deletions on chromosome 3 are common Hi this disease and are thought to indicate the presence of a tumor suppressor gene Hi this region.
  • Activating mutations Hi K- ras are commonly found in lung cancer and are the basis of one of the mouse models for the disease.
  • SEQ ED NO:64 was downregulated by at least two-fold in lung cancer tissue in two separate experiments. In one experiment, normal lung tissue from a 68 year-old female was compared to lung tumor from the same donor (Roy Castle International Centre for Lung Cancer Research, Liverpool, UK). In another experiment, grossly uninvolved lung tissue from a 71 year-old female was compared to lung adenocarcinoma tissue from the same donor.
  • SEQ TD NO:64 exhibited significant differential expression patterns usmg microa ⁇ ay techniques, and further estabUsh its utiUty as a diagnostic marker or therapeutic agent which may be useful for lung cancer and other diseases or disorders involving enzymes.
  • SEQ ED NO:81, SEQ ED NO:85, SEQ ED NO:89, SEQ ED NO:91, SEQ ED NOJOl, SEQ ED NOJ02, SEQ ED NOJ05, SEQ ED NOJ13, and SEQ ED NOJ14 exhibited differential expression in tumorous tissue as compared to non-tumorous, healthy tissue, as determined by microa ⁇ ay analysis.
  • Array elements that exhibited about at least a two-fold change Hi expression and a signal intensity over 250 units, a signal-to-background ratio of a least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics).
  • SEQ TD NO:81, SEQ ED NO:91, SEQ ED NOJ01, SEQ LD NOJ02, SEQ ED NO:105, and SEQ ED NO:114 was decreased by at least two-fold when comparing uninvolved, normal human colon tissue versus adenocarcinoma colon tumor tissue obtained from the same donor, as determmed by microarray analysis. Therefore, SEQ ED NO:81, SEQ ED NO:91, SEQ ED NOJ01, SEQ ED NO: 102, SEQ ED NO: 105, and SEQ ED NO: 114 are useful Hi monitoring treatment of, and diagnostic assays for, colon cancer.
  • SEQ TD NO:85, SEQ LD NO: 89, and SEQ ED NO:l 13 were decreased by at least two-fold when comparing uninvolved, normal human lung tissue to squamous ceU carcinoma and adenocarcinoma tumor tissue obtained from the same donor, as determined by microa ⁇ ay analysis. Therefore, SEQ ED NO:85, SEQ ED NO: 89, and SEQ ED NO:113 are useful Hi monitoring treatment of, and diagnostic assays for lung cancer.
  • SEQ ED NO: 89 The expression of SEQ ED NO: 89 was decreased by at least two-fold when comparing uninvolved, normal human breast tissue to invasive lobular carcinoma in situ tissue obtained from the same donor, as determmed by microarray analysis. Therefore, SEQ ED NO: 89 is useful H monitoring treatment of, and diagnostic assays for breast cancer.
  • SEQ TD NO: 89 and SEQ ED NO 13 were decreased by at least two-fold when comparing uninvolved, normal human ovary tissue to ovarian adenocarcinoma tissue obtained from the same donor, as determined by microa ⁇ ay analysis. Therefore, SEQ ED NO: 89 and SEQ ED NO: 113 are useful Hi monitoring treatment of, and diagnostic assays for ovarian cancer.
  • SEQ ED NO:99, SEQ ED NO: 104, and SEQ ED NO: 112 exhibited differential expression Hi Tangier-derived fibroblast ceUs as compared to normal fibroblast cells, as determined by microa ⁇ ay analysis.
  • a ⁇ ay elements that exhibited about at least a two-fold change Hi expression and a signal intensity over 250 units, a signal-to-background ratio of a least 2.5, and an element spot size of at least 40% were identified as differentiaUy expressed using the GEMTOOLS program (Incyte Genomics).
  • SEQ ED NO:99 The expression of SEQ ED NO:99, SEQ ED NO: 104, and SEQ ED NO: 112 was decreased by at least two-fold Hi Tangier disease-derived fibroblasts compared to normal fibroblasts.
  • both types of ceUs were cultured Hi the presence of cholesterol and compared with the same ceU type cultured Hi the absence of cholesterol.
  • Human fibroblasts were obtained from skin explants from both normal subjects and two patients with homozygous Tangier disease.
  • CeU Unes were knmortaUzed by transfection with human papiUomavirus 16 genes E6 and E7 and a neomycin resistance selectable marker.
  • TD-derived ceUs are deficient Hi an assay of apoA-I mediated tritiated cholesterol efflux. Therefore, SEQ TD NO:99, SEQ ED NOJ04, and SEQ ED NOJ12 are useful Hi diagnostic assays for Tangier disease.
  • Sequences complementary to the ENZM-encoding sequences, or any parts thereof, are used to detect, decrease, or mhibit expression of naturaUy occurring ENZM.
  • oUgonucleotides comprising from about 15 to 30 base paks is described, essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oUgonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the codmg sequence of ENZM.
  • a complementary oUgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the codmg sequence.
  • a complementary oUgonucleotide is designed to prevent ribosomal binding to the ENZM-encoding transcript.
  • ENZM is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned mto an appropriate vector containing an antibiotic resistance gene and an inducible promoter that dkects high levels of cDNA transcription.
  • promoters include, but are not Umited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express ENZM upon induction with isopropyl beta-D- thiogalactopyranoside (EPTG).
  • ENZM Hi eukaryotic ceUs is achieved by infecting insect or mammaUan cell Unes with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculo virus.
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculo virus is replaced with cDNA encoding ENZM by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Vkal infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculo virus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases.
  • baculovkus Infection of the latter requkes additional genetic modifications to baculovkus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937- 1945).
  • ENZM is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude ceU lysates.
