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WO2003070926A1 - Nouvelles sequences nucleotidiques codant pour les polypeptides de la phb:polyprenyltransferase humaine - Google Patents

Nouvelles sequences nucleotidiques codant pour les polypeptides de la phb:polyprenyltransferase humaine Download PDF

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WO2003070926A1
WO2003070926A1 PCT/SE2003/000285 SE0300285W WO03070926A1 WO 2003070926 A1 WO2003070926 A1 WO 2003070926A1 SE 0300285 W SE0300285 W SE 0300285W WO 03070926 A1 WO03070926 A1 WO 03070926A1
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polypeptide
polyprenyltransferase
nucleic acid
seq
phb
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Isabel Climent-Johansson
Staffan Lake
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Swedish Orphan Biovitrum AB
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Biovitrum AB
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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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to new nucleotide sequences, to PHB -.polyprenyltransferase (EC 2.5.1) polypeptides encoded by the nucleotide sequences, as well as to methods for identifying agents modulating the activity of PHB polyprenyltransferase. Such agents are expected to be useful in the treatment of medical conditions relating to insulin resistance, in particular type II diabetes.
  • Insulin that is synthesized in the beta cells of the islets of Langerhans of the pancreas is one of the major hormones influencing metabolism. Insulin primarily regulates the direction of metabolism, shifting many processes toward the storage of substrates and away from their degradation (for reviews, see e.g. Shepherd, P.R. et al. (1998) Biochem. J. 333: 471-490; Alessi, D. R. & Downes, C. P. (1998) Biochim. Biophys. Acta 1436: 151-164). Insulin acts to increase the transport of glucose and amino acids as well as key minerals such as potassium, magnesium, and phosphate from the blood into cells. It also regulates a variety of enzymatic reactions within the cells, all of which have a common overall direction, namely the synthesis of large molecules from small units.
  • Insulin regulates a wide range of biological processes, including glucose transport, glycogen synthesis, protein synthesis, cell growth, and gene expression. It exerts its biological function by a well-characterized signal transduction mechanisms downstream of the insulin receptor involving a cascade of kinase/phosphatase reactions leading to changes in cellular physiology and patterns of gene expression (Saltiel A, & Kahn, CK (2001) Nature 414:799-806). Binding if insulin to its cell surface transmembrane receptor stimulates receptor autophosphorylation and activation of the intrinsic tyrosine kinase activity, which results in phosphorylation of several cytosolic docking proteins called insulin receptor substrates (IRSs).
  • IFSs insulin receptor substrates
  • IRSs bind to various effector molecules including the 85 kDa regulatory subunit of PI3K. This localizes the 110 kDa catalytic domain of PI3K to the plasma membrane.
  • the activated PI3K phosphorylates membrane bound phosphoinositides (Ptdlns), generating PtdIns(3,4)P2 and Ptdh ⁇ s(3,4,5)P3.
  • Ptdlns membrane bound phosphoinositides
  • Ptdlns membrane bound phosphoinositides
  • Ptdlns membrane bound phosphoinositides
  • Ptdlns membrane bound phosphoinositides
  • Ptdh protein kinase B
  • the kinases which phosphorylate PKB, are themselves targets for lipid products of PI3K and are therefore also localized to the membrane. These kinases are called phosphoinositide-dependent protein kinases (PDK1 and PDK2). Activated PKB dissociates from the membrane and moves to the nucleus and other subcellular compartments. PKB is thought to phosphorylate a variety of different protein substrates, so accounting for the different regulatory events in which it is involved (Coffer, PJ et al. (1998) Biochem J. 335:1-13).
  • glycogen synthase kinase-3 (GSK-3)
  • the heart isoform of 6-phosphofructo-2 -kinase the BCL-2 family member BAD
  • FKHRL1 the forkhead transcription factor
  • a deficiency in the action of insulin causes severe impairment in (i) the storage of glucose in the form of glycogen and the oxidation of glucose for energy; (ii) the synthesis and storage of fat from fatty acids and their precursors and the completion of fatty-acid oxidation; and (iii) the synthesis of proteins from amino acids.
  • Type I diabetes insulin-dependent diabetes mellitus
  • NIDDM insulin-dependent diabetes mellitus
  • Type II diabetes non-insulin-dependent diabetes mellitus
  • Glucose homeostasis depends upon a balance between glucose production by the liver and glucose utilization by insulin-dependent tissues, such as fat and muscle, and insulin-independent tissues, such as brain and kidney.