  • GST a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobiUzed glutathione under conditions that maintain protem activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticaUy cleaved from ENZM at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protem expression and purification are discussed Hi Ausubel et al. (supra, ch. 10 and 16). Purified ENZM obtained by these methods can be used dkectly Hi the assays shown Hi Examples XV ⁇ , XVm, and XLX, where appUcable. XIV. Functional Assays
  • ENZM function is assessed by expressing the sequences encodmg ENZM at physiologicaUy elevated levels Hi mammaUan ceU culture systems.
  • cDNA is subcloned mto a mammaUan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice mclude PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalo virus promoter.
  • recombinant vector 5-10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endotheUal or hematopoietic cell line, using either Uposome formulations or electroporation. 1-2 ⁇ g of an additional plasmid containing sequences encodmg a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected ceUs and is a reUable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protem
  • FCM Flow cytometry
  • the influence of ENZM on gene expression can be assessed using highly purified populations of ceUs transfected with sequences encodmg ENZM and either CD64 or CD64-GFP.
  • CD64 and CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobuUn G (IgG).
  • Transfected cells are efficiently separated from nontransfected ceUs using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY).
  • mRNA can be purified from the ceUs using methods weU known by those of skiU Hi the art. Expression of mRNA encodmg ENZM and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques.
  • the ENZM amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a co ⁇ esponding oUgopeptide is synthesized and used to raise antibodies by means known to those of skiU in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophiUc regions are well described in the art (Ausubel et al., supra, ch. 11).
  • oUgopeptides of about 15 residues Hi length are synthesized usmg an ABI 431 A peptide synthesizer (AppUed Biosystems) usmg FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleHmdobenzoyl-N-hydroxysuccmimide ester (MBS) to increase immunogenicity (Ausubel et al., supra). Rabbits are immunized with the oUgopeptide-KLH complex in complete Freund's adjuvant.
  • Resulting antisera are tested for antipeptide and anti-ENZM activity by, for example, binding the peptide or ENZM to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Media containing ENZM are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of ENZM (e.g., high ionic strength buffers Hi the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/ENZM binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chao trope, such as urea or thiocyanate ion), and ENZM is coUected.
  • ENZM or biologicaUy active fragments thereof, are labeled with 125 I Bolton-Hunter reagent (Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539).
  • Candidate molecules previously arrayed Hi the weUs of a multi-weU plate are incubated with the labeled ENZM, washed, and any weUs with labeled ENZM complex are assayed. Data obtained using different concentrations of ENZM are used to calculate values for the number, affinity, and association of ENZM with the candidate molecules.
  • molecules interacting with ENZM are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or usmg commerciaUy available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).
  • ENZM may also be used Hi the PAT ⁇ CALLENG process (CuraGen Corp., New Haven CT) which employs the yeast two-hybrid system Hi a high-throughput manner to determine all interactions between the protems encoded by two large Ubraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • ENZM activity is demonstrated through a variety of specific enzyme assays; some of which are outlined below.
  • ENZM oxidoreductase activity is measured by the increase in extinction coefficient of
  • NAD(P)H coenzyme at 340 nmfor the measurement of oxidation activity or the decrease Hi extinction coefficient of NAD(P)H coenzyme at 340 nmfor the measurement of reduction activity (Dalziel, K. (1963) J. Biol. Chem. 238:2850-2858).
  • One of three substrates may be used: Asn- ⁇ Gal, biocytidine, or ubiquinone-10.
  • the respective subunits of the enzyme reaction, for example, cytochrome -b oxidoreductase and cytochrome c, are reconstituted.
  • the reaction mixture contams a)l-2 mg/ml ENZM; and b) 15 mM substrate, 2.4 mM NAD(P) + in 0.1 M phosphate buffer, pH 7.1 (oxidation reaction), or 2.0 mM NAD(P)H, in 0.1 M Na j HP j buffer, pH 7.4 ( reduction reaction); Hi a total volume of 0.1 ml.
  • Changes in absorbance at 340 nm (A 340 ) are measured at 23.5 °C using a recording spectrophotometer (Shimadzu Scientific Instruments, Inc., Pleasanton, CA).
  • ENZM activity is proportional to the amount of NAD(P)H present in the assay.
  • Aldo/keto reductase activity of ENZM is proportional to the decrease in absorbance at 340 nm as NADPH is consumed (or increased absorbance if NADPH is produced, i.e., if the reverse reaction is monitored).
  • a standard reaction mixture is 135 mM sodium phosphate buffer (pH 6.2-7.2 depending on enzyme), 0.2 mM NADPH, 0.3 M Utbium sulfate, 0.5-2.5 mg ENZM and an appropriate level of substrate. The reaction is incubated at 30 °C and the reaction is momtored contmuously with a spectrophotometer.
  • ENZM activity is calculated as mol NADPH consumed / mg of ENZM.
  • Acyl-CoA dehydrogenase activity of ENZM is measured using an anaerobic electron transferring flavoprotein (ETF) assay.
  • the reaction mixture comprises 50 mM Tris-HCl (pH 8.0), 0.5% glucose, and 50 ⁇ M acyl-CoA substrate (i.e., isovaleryl-CoA) that is pre-warmed to 32 °C
  • the mixture is depleted of oxygen by repeated exposure to vacuum foUowed by layering with argon.
  • Trace amounts of oxygen are removed by the addition of glucose oxidase and catalase foUowed by the addition of ETF to a final concentration of 1 ⁇ M.
  • the reaction is initiated by addition of purified ENZM or a sample containing ENZM and exciting the reaction at 342 nm. Quenching of fluorescence caused by the transfer of electrons from the substrate to ETF is monitored at 496 nm.
  • 1 unit of acyl-CoA dehydrogenase activity is defined as the amount of ENZM requked to reduce 1 ⁇ mol of ETF per minute (Reinard, T. et al. (2000) J. Biol. Chem. 275:33738-33743).
  • Substrate (e.g., ethanol) and ENZM are then added to the reaction.
  • Aldehyde dehydrogenase activity of ENZM is measured by deterrnining the total hydrolase + dehydrogenase activity of ENZM and subtracting the hydrolase activity.
  • Hydrolase activity is first determined in a reaction mixture containing 0.05 M Tris-HCl (pH 7.8), 100 mM 2-mercaptoethanol, and 0.5-18 ⁇ M substrate, e.g., 10-HCO-HPteGlu (10-formyltetrahydrofolate; HPteGlu, tetrahydrofolate) or 10-FDDF (10-formyl-5,8-dideazafolate).
  • 10-HCO-HPteGlu 10-formyltetrahydrofolate
  • HPteGlu tetrahydrofolate
  • 10-FDDF 10-FDDF
  • the reaction is monitored and read against a blank cuvette, containing all components except enzyme.
  • the appearance of product is measured at either 295 nm for 5,8-dideazafolate or 300 nm for HPteGlu using molar extinction coefficients of 1.89xl0 4 and 2.17xl0 4 for 5,8-dideazafolate and HPteGlu, respectively.
  • the addition of NADP + to the reaction mixture aUows the measurement of both dehydrogenase and hydrolase activity (assays are performed as before). Based on the production of product Hi the presence of NADP + and the production of product in the absence of the cofactor, aldehyde dehydrogenase activity is calculated for ENZM.
  • aldehyde dehydrogenase activity is assayed using propanal as substrate.
  • the reaction mixture contams 60 mM sodium pyrophosphate buffer (pH 8.5), 5 mM propanal, 1 mM NADP " ⁇ and ENZM in a total volume of 1 ml.
  • Activity is determined by the increase Hi absorbance at 340 nm, resulting from the generation of NADPH, and is proportional to the aldehyde dehydrogenase activity Hi the sample (Krupenko, S.A. et al. (1995) J. Biol. Chem. 270:519-522).
  • 6-phosphogluconate dehydrogenase activity of ENZM is measured by incubating purified ENZM, or a composition comprising ENZM, Hi 120 mM triethanolamine (pH 7.5), 0.1 mM EDTA, 0.5 mM NADP + , and 10-150 ⁇ M 6-phosphogluconate as substrate at 20-25 °C
  • the production of NADPH is measured fluorimetricaUy (340 nm excitation, 450 nm emission) and is indicative of 6-phosphogluconate dehydrogenase activity.
  • the production of NADPH is measured photometricaUy, based on absorbance at 340 nm.
  • the molar amount of NADPH produced Hi the reaction is proportional to the 6-phosphogluconate dehydrogenase activity in the sample (Tetaud et al., supra).
  • Ribonucleotide diphosphate reductase activity of ENZM is determined by incubating purified ENZM, or a composition comprising ENZM, along with dithiothreitol, Mg ++ , and ADP, GDP, CDP, or UDP substrate.
  • the product of the reaction, the co ⁇ esponding deoxyribonucleotide, is separated from the substrate by thin-layer chromatography.
  • the reaction products can be distinguished from the reactants based on rates of migration.
  • the use of radiolabeled substrates is an alternative for increasing the sensitivity of the assay.
  • the amount of deoxyribonucleotides produced Hi the reaction is proportional to the amount of ribonucleotide diphosphate reductase activity Hi the sample (note that this is true only for pre-steady state kinetic analysis of ribonucleotide diphosphate reductase activity, as the enzyme is subject to negative feedback inhibition by products) (Nutter and Cheng, supra).
  • Dihydrodiol dehydrogenase activity of ENZM is measured by incubating purified ENZM, or a composition comprismg ENZM, in a reaction mixture comprising 50 mM glycine (pH 9.0), 2.3 mM NADP + , 8% DMSO, and a trans-dihydrodiol substrate, selected from the group mcluding but not Umited to, ( ⁇ )-trans-naphthalene-l,2-dihydrodiol, ( ⁇ )-trans-phenanthrene-l,2-dihydrodiol, and ( ⁇ )-trans- chrysene-l,2-dihydrodiol.
  • the oxidation reaction is monitored at 340 nm to detect the formation of NADPH, which is indicative of the oxidation of the substrate.
  • the reaction mixture can also be analyzed before and after the addition of ENZM by ckcular dichroism to determine the stereochemistry of the reaction components and determine which enantiomers of a racemic substrate composition are oxidized by the ENZM (Penning, supra).
  • Glutathione S-transferase (GST) activity of ENZM is deteimined by measuring the ENZM catalyzed conjugation of GSH with l-chloro-2,4-dHHtrobenzene (CDNB), a common substrate for most GSTs.
  • ENZM is incubated with 1 mM CDNB and 2.5 mM GSH together in 0.1M potassium phosphate buffer, pH 6.5, at 25 °C The conjugation reaction is measured by the change Hi absorbance at 340 nm using an ultraviolet spectrophometer.
  • ENZM activity is proportional to the change Hi absorbance at 340 nm.
  • 15-oxoprostaglandin 13 -reductase (PGR) activity of ENZM is measured foUowing the separation of contamkiating 15-hydroxyprostaglandin dehydrogenase (15-PGDH) activity by DEAE chromatography. FoUowing isolation of PGR containing fractions (or using the purified ENZM), activity is assayed Hi a reaction comprising 0.1 M sodium phosphate (pH 7.4), 1 mM 2- mercaptoethanol, 20 ⁇ g substrate (e.g., 15-oxo derivatives of prostaglandins PGE ⁇ PGE 2 , and PGE 2 ⁇ ), and 1 mM NADH (or a higher concentration of NADPH).
  • PGR 15-oxoprostaglandin 13 -reductase
  • ENZM is added to the reaction which is then incubated for 10 min at 37 °C before termination by the addition of 0.25 ml 2 N NaOH.
  • the amount of 15-oxo compound remaining in the sample is determined by measuring the maximum absorption at 500 nm of the terminated reaction and comparing this value to that of a terminated control reaction that received no ENZM.