  • Coenzyme Q also known as ubiquinone
  • CoQ is the only endogenously synthesized lipid with a redox function in mammals. It is present in all cellular membranes. It has a well-recognized function in energy transduction on the mitochondria inner membrane where acts as electron transporter between NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and bcl complex (complex III)
  • complex I NADH dehydrogenase
  • complex II succinate dehydrogenase
  • complex III complex
  • Another function of CoQ is in mediation and preservation of antioxidant functions in cells (Ernster, L. (1993) in Active oxygens, lipid peroxides, and antioxidants Biochemical and clinical aspects of coenzyme Q, pp 1-38, CRC Press, Boca Raton.).
  • the mevalonate pathway is a sequence of cellular reactions that leads to farnesyl-PP, the common substrate for the synthesis of cholesterol, dolichol, dolichol-P and CoQ as well as prenylation of proteins (Gmnler, J . et al. (1994) Biochim Biophys Acta 1212:259-277). Most of the studies of CoQ biosynthesis have been done on bacterial and yeast and none of the enzymes involved have been either purified or cloned from mammals.
  • the first step unique to CoQ biosynthesis involves the synthesis from IPP of an all-trans polyprenyl diphosphate where the chain length is a species-specific phenomenon (Olson RE. and Rudney H.
  • Fig. 1 is a graph depicting the relative expression levels of human l o PHB polyprenyltransferase in different human tissues.
  • Fig. 2 is a graph depicting the effect of human PHB polyprenyltransferase on glucose uptake, when overexpressed in differentiated rat skeletal muscle L6 cells.
  • Fig. 3 is a graph depicting complementation of the yeast null mutant strain CEN ⁇ Coq2 (O) by human PHB polyprenyltransferase (D) and yeast coq2 ( ⁇ ).
  • Fig. 4 illustrates the effect of human PHB polyprenyltransferase on growth complementation of recombinant yeast cells.
  • the invention provides an isolated nucleic acid molecule that 0 encodes a PHB polyprenyltransferase polypeptide described herein or a fragment thereof.
  • the isolated nucleic acid molecule includes a nucleotide sequence that is at least 60% identical to the nucleotide sequence shown in SEQ ID NO:l. More preferably, the nucleotide sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to the nucleotide 5 sequence shown in SEQ ID NO: 1.
  • nucleotide sequence that is longer than or equivalent in length to the reference sequence, e.g., SEQ ID NO:l
  • the comparison is made with the full length of the reference sequence.
  • the nucleotide sequence is shorter that the reference sequence, e.g., shorter than SEQ ID NO:l
  • the comparison is made to a segment of the reference sequence of the same 0 length (excluding any loop required by the homology calculation).
  • the nucleotide sequence encodes a polypeptide having polyprenyltransferase activity.
  • the nucleotide sequence is identical to SEQ ID NO:l.
  • the invention features an isolated nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that is at least about 60% identical to a sequence shown as SEQ ID NO:2 or a fragment thereof.
  • the amino acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:2 and has a polyprenyltransferase activity described herein.
  • the amino acid sequence can be identical to the sequence of SEQ ID NO:2.
  • nucleic acid molecule comprising a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:l, the complete complement of SEQ ID NO:l, or a segment thereof as described herein.
  • High stringency conditions refers to hybridization in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65°C.
  • the nucleotide sequence encodes a polypeptide having polyprenyltransferase activity.
  • nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising a functional domain of the polypeptide of SEQ ID NO:2 described herein, e.g., a transmembrane domain or a UbiA domain of SEQ ID NO:2.
  • the nucleotide sequence encodes a polypeptide having polyprenyltransferase activity.
  • nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising at least 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, or more contiguous amino acid residues of SEQ ID NO:2.
  • the polypeptide comprises an immuno genie fragment of at least 20 amino acids of SEQ ID NO:2.
  • the nucleotide sequence encodes a polypeptide having polyprenyltransferase activity.
  • an "isolated nucleic acid” is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a .
  • a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and
  • a DNA library such as a cDNA or genomic DNA library.
  • % identity of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin and Altschul, PNAS USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990).
  • GappedBLAST is utilized as described in Altschul et al (Nucleic Acids Res. 25:3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST are used to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention.
  • the nucleic acid molecules according to the present invention include, for example, cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. RNA transcribed from DNA is also encompassed by the present invention.
  • the nucleic acid molecule has a nucleotide sequence identical with SEQ ID NO:l of the Sequence Listing.
  • the nucleic acid molecule according to the invention is not to be limited strictly to the sequence shown as SEQ ID NO:l.
  • the invention also encompasses nucleic acid molecules which are substantially identical to SEQ ID NO:l, as well as nucleic acid molecules carrying modifications like substitutions, small deletions, insertions or inversions, which nevertheless encode proteins having substantially the biochemical activity of the PHB polyprenyltransferase polypeptide according to the invention.