  • 1 unit of enzyme is defined as the amount requked to catalyze the oxidation of 1 ⁇ mol substrate per minute and is proportional to the amount of PGR activity Hi the sample.
  • Choline dehydrogenase activity of ENZM is identified by the abiUty of E. coli, transformed with an ENZM expression vector, to grow on media containing choline as the sole carbon and nitrogen source. The abiUty of the transformed bacteria to thrive is indicative of choUne dehydrogenase activity (Magne ⁇ steras, M. (1998) Proc. Natl. Acad. Sci. USA 95:11394-11399
  • ENZM thioredoxin activity is assayed as described (Luthman, M. (1982) Biochemistry 21:6628-6633).
  • Thioredoxins catalyze the formation of disulfide bonds and regulate the redox envkonment in ceUs to enable the necessary thio disuUide exchanges.
  • One way to measure the thioLdisulfide exchange is by measuring the reduction of insulin Hi a mixture containing 0.1 M potassium phosphate, pH 7.0, 2 mM EDTA, 0.16 ⁇ M insuUn, 0.33 mM DTT, and 0.48 mM NADPH. Different concentrations of ENZM are added to the mixture, and the reaction rate is foUowed by monitoring the oxidation of NADPH at 340 nM.
  • ENZM transferase activity is measured through assays such as a methyl transferase assay Hi which the transfer of radiolabeled methyl groups between a donor substrate and an acceptor substrate is measured (Bokar, J.A. et al. (1994) J. Biol. Chem. 269:17697-17704).
  • Reaction mixtures (50 ⁇ l final volume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgC ⁇ , 10 mM dithiothreitol, 3% polyvinylalcohol, 1.5 ⁇ Ci [met/ry/- 3 H]AdoMet (0.375 ⁇ M AdoMet) (DuPont-NEN), 0.6 ⁇ g ENZM, and acceptor substrate (0.4 ⁇ g [ 35 S]RNA or 6-mercaptopurine (6-MP) to 1 mM final concentration). Reaction mixtures are incubated at 30 °C for 30 minutes, then at 65 °C for 5 minutes. The products are separated by chromatography or electrophoresis and the level of methyl transferase activity is dete ⁇ rHned by quantification of methyl- 3 ⁇ l recovery.
  • Ammotransferase activity of ENZM is assayed by incubating samples contammg ENZM for 1 hour at 37°C Hi the presence of 1 mM L-kynurenine and 1 mM 2-oxoglutarate in a final volume of 200 ⁇ l of 150 mM Tris acetate buffer (pH 8.0) containing 70 ⁇ M PLP.
  • the formation of kynurenic acid is quantified by HPLC with spectrophotometric detection at 330 nm using the appropriate standards and controls weU known to those skiUed in the art.
  • L-3-hydroxykynurenine is used as substrate and the production of xanthurenic acid is determined by HPLC analysis of the products with UV detection at 340 nm.
  • the production of kynurenic acid and xanthurenic acid, respectively, is indicative of aminotransferase activity (BuchU et al., supra).
  • ammotransferase activity of ENZM is measured by dete ⁇ nining the activity of purified ENZM or crude samples containing ENZM toward various amino and oxo acid substrates under single turnover conditions by monitoring the changes Hi the UV/VLS absorption spectrum of the enzyme-bound cofactor, pyridoxal 5'-phosphate (PLP).
  • the reactions are performed at 25°C in 50 mM 4-methylmorpholine (pH 7.5) containing 9 ⁇ M purified ENZM or ENZM containing samples and substrate to be tested (amino and oxo acid substrates).
  • the half-reaction from amino acid to oxo acid is followed by measuring the decrease Hi absorbance at 360 nm and the increase in absorbance at 330 nm due to the conversion of enzyme-bound PLP to pyridoxamine 5' phosphate (PMP).
  • the specificity and relative activity of ENZM is determined by the activity of the enzyme preparation against specific substrates (Vacca, supra).
  • ENZM chitinase activity is determined with the fluorogenic substrates 4-methylumbeUiferyl chitotriose, methylumbeUiferyl chitobiose, or methylumbeUiferyl N-acetylglucosamine.
  • Purified ENZM is incubated with 0.5uM substrate at pH 4.0 (0.1M citrate buffer), pH 5.0 (0.1M phosphate buffer), or pH 6.0 (0. IM Tris-HCL). After various times of incubation, the reaction is stopped by the addition of 0.1M glycine buffer, pH 10.4, and the concentration of free methylumbelliferone is determined fluorometricaUy.
  • Chitinase B from Serratia marcescens may be used as a positive control (Hakala, supra).
  • ENZM isomerase activity is determmed by measuring 2-hydroxyhepta-2,4-diene,l,7 dioate isomerase (HHDD isomerase) activity, as described by Garrido-Peritie ⁇ a, A. and R.A. Cooper (1981; Eur. J. Biochem. 17:581-584).
  • the sample is combined with 5-carboxymethyl-2-oxo-hex-3-ene-l,5, dioate (CMHD), which is the substrate for HHDD isomerase.
  • CMHD concentration is monitored by measuring its absorbance at 246 nm. Decrease in absorbance at 246 nm is proportional to HHDD isomerase activity of ENZM.
  • ENZM isomerase activity such as peptidyl prolyl cis/trans isomerase activity can be assayed by an enzyme assay described by Rahfeld (supra). The assay is performed at 10 °C in 35 mM HEPES buffer, pH 7.8, containing chymotrypsin (0.5 mg/ml) and ENZM at a variety of concentrations. Under these assay conditions, the substrate, Suc-Ala-Xaa-Pro-Phe-4-NA, is Hi equiUbrium with respect to the prolyl bond, with 80-95% Hi trans and 5-20% in cis conformation.
  • peptidyl prolyl cis-trans isomerase activity of ENZM can be assayed using a chromogenic peptide in a coupled assay with chymotrypsin (Fischer, G. et al. (1984) Biomed. Biochim. Acta 43:1101-1111).
  • UDP glucuronyltransferase activity of ENZM is measured using a colorimetric determination of free amine groups (Gibson, G.G. and P. Skett (1994) Introduction to Drug MetaboUsm, Blackie Academic and Professional, London).
  • An amme-containing substrate such as 2-aminophenol
  • a reaction buffer containing the necessary cofactors (40 mM Tris pH 8.0, 7.5 mM MgC ⁇ , 0.025% Triton X-100, 1 mM ascorbic acid, 0.75 mM UDP-glucuronic acid).
  • the reaction is stopped by addition of ice-cold 20% trichloroacetic acid in 0.1 M phosphate buffer pH 2.7, incubated on ice, and centrifuged to clarify the supernatant. Any unreacted 2-aminophenol is destroyed Hi this step.
  • Adenylosuccinate synthetase activity of ENZM is measured by synthesis of AMP from EMP.
  • EMP concentration is monitored spectrophotometrically at 248 nm at 23°C (Wang, W. et al. (1995) J. Biol. Chem. 270:13160-13163).
  • the increase in EMP concentration is proportional to ENZM activity.
  • AMP binding activity of ENZM is measured by combining the sample with
  • radioactivity retained in the gel is proportional to ENZM activity.
  • xenobiotic carboxyUc acid:CoA Ugase activity of ENZM is measured by combining the sample with ⁇ 33 P-ATP and measuring the formation of ⁇ - 33 P- pyrophosphate with time
  • Protein phosphatase (PP) activity can be measured by the hydrolysis of P-nitrophenyl phosphate (PNPP).
  • ENZM is incubated together with PNPP Hi HEPES buffer pH 7.5, Hi the presence of 0.1% ⁇ -mercaptoethanol at 37 °C for 60 min. The reaction is stopped by the addition of 6 ml of 10 N NaOH (Diamond, R.H. et al. (1994) Mol. CeU. Biol. 14:3752-62).
  • acid phosphatase activity of ENZM is demonstrated by incubating ENZM containing extract with 100 ⁇ l of 10 mM PNPP in 0.1 M sodium citrate, pH 4.5, and 50 ⁇ l of 40 mM
  • Hi Ught absorbance is proportional to the activity of ENZM in the assay.
  • ENZM activity is determined by measuring the amount of phosphate removed from a phosphorylated protein substrate. Reactions are performed with 2 or 4 nM ENZM Hi a final volume of 30 ⁇ l containing 60 mM Tris, pH 7.6, 1 mM EDTA, 1 mM EGTA, 0.1 %
  • Reactions are initiated with substrate and incubated at 30° C for 10-15 min. Reactions are quenched with 450 ⁇ l of 4% (w/v) activated charcoal Hi 0.6 M HCl, 90 mM Na 4 P 2 O 7 , and 2 mM NaH 2 PO 4 , then centrifuged at 12,000 x g for 5 min. Acid-soluble 32 Pi is quantified by Uquid scintiUation counting (Sinclair, C et al. (1999) J. Biol. Chem. 274:23666-23672).
  • the adenosine deaminase activity of ENZM is determined by measuring the rate of deamination that occurs when adenosine substrate is incubated with ENZM. Reactions are performed with a predete ⁇ nined amount of ENZM in a final volume of 3.0 ml containing 53.3 mM potassium phosphate and 0.045 mM adenosine. Assay reagents excluding ENZM are mixed Hi a quartz cuvette and equiUbrated to 25° C Reactions are initiated by the addition of ENZM and are mixed immediately by inversion.
  • the decrease Hi Ught absorbance at 265 nm resulting from the hydrolysis of adenosine to inosine is measured using a spectrophotometer.
  • the decrease Hi the A 265 noir diagram, is recorded for approximately 5 minutes.
  • the decrease Hi Ught absorbance is proportional to the activity of ENZM H the assay.
  • ENZM hydrolase activity is measured by the hydrolysis of appropriate synthetic peptide substrates conjugated with various chromogenic molecules in which the degree of hydrolysis is quantified by spectrophotometric (or fluorometric) absorption of the released chromophore (Beynon and Bond, supra, pp.25-55).
  • Peptide substrates are designed according to the category of protease activity as endopeptidase (serine, cysteine, aspartic proteases), aminopeptidase (leucine ai ⁇ iinopeptidase), or carboxypeptidase (Carboxypeptidase A and B, procoUagen C-proteinase).
  • An assay for carbonic anhydrase activity of ENZM uses the fluorescent pH indicator 8- hydroxypyrene-l,3,6-trisulfonate (pyranme) in combination with stopped-flow fluorometry to measure carbonic anhydrase activity (Shingles, et al. 1997, Anal. Biochem. 252:190-197).
  • a pH 6.0 solution is mixed with a pH 8.0 solution and the initial rate of bicarbonate dehydration is measured. Addition of carbonic anhydrase to the pH 6.0 solution enables the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. Shingles et al.
  • Decarboxylase activity of ENZM is measured as the release of CO 2 from labeled substrate.
  • oinitbine decarboxylase activity of ENZM is assayed by measuring the release of CO 2 from L-tl- 14 C]-ornithH ⁇ e (Reddy, S.G et al. (1996) J. Biol. Chem. 271:24945-24953).
  • Activity is measured Hi 200 ⁇ l assay buffer (50 mM Tris/HCl, pH 7.5, 0.1 mM EDTA, 2 mM dithiothreitol, 5 mM NaF, 0.1% Brij35, 1 mM PMSF, 60 ⁇ M pyridoxal-5 -phosphate) containing 0.5 mM L-ormthine plus 0.5 ⁇ Ci L-[l- 14 C]ornithine. The reactions are stopped after 15-30 mmutes by addition of 1 M citric acid, and the 14 CO 2 evolved is trapped on a paper disk filter saturated with 20 ⁇ l of 2 N NaOH. The radioactivity on the disks is determined by Uquid scintiUation spectography.