  • substantially identical indicates that two or more nucleotide sequences share a majority of their sequence. Generally, this will be at least about 90% of their sequence and preferably about 95% of their sequence. Another indication that sequences are substantially identical is if they hybridize under stringent conditions (see, e.g., Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1985). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • stringent conditions will be those in which the salt concentration is at least about 0.2 molar at pH 7 and the temperature is at least about 60°C.
  • nucleic acid molecule which nucleotide sequence is degenerate, because of the genetic code, to the nucleotide sequence shown as SEQ ID NO:l.
  • the invention features a substantially pure polypeptide having a sequence shown as SEQ ID NO:2.
  • the invention also includes a polypeptide, or fragment thereof, that differs from the corresponding sequence shown as SEQ ID NO:2.
  • the differences are, preferably, differences or changes at a non-essential residue or a conservative substitution.
  • the polypeptide includes an amino acid sequence at least about 60% identical to a sequence shown as SEQ ID NO:2 or a fragment thereof.
  • the amino acid sequence is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:2 and has a polyprenyltransferase activity described herein.
  • the amino acid sequence can be identical to SEQ ID NO:2.
  • Preferred polypeptide fragments of the invention are at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of the sequence shown as SEQ ID NO:2 and have a polyprenyltransferase activity described herein.
  • the fragment can be merely an immunogenic fragment, e.g., a fragment that can be used to raise monoclonal and/or polyclonal antibodies that specifically bind to a polypeptide of the sequence of SEQ ID NO:2.
  • the invention encompasses polypeptides carrying modifications such as substitutions, small deletions, insertions or inversions, which polypeptides nevertheless have substantially the biological activities of PHB polyprenyltransferase.
  • a polypeptide encoded by a nucleic acid molecule described herein is also included in the invention.
  • a substantially pure polypeptide comprising a functional domain of the polypeptide of SEQ ID NO:2 described herein, e.g., a transmembrane domain or a UbiA domain of SEQ ID NO:2.
  • the polypeptide has polyprenyltransferase activity.
  • a substantially pure polypeptide comprising at least 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, or more contiguous amino acid residues of SEQ ID NO:2.
  • the polypeptide comprises an immunogenic fragment of at least 20 amino acids of SEQ ID NO:2.
  • the polypeptide has polyprenyltransferase activity.
  • substantially pure as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules.
  • the substantially pure polypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight. Purity can be measured by any appropriate standard method known in the art, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a further aspect of the invention is a vector, for instance a replicable expression vector, which carries and is capable of mediating the expression of a nucleic acid molecule according to the invention.
  • replicaable means that the vector is able to replicate in a given type of host cell into which is has been introduced.
  • vectors are viruses such as bacteriophages, cosmids, plasmids and other recombination vectors.
  • Nucleic acid molecules can be inserted into vector genomes by methods well known in the art.
  • a cultured host cell comprising a vector according to the invention.
  • a host cell can be a prokaryotic cell, a unicellular eukaryotic cell or a cell derived from a multicellular organism.
  • the host cell can thus e.g. be a bacterial cell such as an E. coli cell; a cell from yeast such as Saccharomyces cerevisiae or Pichiapastoris, or a mammalian cell.
  • the methods employed to effect introduction of the vector into the host cell are standard methods well known to a person familiar with recombinant DNA methods.
  • Yet another aspect of the invention is a method for production of a polypeptide described herein, the method comprising culturing a host cell described herein under conditions whereby said polypeptide is produced, and recovering the polypeptide.
  • the medium used to grow the cells may be any conventional medium suitable for the purpose.
  • a suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above.
  • the methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA.
  • the recombinant polypeptide expressed by the cells may optionally be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.
  • the invention provides a method for identifying an agent useful for the treatment of a medical condition relating to insulin resistance, the method comprising the steps (i) contacting a candidate agent with a human PHB polyprenyltransferase polypeptide according to the invention; and (ii) determining whether said candidate agent modulates the biological activities of the said polypeptide, such modulation being indicative for an agent useful for the treatment of a medical condition relating to insulin resistance.
  • the said medical condition relating to insulin resistance is typically a medical condition associated with reduced glucose uptake, such as type II diabetes.
  • the method according to the invention could comprise the steps (i) contacting a candidate agent with a nucleic acid molecule according to claim 1 encoding a human PHB polyprenyltransferase polypeptide; and (ii) determining whether said candidate agent modulates the expression of the said nucleic acid molecule, such modulation being indicative for an agent useful for the treatment of a medical condition relating to insulin resistance.
  • the invention also features a method of identifying an agent that modulates an activity of a PHB polyprenyltransferase, the method comprising: (i) contacting a polypeptide described herein (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2) with a candidate agent; (ii) measuring a polyprenyltransferase activity of the polypeptide in the presence of the candidate agent; and (iii) comparing the polyprenyltransferase activity of the polypeptide in the presence of the candidate agent with the PHB polyprenyltransferase activity of the polypeptide in the absence of the candidate agent, to thereby determine whether the candidate agent modulates an activity of a PHB polyprenyltransferase.
  • the invention also features a method of identifying an agent that binds to a
  • PHB polyprenyltransferase the method comprising: (i) contacting a polypeptide described herein (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2) with a candidate agent; and (ii) determining that the candidate agent binds to the polypeptide, to thereby identify a candidate agent that binds to a PHB polyprenyltransferase.
  • a polypeptide described herein e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2
  • the invention also features a method of identifying an agent that modulates expression of a nucleic acid encoding a PHB polyprenyltransferase, the method comprising: (i) contacting a nucleic acid molecule comprising a nucleotide sequence described herein (e.g., the nucleotide sequence of SEQ ID NO:l) with a candidate agent; (ii) measuring expression of the nucleic acid molecule in the presence of the candidate agent; and (iii) comparing the expression of the nucleic acid molecule in the presence of the candidate agent with expression of the nucleic acid molecule in the absence of the candidate agent, to thereby determine whether the candidate agent modulates expression of a nucleic acid encoding a PHB polyprenyltransferase.
  • hPHB:polyprenyltransferase biological activity can be used for identifying modulating agents.
  • Such assays can be cell-based, cell-free, or utilize cell extracts or purified enzyme. Suitable assays are typically radioactive; fluorescence-based; or based on other methods that can be employed by a person skilled in the art, and require the measurement, direct or indirectly, of an PHB:polyprenyltransferse biological activity. Methods that can be use to quantitatively measure PHB:PPT activity include for example measuring a radiolabeled product, derived from a labeled substrate, or measuring pyrophosphate, the second product produced by the enzyme activity.
  • Suitable assays could also involve quantification of a binding interaction between the enzyme and one or several reagents.
  • screening may employ a cell-based assay (e.g. genetically modified yeast cells) in which the human PHB polyprenyltransferase gene is expressed from a vector, and cell growth under non-fermentable conditions is determined.
  • a cell-based assay e.g. genetically modified yeast cells
  • a transfection assay can be another form of a useful screening assay for identifying an effective agent.
  • a nucleic acid containing a gene such as a reporter gene that is operably linked to a PHB polyprenyltransferase promoter, or an active fragment thereof, is transfected into the desired cell type.
  • a test level of reporter gene expression is assayed in the presence of a candidate agent and compared to a control level of expression.
  • An effective agent is identified as an agent that results in a test level of expression that is different than a control level of reporter gene expression, which is the level of expression determined in the absence of the agent.
  • This invention also relates to the use of PHB polyprenyltransferase polynucleotides for use as diagnostic reagents. Detection of a mutated form of PHB polyprenyltransferase gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered expression of PHB polyprenyltransferase.
  • such a disease could be relating to insulin resistance and /or reduced glucose uptake, such as type II diabetes.
  • Individuals carrying mutations in the PHB polyprenyltransferase gene may be detected at the DNA level by a variety of techniques known to the person skilled in the art.
  • the invention features a method for diagnosis of a medical condition relating to insulin resistance, the method comprising detecting in a subject an altered expression or activity of PHB polyprenyltransferase.
  • the invention also features a method for diagnosis of a medical condition relating to insulin resistance, the method comprising detecting in a subject a mutated form of a PHB polyprenyltransferase gene.
  • diseases resulting from under-expression, over-expression or altered expression of PHB polyprenyltransferase can be diagnosed by methods comprising determining from a sample derived from a subject an altered, i.e. abnormally decreased or increased, level of PHB polyprenyltransferase polypeptide or PHB polyprenyltransferase mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as an PHB polyprenyltransferase polypeptide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
  • the invention features a method of modulating PHB polyprenyltransferase expression or activity in a cell, the method comprising contacting a cell expressing PHB polyprenyltransferase with an amount of a polypeptide described herein (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2) or a nucleic acid encoding the polypeptide sufficient to modulate the expression or activity of PHB polyprenyltransferase in the cell.
  • a polypeptide described herein e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2
  • a nucleic acid encoding the polypeptide sufficient to modulate the expression or activity of PHB polyprenyltransferase in the cell.
  • the invention features a method for the treatment or prophylaxis of a medical condition relating to insulin resistance (e.g., a medical condition described herein), the method comprising administering to a patient in need of such treatment or prophylaxis an amount of a polypeptide described herein (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2) or a nucleic acid encoding the polypeptide effective to treat or prevent a medical condition relating to insulin resistance, and a pharmaceutically acceptable carrier.