  • the amount of 14 CO 2 released is proportional to ormthine decarboxylase activity of ENZM.
  • AdoHCYase activity of ENZM Hi the hydrolytic direction is performed spectroscopicaUy by measuring the rate of the product (homocysteine) formed by reaction with 5,5'-Dithiobis(2-nitiObenzoic acid) (DTNB).
  • Enzyme activity is defined as the amount of enzyme that can hydrolyze 1 ⁇ mol of 5-Adenosyl-L-homocysteme/mH ⁇ ute (Yuan, C-S et al. (1996) J. Biol. Chem. 271:28009-28015).
  • AdoHCYase activity of ENZM can be measured in the synthetic dkection as the production of S-adenosyl homocysteine usmg 3-deazaadenosine as a substrate (Sganga et al. supra). Briefly,
  • ENZM is mcubated in a 100 ⁇ l volume containing 0.1 mM 3-deazaadenosine, 5 mM homocysteine, 20 mM HEPES (pH 7.2).
  • the assay mixture is incubated at 37 °C for 15 minutes.
  • the reaction is terminated by the addition of 10 ⁇ l of 3 M perchloric acid.
  • the mixture is centrifuged for 5 mmutes at 18,000 x g in a microcentrifuge at 4°C The supernatant is removed, neutraUzed by the addition of 1 M potassium carbonate, and centrifuged again.
  • a 50 ⁇ l aUquot of supernatant is then chromatographed on an Altex Ultrasphere ODS column (5 ⁇ m particles, 4.6 x 250 mm) by isocratic elution with 0.2 M ammonium dihydrogen phosphate (Aldrich) at a flow rate of 1 ml/min. Protein is determmed by the bicinchoninic acid assay (Pierce).
  • AdoHCYase activity of ENZM can be measured Hi the synthetic dkection by a TLC method (Hershfield, M.S. et al. (1979) J. Biol. Chem. 254:22-25).
  • 50 ⁇ M [8 "14 C]adenosine is incubated with 5 molar equivalents of NAD + for 15 minutes at 22 °C
  • Assay samples containing ENZM Hi a 50 ⁇ l final volume of 50 mM potassium phosphate buffer, pH 7.4, 1 mM DTT, and 5 mM homocysteine are mixed with the preincubated [8" 14 C]adenosine/NAD + to initiate the reaction.
  • the reaction is mcubated at 37 °C, and 1 ⁇ l samples are spotted on TLC plates at 5 minute intervals for 30 minutes.
  • the chromatograms are developed Hi butanol-1/glacial acetic acid/water (12:3:5, v/v) and dried. Standards are used to identify substrate and products under ultraviolet Ught.
  • the complete spots containing [ 14 C]adenosine and [ 14 C]SAH are then detected by exposing x-ray film to the TLC plate.
  • the radiolabeled substrate and product are then cut from the chromatograms and counted by Uquid scintiUation spectrometry.
  • Asparaginase activity of ENZM can be measured in the hydrolytic dkection by determinkig the amount of radiolabeled L-aspartate released from 0.6 mM N 4 - ⁇ '-N-acetylglucosaminyl-L- asparagine substrate when it is mcubated at 25 °C with E ⁇ ZM in 50 mM phosphate buffer, pH 7.5 (Kaartinen, V. et al. (1991) J. Biol. Chem. 266:5860-5869).
  • E ⁇ ZM Hi the hydrolytic dkection is performed spectroscopicaUy by monitoring the appearance of the product (CoASH) formed by reaction of substrate (acyl-CoA) and E ⁇ ZM with 5,5'-Dithiobis(2-nitrobenzoic acid) (DT ⁇ B).
  • the final reaction volume is 1 ml of 0.05 M potassium phosphate buffer, pH 8, containing 0.1 mM DT ⁇ B, 20 ⁇ g/ml bovine serum albumin, 10 ⁇ M of acyl-CoA of different lengths (C6-C0A, ClO-CoA, C14-CoA and CI8-C0A, Sigma), and E ⁇ ZM.
  • reaction mixture is incubated at 22 °C for 7 minutes. Hydrolytic activity is monitored spectrophotometricaUy by measuring absorbance at 412 nm (Poupon, V. et al. (1999) J. Biol. Chem. 274:19188-19194).
  • E ⁇ ZM activity of E ⁇ ZM can be measured spectrophotometricaUy by determining the amount of solubiUzed R ⁇ A that is produced as a result of incubation of R ⁇ A substrate with E ⁇ ZM.
  • 5 ⁇ l (20 ⁇ g) of a 4 mg/ml solution of yeast tR ⁇ A (Sigma) is added to 0.8 ml of 40 mM sodium phosphate, pH 7.5, containing E ⁇ ZM.
  • the reaction is mcubated at 25 °C for 15 minutes.
  • the reaction is stopped by addition of 0.5 ml of an ice-cold fresh solution of 20 mM lanthanum nitrate plus 3% perchloric acid.
  • the stopped reaction is mcubated on ice for at least 15 min, and the insoluble tR ⁇ A is removed by centrifugation for 5 min at 10,000 g.
  • SolubiUzed tR ⁇ A is determined as UV absorbance (260 nm) of the remaining supernatant, with A 260 of 1.0 co ⁇ esponding to 40 ⁇ g of solubiUzed R ⁇ A (Rosenberg, H.F. et al. (1996) Nucleic Acids Research 24:3507-3513).
  • ENZM activity can be determined as the abiUty of ENZM to cleave 32 P internaUy labeled T. thermophila pre-tRNA Gbl .
  • ENZM and substrate are added to reaction vessels and reactions are carried out Hi MBB buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 ) for 1 hour at 37 °C Reactions are terminated with the addition of an equal volume of sample loading buffer (SLB: 40 mM EDTA, 8 M urea, 0.2% xylene cyanol, and 0.2% bromophenol blue).