  • a polypeptide described herein e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO:2
  • a nucleic acid encoding the polypeptide effective to treat or prevent a medical condition relating to insulin resistance
  • the invention features a method for the treatment or prophylaxis of a medical condition relating to insulin resistance (e.g., a medical condition described herein), the method comprising administering to a patient in need of such treatment or prophylaxis an amount of an agent identified by a screening method described herein that is effective to treat or prevent a medical condition relating to insulin resistance, and a pharmaceutically acceptable carrier.
  • a medical condition relating to insulin resistance e.g., a medical condition described herein
  • standard protocols and “standard procedures”, when used in the context of molecular biology techniques, are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994, or Sambrook, J., Fritsch, E.F. and Maniatis, T, Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.
  • P element-mediated mutagenesis is a widely used technology in Drosophila genetics (Cooley, L. et al. (1988) Science 239: 1121-1128; Robertson, H.M. et al. (1988) Genetics 118: 461-470).
  • the P element is a well-characterized transposable element, which can introduce heritable loss of function mutations into a wide array of genes. Coupled with genomic annotation of the P element insertion site, P element libraries provide a valuable reverse genetics tool. Genetic screens using libraries of P insertion mutants in known genes enable a rapid scanning of the genome to identify potential modifier genes.
  • Impaired insulin receptor signaling has phenotypic manifestation of smaller cell size (Huang, H. et al. (1999) Development 126: 5365-5372).
  • a genetic screen was performed to identify modifiers of insulin receptor signaling, using a library of P insertion mutagenized Drosophila lines. In this screen, the small eye phenotypic manifestation was used as read out.
  • the D. melanogaster gene CG9613 (GenBank Accession No. AE003678_27) was identified as a weak but consistent enhancer of the dominant negative insulin receptor phenotype.
  • the D. melanogaster predicted CG9613 protein sequence was used to search human genomic and EST databases, using the BLAST algorithm (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402).
  • a human protein sequence was predicted from human genomic DNA sequence using the Gene Wise algorithm (Birney, E. & Durbin, R. (2000) Genome Res. 10: 547-548).
  • the predicted human PHB polyprenyltransferase sequence is partially supported by a known EST sequence (GenBank Accession No. AF091086).
  • the human PHB polyprenyltransferase amino acid sequence claimed in this invention is 46% identical to the D.
  • melanogaster protein CG9613 melanogaster protein CG9613, and about 30% and 22 % identical to the yeast and bacterial protein homologues, respectively.
  • the identity to a putative PHB polyprenyltransferase protein from mouse is about 60%.
  • the 3 '-RACE, 50 ⁇ l PCR-reaction were made according to the manual using 5 ⁇ l universal primer mix (UPM), and 1 ⁇ l "FOMA 354" primer (SEQ ID NO:3).
  • the PCR reactions were performed using Advantage2 Polymerase Mix, Clontech (Cat. No. 8430-2) in a PE GeneAmp System 9700.
  • the PCR program was as follows: 5 cycle of 94°C 5 seconds, 72°C 3 minutes and 5 cycles of 94°C 5 minutes, 70°C 10 seconds, 72°C 3 minutes and finally 25 cycles for 94°C 5 seconds, 68°C 10 seconds, 72°C 3 minutes, and cooling to 4°C.
  • the purified PCR- fragment containing the 5 '-end of PHB polyprenyltransferase was joined together in a PCR reaction with the plasmid DNA containing the 3 '-part of this gene described above.
  • the PCR was performed as before, except for the use of 5 ⁇ l UPM from the SMARTTM RACE cDNA Amplification Kit, Clontech (Cat. No. K1811- 1) instead of FOMA 355.
  • a PCR product of 1700bp was purified with QIAGEN Mini Elute PCR Purification Kit (Cat. No. 28004).
  • PHB polyprenyltransferase for mammalian expression was made by using Gateway Cloning Technology from Life Technologies. Gateway compatible primers were designed FOMA377 and FOMA 376 and PCR was performed using sequenced plasmid above as a template. The PCR was performed in 50 ⁇ l using Advantage-GC 2PCR DNA polymerase2 Mix Clontech (Cat. No.
  • pBV130 The resulting entry clone was designated pBV130, and after replacement of five amino acid substitutions the insert was confirmed by sequencing as above (# BO 120) which gave the human PHB polyprenyltransferase nucleotide sequence (SEQ ID NO: 1).
  • SEQ ID NO: 1 A mammalian expression clone with native PHB polyprenyltransferase, designated pBV134, was constructed according to the manufacturer's instructions using the destination vector pDEST12.2 (Life Technologies; Cat. No.l 1812-011).
  • MTE- Array Multiple Tissue Expression Array
  • pBV130 The PHB polyprenyltransferase cDNA clone, pBV130, was digested with the restriction enzymes Dralll and Hz ⁇ dll. The resulting DNA fragment was separated on a 2% agarose gel. The 283 base pairs fragment was exercised from the gel and purified using QIAEX II Agarose Gel Extraction Kit (QIAGEN, Cat. No. 20021).
  • Glucose uptake was measured as described by Hundal, H.S. et al. (1994) Biochem. J. 297 (Pt 2): 289-295. Briefly, after incubation with hormone for 45 minutes, if not otherwise stated, cell monolayers were rinsed with glucose free PBS. Glucose uptake was quantified by incubating the cells in the presence of 1 ⁇ Ci/ml -1H-2-deoxy- glucose in PBS for 8 minutes. Non-specific uptake was determined by quantifying cell- associated radioactivity in the presence of 10 ⁇ M cytochalasin B. Uptake of 2-deoxy- glucose was terminated by rapidly aspirating the medium, followed by three successive washes of cell monolayers with ice cold PBS. The cells were lysed in 0.5 M NaOH, followed by liquid scintillation counting. Rates of transport were normalized for protein content in each well.
  • Rat skeletal muscle L6 cells were transient transfected with the pBV134 vector expressing native human PHB polyprenyltransferase. As control the same cells were transfected in parallel with a pDEST12.2 vector (Life Technologies; Cat. No.11812- 011). Transfection was done using lipofectamine following the manufacturer's recommendation (Invitrogen; LipofectAMINE 2000 Cat. No. 11668).
  • Fig. 2 The results indicate that overexpression of PHB polyprenyltransferase in muscle cells induces an insulin resistance since no increase of glucose uptake by the muscle cells after insulin stimulation was observed compared to control experiment.
  • EXAMPLE 5 Prediction of a coding region, sorting signals and localization sites
  • SEQ ID NO:l the nucleotide sequence (SEQ ID NO:l) was analyzed by the ESTScan method (http://www.ch.embnet.org/software/ESTScanhelp.html). This prediction suggested a translated protein starting at methionine-38 of SEQ ID NO:2.
  • previously known human DNA sequences having similarity to SEQ ID NO:l predict a protein with an ORF starting after the fourth (GenBank accession Nos. AAY27589 and BC008804) or the fifth (AAG93279) encoded methionine in SED ID NO:2.
  • TM-HMM is a method to model and predict the localization and orientation of alpha helices in membrane-spanning proteins (Sonnhammer et al. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. ISMB 6: 175-182). Seven to eight transmembrane segments were identified in the protein shown as SEQ ID NO:2.
  • the PSORT II tool (available at http://psort.nibb.ac.jp) is used to predict the subcellular localization sites of proteins from their amino acid sequences.
  • the analysis done on SEQ ID NO.2 predicted a protein of mitochondrial 47% and 39% endoplasmic reticulum localization with no N-terminal signal peptide nor KKXX-like motif in the C-terminus. This is in agreement with the general view for other PHB polyprenyltransferases.
  • the yeast homologue protein has been shown to be mitochondrial (Ashby, MN, et al. (1992) J. Biol. Chem.
  • Pfam http:/p fam.wustl.edu
  • UbiA domain which is also present in several other polyprenyltransferases.
  • the dbSNP database http://www.ncbi.nlm.nih.gov/blast/Blast.cgi was screened using the PHB polyprenyltransferase sequence as query. No polymorphism in the PHB polyprenyltransferase gene between nucleotide 151 to 1263 was identified.
  • EXAMPLE 6 Conservation of biochemical function between the yeast and human PHB polyprenyltransferase proteins To study the functional activity of the human PHB polyprenyltransferase protein, we introduced a plasmid containing the human PHB polyprenyltransferase cDNA (SEQ ID NO:l) into S. cerevisiae yeast cells having a disrupted PHB polyprenyltransferase (coq2) gene. Compared to wild type yeast cells, this coq2- truncated strain cannot grow on a non-fermentable carbon source (i.e., glycerol or ethanol), but growth is restored after complementation by the human homologue protein.
  • a non-fermentable carbon source i.e., glycerol or ethanol
  • the human PHB polyprenyltransferase gene was cloned in a yeast expression vector using pYES2.1 TOPO TA cloning kit from Invitrogen (Catalog No. K4150-01). Construction began with the PCR amplification of the gene using oligonucleotides designated SOCY-224 (SEQ ID NO:8) and SOCY-225 (SEQ ID NO:9). PCR was performed in 50 ⁇ l using pfu-turbo DNA Polymerase, Stratagene (Cat. No. 600252-51), 2 ⁇ l each of the primers (10 ⁇ M), 5 % DMSO, 160 ⁇ M dATP, and pBV134 as template DNA.