  • the reaction products are separated by electrophoresis on 8 M urea, 6% polyacrylamide gels and analyzed using detection instruments and software capable of quantification of the products.
  • ENZM activity is defined as the amount of enzyme requked to cleave 10% of 28 fmol of T. thermophila pre-tRNA Gln to mature products in 1 hour at 37 °C (True, H.L. et al. (1996) J. Biol. Chem. 271:16559-16566).
  • cleavage of 32 P internally labeled substrate tRNA by ENZM can be determined in a 20 ⁇ l reaction mixture containing 30 mM HEPES-KOH (pH 7.6), 6 mM MgCl 2 , 30 mM KCl, 2 mM DTT, 25 ⁇ g/ml bovine serum albumin, 1 unit/ ⁇ l rRNasin, and 5,000-50,000 cpm of gel-purified substrate RNA. 3.0 ⁇ l of ENZM is added to the reaction mixture, which is then mcubated at 37 °C for 30 mmutes.
  • the reaction is stopped by guanidHHum/phenol extraction, precipitated with ethanol in the presence of glycogen, and subjected to denaturing polyacrylamide gel electrophoresis (6 or 8% polyacrylamide, 7 M urea) and autoradiography (Rossmanith, W. et al. (1995) J. Biol. Chem. 270:12885-12891).
  • the ENZM activity is proportional to the amount of cleavage products detected.
  • ENZM activity can be measured by dete ⁇ r ning the amount of free adenosine produced by the hydrolysis of AMP, as described by Sala-Newby et al., supra.
  • ENZM is mcubated with AMP Hi a suitable buffer for 10 mmutes at 37 °C Free adenosine is separated from AMP and measured by reverse phase HPLC.
  • ENZM activity is measured by the hydrolysis of ADP-ribosylarginine
  • Epoxide hydrolase activity of ENZM can be dete ⁇ nined with a radiometric assay utiUzing [LPj-labeled trans- stilbene oxide (TSO) as substrate.
  • TSO trans- stilbene oxide
  • ENZM is preincubated Hi Tris-HCl pH 7.4 buffer H a total volume of 100 ⁇ l for 1 minute at 37 °C 1 ⁇ l of [ffj-labeled TSO (0.5 ⁇ M in EtOH) is added and the reaction mixture is incubated at 37 °C for 10 minutes. The reaction mixture is extracted with 200 ⁇ l n-dodecane. 50 ⁇ l of the aqueous phase is removed for quantification of diol product H a Uquid scintiUation counter (LSC).
  • ENZM activity is calculated as nmol diol product/rmn/mg protem (GiU, S.S. et al. (1983) Analytical Biochemistry 131:273-282).
  • Lysophosphatidic acid acyltransferase activity of ENZM is measured by incubating samples containing ENZM with 1 mM of the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 50 ⁇ m LPA, and 50 ⁇ m acyl-CoA Hi 100 mM Tris-HCl, pH 7.4. The reaction is initiated by addition of acyl- CoA, and aUowed to reach equiUbrium. Transfer of the acyl group from acyl-CoA to LPA releases free CoA, which reacts with DTNB. The product of the reaction between DTNB and free CoA absorbs at 413 nm. The change in absorbance at 413 nm is measured using a spectrophotometer, and is proportional to the lysophosphatidic acid acyltransferase activity of ENZM in the sample.
  • DTNB thiol reagent 5,5'-dithiobis(
  • N-acyltransferase activity of ENZM is measured using radiolabeled ammo acid substrates and measuring radiolabel mcorporation into conjugated products.
  • ENZM is mcubated in a reaction buffer containing an unlabeled acyl-CoA compound and radiolabeled ammo acid, and the radiolabeled acyl- conjugates are separated from the unreacted amino acid by extraction mto n-butanol or other appropriate organic solvent.
  • a reaction buffer containing an unlabeled acyl-CoA compound and radiolabeled ammo acid
  • bile acid-CoA amino acid N-acyltransferase activity by incubating the enzyme with cholyl-CoA and 3 H-glycine or 3 H-taurine, separating the tritiated cholate conjugate by extraction into n-butanol, and measuring the radioactivity Hi the extracted product by scintiUation.
  • N- acyltransferase activity is measured using the spectrophotometric determination of reduced CoA (Co ASH) described below.
  • N-acetyltransferase activity of ENZM is measured usmg the transfer of radiolabel from [ 14 C]acetyl-CoA to a substrate molecule (for example, see Deguchi, T. (1975) J. Neurochem.
  • a newer spectrophotometric assay based on DTNB reaction with Co ASH may be used. Free thiol-containing Co ASH is formed during N-acetyltransferase catalyzed transfer of an acetyl group to a substrate. Co ASH is detected using the absorbance of DTNB conjugate at 412 nm (De AngeUs, J. et al. (1997) J. Biol. Chem. 273:3045-3050). ENZM activity is proportional to the rate of radioactivity mcorporation into substrate, or the rate of absorbance mcrease in the spectrophotometric assay.
  • Galactosyltransferase activity of ENZM is determined by measuring the transfer of galactose from UDP-galactose to a GlcNAc-terminated oUgosaccharide chain Hi a radioactive assay.
  • the ENZM sample is mcubated with 14 ⁇ l of assay stock solution (180 mM sodium cacodylate, pH 6.5, 1 mg/ml bovine serum albumin, 0.26 mM UDP-galactose, 2 ⁇ l of UDP-[ H]galactose), 1 ⁇ l of MnCl 2 (500 mM), and 2.5 ⁇ l of GlcNAc ⁇ O- (CH 2 ) 8 -CO 2 Me (37 mg/ml in dimethyl sulfoxide) for 60 minutes at 37 °C
  • the reaction is quenched by the addition of 1 ml of water and loaded on a C18 Sep-Pak cartridge (Waters), and the column is washed twice with 5 ml of water to remove unreacted UDP-[ 3 H]galactose.