  • the Perkin Elmer Gene Amp PCR system 9700 was used with the following program: 95°C, 4 min; (95°C 30 s, 50°C 30 s, 72°C 1.25 min) x 25; and 72°C, 10 min; followed by cooling to 4°C. 7 ⁇ l of the reaction mixture was loaded on a 1.2% E-Gel, and a fragment of approximately 1265 base pairs was cut out from the gel, DNA was extracted by centrifugation at 13,000 rpm during 5 minutes in a SPIN-X filter (Costar, Cat. No. 8163). Afterwards, the DNA was incubated with Taq DNA Polymerase (Boehringer, Cat.
  • the yeast PHB polyprenyltransferase (Coq2) gene was cloned from Saccharomyces cerevisiae Poly A RNA (Clontech Catalog No. 6999-1) using the LifeTechnologies ThermoScript RT-PCR system following the manufacturer's instructions (Cat. No. 11146-016). The best result was obtained using 100 ng of yeast Poly A + RNA and the resulting cDNA was used a template to PCR amplify the Coq2 gene using primers designated SOCY-221 (SEQ ID NO: 10) and SOCY-226 (SEQ D NO: 11).
  • the PCR was performed in 50 ⁇ l using 2 ⁇ l of cDNA, and PLATINIUM Taq DNA Polymerase High Fidelity according to manufacture (Gibco BRL, Cat. No. 11304- 011).
  • the Perkin Elmer Gene Amp PCR system 9700 was used with the following program: 94°C, 2 in; (94°C 30 s, 55°C 30 s, 68°C 1.25 min) x 40, followed by cooling to 4°C.
  • a 10 ⁇ l reaction aliquot was loaded on a 1.2% E-Gel, a fragment of approximately 1250 base pairs was cut out from the gel and the DNA was extracted by ceritrifugation at 13,000 rpm during 5 minutes in a SPIN-X filter.
  • the cloning of the yeast coq2 gene in pYes2.1/V5-His plasmid was done as described above, and the resulting plasmid was named pBV275.
  • the yeast null mutant strain CEN ⁇ Coq2 (MATa, his3- ⁇ l, leu2-3,112, tpr-289, ura3-52, MAL2-8 c, MAL3, SUC3, COQ2::HIS3) (Hsu, AY et al. (2000) Biochem. Biophys. Acta 1484: 287-297) which carries a truncated Coq2 gene was grown overnight in 10 ml of YPD medium to stationary phase.
  • the cells were made competent and transformed with pBV277 and pBV275 plasmid DNA following Invitrogen protocols (Invitrogen, pYes2.1 TOPO TA cloning Kit, Instruction Manual, 00092 25- 0261). Isolated recombinant yeast colonies carrying the gene for yeast Coq2 and human PHB polyprenyltransferase were obtained after growth in SD/-ura plates. Before the complementation experiments the Rho + genotype of these recombinant yeast cells was determine by mating with the JM8 yeast strain which is Rho " (MATa, adel,r°) (McEwen, J.E. et al. (1986) J. Biol. Chem. 261: 11872-11879).
  • Diploid cells were selected by 2-3 days growth in YNB-Glucose medium. Cells were replica plated into YPGlyc medium. After 2-3 days at 30°C, recombinant yeast expressing either yeast Coq2 or human PHB:PPT proteins grew, indicating that the yeast null mutant strain CEN ⁇ Coq2 in both cases was Rho .
  • EXAMPLE 7 Analysis of minimal sequence requirement for enzymatic activity of human PHB polyprenyltransferase
  • PHB polyprenyltransferase protein we introduced in separate experiments a plasmid containing either the full-length human PHB polyprenyltransferase cDNA (SEQ ID NO: 1) or two N-terminal deleted forms, 50 and 127 amino acids, respectively, into S. cerevisiae yeast cells having a disrupted PHB polyprenyltransferase (coq2) gene.
  • S. cerevisiae yeast cells having a disrupted PHB polyprenyltransferase (coq2) gene.
  • this co ⁇ 2-truncated strain cannot grow on a non- fermentable carbon source (i.e., glycerol or ethanol), but growth can be restored after complementation by the functionally active human homologue protein.
  • the plasmid carrying the full-length human PHB polyprenyltransferase and the yeast Coq2 gene used as control were prepared as described in Example 6.
  • two new plasmids were prepared, containing a sequence deletion of 50 ( ⁇ l-50) and 127 ( ⁇ l- 127) N-terminal amino acids of the human PHB polyprenyltransferase protein.
  • the deleted genes were cloned in a yeast expression vector using pYES2.1 TOPO TA cloning kit from Invitrogen (Catalog No. K4150-01).
  • PCR was performed in 50 ⁇ l using pfu-turbo DNA Polymerase, Stratagene (Cat. No. 600252-51), 2 ⁇ l each of the primers (10 ⁇ M), 5 % DMSO, 160 ⁇ M dATP, and pBV134 as template DNA.
  • the Perkin Elmer Gene Amp PCR system 9700 was used with the following program: 95°C, 4 min; (95°C 30 s, 50°C 30 s, 72°C 1.25 min) x 25; and 72°C, 10 min; followed by cooling to 4°C. 7 ⁇ l of each reaction mixture were loaded on a 1.2% E-Gel, and a fragment of approximately 1115 bp and 884 bp were cut out from the gel, DNA was extracted by centrifugation at 13,000 rpm during 5 min in a SPTN-X filter (Costar, Cat.No. 8163). Afterwards, the DNAs were incubated with Taq DNA Polymerase (Boehringer, Cat.
  • the resulting plasmids carrying the ( ⁇ l-50) and 127 ( ⁇ l-127) deleted human PHB polyprenyltransferase genes were named pBV601 and pBV600, respectively.
  • the yeast null mutant strain CEN ⁇ Coq2 (MATa, his3- ⁇ l, leu2-3, 112, tpr-289, ura3-52, MAL2-8 c, MAL3, SUC3, COQ2::HIS3) (Hsu, AY et al. (2000) Biochem. Biophys. Acta 1484: 287-297) which carries a truncated Coq2 gene was grown overnight in 10 ml of YPD medium to stationary phase.
  • the cells were made competent and transformed separately with pBV277, pBV275, pBV600 and pBV601 plasmid DNA following Invitrogen protocols (Invitrogen, pYes2.1 TOPO TA cloning Kit, Instruction Manual, 00092 25-0261). Isolated recombinant yeast colonies carrying the gene for yeast Coq2 and full-length, ( ⁇ l-50) and ( ⁇ l-127) human PHB polyprenyltransferase were obtained after growth in SD/-ura plates. Before the complementation experiments the Rho genotype of these recombinant yeast cells was determine by mating with the JM8 yeast strain, which is Rho " (MATa, adel, rO) (McEwen, J.E. et al.
  • Diploid cells were selected by 2-3 days growth in YNB-Glucose medium. Cells were replica plated into YPGlyc medium. After 2-3 days at 30°C, recombinant yeast expressing yeast Coq2 or all human PHB: PPT proteins grew, indicating that the yeast null mutant strain CEN ⁇ Coq2 in both cases was Rho + .

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Abstract

La présente invention concerne de nouvelles séquences nucléotidiques, les polypeptides de la PHB :polyprényltransférase humaine (EC 2.5.1) codés par les séquences nucléotidiques et des procédés d'identification d'agents modulant l'activité de la PHB:polyprényltransférase humaine. De tels agents devraient être utiles dans le traitement d'états médicaux liés à la résistance à l'insuline, notamment le diabète de type II.
PCT/SE2003/000285 2002-02-21 2003-02-21 Nouvelles sequences nucleotidiques codant pour les polypeptides de la phb:polyprenyltransferase humaine Ceased WO2003070926A1 (fr)

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WO2005082400A1 (fr) * 2004-02-27 2005-09-09 Leangene Ab Proteines therapeutiques traitant des etats medicaux associes a l'obesite et/ou la resistance a l'insuline

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Publication number Priority date Publication date Assignee Title
WO2002055664A2 (fr) * 2001-01-12 2002-07-18 Exelixis, Inc. Modulation de la signalisation du recepteur de l'insuline

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WO2002055664A2 (fr) * 2001-01-12 2002-07-18 Exelixis, Inc. Modulation de la signalisation du recepteur de l'insuline

Non-Patent Citations (5)

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Title
ARUN GUPTA ET AL.: "(37) 4-Hydroxybenzoate polyprenyltransferase from rat liver", METHODS IN ENZYMOLOGY, vol. 110, 1985, pages 327 - 334, XP002966443 *
DATABASE BIOSIS [online] MCCARTY M.F.: "Can correction of sub-optimal coenzyme Q status improve beta-cell function in type II diabetes?", XP002966444, Database accession no. PREV199900343016 *
DATABASE GENBANK [online] January 2002 (2002-01-01), STRAUSBERG R., XP002966447, accession no. EBI Database accession no. (Q96H96) *
MEDICAL HYPOTHESES, vol. 52, no. 5, 1999, pages 397 - 400 *
MELZER MARTIN ET AL.: "Characterization of polyprenyldiphosphate: 4-Hydroxybenzoate polyprenyltransferase from escherichia coli", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1212, 1994, pages 93 - 102, XP002966445 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005082400A1 (fr) * 2004-02-27 2005-09-09 Leangene Ab Proteines therapeutiques traitant des etats medicaux associes a l'obesite et/ou la resistance a l'insuline

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