  • the [ 3 H]galactosylated GlcNAc ⁇ O-(CH 2 ) 8 -CO 2 Me remains bound to the column during the water washes and is eluted with 5 ml of methanol. Radioactivity in the eluted material is measured by Uquid scintiUation counting and is proportional to galactosyltransferase activity of ENZM in the starting sample. Phosphoribosyltransferase activity of ENZM is measured as the transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (PRPP) to a purine or pyrimidine base.
  • PRPP phosphoribosylpyrophosphate
  • Assay mixture (20 ⁇ l) containing 50 mM Tris acetate, pH 9.0, 20 mM 2-mercaptoethanol, 12.5 mM MgCl 2 , and 0.1 mM labeled substrate, for example, [ 14 C]uracil, is mixed with 20 ⁇ l of ENZM diluted in 0.1 M Tris acetate, pH 9.7, and 1 mg/ml bovine serum albumin. Reactions are preheated for 1 min at 37 °C, initiated with 10 ⁇ l of 6 mM PRPP, and incubated for 5 min at 37 °C The reaction is stopped by heating at 100 °C for 1 min.
  • the product [ 14 C]UMP is separated from [ 14 C]uracil on DEAE-ceUulose paper (Turner, R.J. et al. (1998) J. Biol. Chem. 273:5932-5938).
  • the amount of [ 14 C]UMP produced is proportional to the phosphoribosyltransferase activity of ENZM.
  • ADP-ribosyltransferase activity of ENZM is measured as the transfer of radiolabel from adenine-NAD to agmatine (Weng, B. et al. (1999) J. Biol. Chem. 274:31797-31803).
  • Purified ENZM is mcubated at 30 °C for 1 hr in a total volume of 300 ⁇ l containing 50 mM potassium phosphate (pH. 7.5), 20 mM agmatine, and 0.1 mM [adenine-U- 14 C]NAD (0.05 mCi). Samples (100 ⁇ l) are appUed to Dowex columns and [ 14 C]ADP-ribosylagmatine eluted with 5 ml of water for Uquid scintiUation countmg. The amount of radioactivity recovered is proportional to ADP-ribosyltransferase activity of ENZM.
  • An ENZM activity assay measures aminoacylation of tRNA in the presence of a radiolabeled substrate.
  • SYNT is incubated with [ 14 C]-labeled arnino acid and the appropriate cognate tRNA (for example, [ 14 C]alanine and tRNA ab ) in a buffered solution.
  • 14 C-labeled product is separated from free [ I4 C]amino acid by chromatography, and the incorporated 14 C is quantified using a scintillation counter.
  • the amount of 14 C-labeled product detected is proportional to the activity of ENZM in this assay (lbba, M. et al. (1997) Science 278:1119-1122).
  • argininosuccinate synthase activity of ENZM is measured based on the conversion of [ 3 H]aspartate to [ 3 H]argH ⁇ H ⁇ osuccinate.
  • ENZM is incubated with a mixture of [ 3 H]aspartate, citruUine, Tris-HCl (pH 7.5), ATP, MgCl 2 , KCl, phosphoenolpyruvate, pyruvate kinase, myokinase, and pyrophosphatase, and allowed to proceed for 60 minutes at 37 °C Enzyme activity was terminated with addition of acetic acid and heating for 30 minutes at 90 °C [ 3 H]argininosuccinate is separated from un-catalyzed [ 3 H]aspartate by chromatography and quantified by Uquid scintiUation spectrometry.
  • the amount of [ 3 H]argininosuccinate detected is proportional to the activity of ENZM in this assay (O'Brien, W. E. (1979) Biochemistry 18:5353-5356).
  • the esterase activity of ENZM is assayed by the hydrolysis of p- nitrophenylacetate (NPA).
  • NPA p- nitrophenylacetate
  • ENZM is incubated together with 0.1 ⁇ M NPA in 0.1 M potassium phosphate buffer (pH 7.25) containing 150 mM NaCl.
  • the hydrolysis of NPA is measured by the mcrease of absorbance at 400 nm with a spectrophotometer.
  • the increase Hi Ught absorbance is proportional to the activity of ENZM (Probst, M.R. et al.

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Abstract

Différents modes de réalisation de l'invention concernent des enzymes humains (ENZM) et des polynucléotides qui identifient et codent pour ENZM. Ces modes de réalisation de l'invention concernent également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. D'autres modes de réalisation concernent des méthodes permettant de diagnostiquer, de traiter ou de prévenir des troubles associés à l'expression aberrante de ENZM.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
CN112662644A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种甘油磷酸二酯磷酸二酯酶突变体及其应用

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* Cited by examiner, † Cited by third party
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US6194167B1 (en) * 1997-02-18 2001-02-27 Washington State University Research Foundation ω-3 fatty acid desaturase

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8871737B2 (en) 2010-09-22 2014-10-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US9278990B2 (en) 2010-09-22 2016-03-08 Alios Biopharma, Inc. Substituted nucleotide analogs
US8980865B2 (en) 2011-12-22 2015-03-17 Alios Biopharma, Inc. Substituted nucleotide analogs
US9605018B2 (en) 2011-12-22 2017-03-28 Alios Biopharma, Inc. Substituted nucleotide analogs
US8916538B2 (en) 2012-03-21 2014-12-23 Vertex Pharmaceuticals Incorporated Solid forms of a thiophosphoramidate nucleotide prodrug
US9856284B2 (en) 2012-03-21 2018-01-02 Alios Biopharma, Inc. Solid forms of a thiophosphoramidate nucleotide prodrug
US9012427B2 (en) 2012-03-22 2015-04-21 Alios Biopharma, Inc. Pharmaceutical combinations comprising a thionucleotide analog
CN112662644A (zh) * 2021-01-19 2021-04-16 华南理工大学 一种甘油磷酸二酯磷酸二酯酶突变体及其应用

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