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WO2001030830A2 - Sequences geniques identifiees par recherche de motif de proteines dans une base de donnees - Google Patents

Sequences geniques identifiees par recherche de motif de proteines dans une base de donnees Download PDF

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Publication number
WO2001030830A2
WO2001030830A2 PCT/US2000/029787 US0029787W WO0130830A2 WO 2001030830 A2 WO2001030830 A2 WO 2001030830A2 US 0029787 W US0029787 W US 0029787W WO 0130830 A2 WO0130830 A2 WO 0130830A2
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Prior art keywords
polypeptide
sequences
elegans
sequence information
sequence
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WO2001030830A3 (fr
Inventor
Anne N. Murphy
Eoin D. Fahy
Sandra E. Wiley
William Clevenger
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Migenix Corp
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Mitokor Inc
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Priority to AU13515/01A priority Critical patent/AU1351501A/en
Publication of WO2001030830A2 publication Critical patent/WO2001030830A2/fr
Publication of WO2001030830A3 publication Critical patent/WO2001030830A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/10—Sequence alignment; Homology search
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the invention relates generally to methods of using biological sequences identified by querying biological sequence databases.
  • the invention relates to molecular cloning and expression of mitochondrial proteins that bind, transport and/or regulate intracellular ions such as calcium ions, and the use of such proteins in screening assays.
  • Mitochondria are organelles that are the main energy source in cells of higher organisms. These organelles provide direct and indirect biochemical regulation of a wide anay of cellular respiratory, oxidative and metabolic processes, including metabolic energy production, aerobic respiration and intracellular calcium regulation.
  • mitochondria are the site of electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis.
  • ETC electron transport chain
  • ATP adenosine triphosphate
  • mitochondria In addition to their role in energy production in growing cells, mitochondria (or at least mitochondrial components) participate in programmed cell death (PCD), also known as apoptosis (see Newmeyer et al., Cell 79:353-364, 1994; Liu et al., Cell 86: 147-157, 1996).
  • PCD programmed cell death
  • Apoptosis is apparently required for normal development of the nervous system and functioning of the immune system.
  • Some disease states are associated with insufficient apoptosis (e.g., cancer and autoimmune diseases) or excessive levels of apoptosis (e.g., stroke and neurodegeneration).
  • Mitochondria contain an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes.
  • the subcompartment within the inner mitochondrial membrane is commonly refened to as the mitochondrial matrix (for review, see, e.g., Ernster et al., 1981 J. Cell Biol.
  • the inner mitochondrial membrane While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (greater than about 10 kD) molecules.
  • Z stands for -2.303 RT/F.
  • the value of Z is -59 at 25°C when ⁇ p and ⁇ m are expressed in mV and ⁇ pH is expressed in pH units (see, e.g., Ernster et al., J. Cell Biol. 91:227s, 1981 and references cited therein).
  • ETC Complex V a process that is coupled stoichiometrically with transport of a proton into the matrix. ⁇ m is also the driving force for the influx of cytosolic Ca 2+ into the mitochondrion.
  • the inner membrane is largely impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the primary means whereby protons can return to the matrix.
  • MMT mitochondrial permeability transition
  • ⁇ m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca 2+ , Na + , K + and H + ) and large (e.g., proteins).
  • small e.g., ionic Ca 2+ , Na + , K + and H +
  • large e.g., proteins
  • Loss of mitochondrial potential also appears to be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases such as Alzheimer's Disease; diabetes mellitus; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome, as well as numerous other mitochondria associated diseases.
  • degenerative diseases such as Alzheimer's Disease; diabetes mellitus; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome, as well
  • Normal mitochondrial function includes regulation of cytosolic free calcium levels by sequestration of excess Ca 2+ within the mitochondrial matrix. Depending on cell type, cytosolic Ca 24" concentration is typically 50-100 nM. In normally functioning cells, when Ca 2+ levels reach 200-300 nM, mitochondria begin to accumulate Ca 2+ as a function of the equilibrium between influx via a Ca 2+ uniporter in the inner mitochondrial membrane and Ca 2+ efflux via both Na + dependent and Na + independent calcium earners, a process driven by the electrochemical gradient of protons described above.
  • such perturbation of intracellular calcium homeostasis is a feature of diseases associated with altered mitochondrial function, regardless of whether the calcium regulatory dysfunction is causative of, or a consequence of, altered mitochondrial function including MPT.
  • Excessive Ca 2+ sequestration in mitochondria may contribute to the pathogenesis of certain diseases associated with altered mitochondrial function, and may be related to MPT, ETC uncoupling or inhibition accompanied by free radical production including generation of reactive oxygen species (ROS) and/or disequilibria among other selective mitochondrial ion transporters such as K /H + and K + /ATP channels.
  • ROS reactive oxygen species
  • Calcium uniporter activity may also play a role, for example, in ischemia-reperfusion injury in a variety of tissues.
  • mitochondrial calcium uniporter calcium transport activity has thus been characterized, including its activation by ADP, inhibition by ATP, Mg; , ruthenium red and its derivative Ru360 (Matlib et al., 1998 J Biol Chem 273:10223; Emerson et al., 1993 J Amer Chem Soc 115:11799) and competitive inhibition by Sr 2+ , Mn 2+ and La + , no specific polypeptide has been identified and confirmed as an authentic mitochondrial calcium uniporter, nor has a gene encoding such a uniporter been determined.
  • mitochondrial calcium uptake is poorly understood at the molecular level, however, and neither the gene(s) encoding the mitochondrial calcium uniporter, nor the protein(s) responsible for such mitochondrial calcium uptake (e g , one or more mitochondrial calcium uniporter polypeptides) have been identified.
  • Previous efforts to define the uniporter involved attempts to isolate mitochondrial calcium uniporter protein(s) that typically yielded molecules that differed functionally from the uniporter (e g, Rosier et al., 1980 FEBS Lett. 109:99; Rosier et al., 1981 Arch Biochem. Biophys.
  • BLAST Basic Local Alignment Search Tool
  • BLAST BLAST
  • BLAST BLAST
  • BLAST BLAST identifies matches and assigns statistical scores of significance for such matches. Further information on BLAST may be found at, for example, http://www.ncbi.nlm.nih.gov/BLAST; Altschul, J. Mol. Biol. 219:555-565, 1991 ; and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 59:10915-10919, 1992.
  • Other known search algorithms include FASTA, PSI-BLAST, ALIGN, MEGALIGN and WISETOOLS.
  • It is an aspect of the invention to provide a recombinant expression construct comprising at least one regulated promoter operably linked to a first nucleic acid encoding a C. elegans p95.6/YN52 polypeptide.
  • the construct further comprises at least one additional nucleic acid sequence that regulates transcription.
  • the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with a polypeptide product of a second nucleic acid sequence.
  • the polypeptide product of the second nucleic acid sequence is an enzyme while in certain embodiments the fusion protein localizes to membranes, which in certain further embodiments are mitochondrial membranes.
  • elegans p95.6/YN52 polypeptide is expressed as a fusion protein with at least one product of a second nucleic acid sequence encoding a polypeptide cleavable by a protease, the C. elegans p95.6/YN52 polypeptide being separable from the fusion protein by cleavage with the protease.
  • the invention provides an isolated host cell comprising a recombinant expression construct comprising at least one regulated promoter operably linked to a first nucleic acid encoding a C. elegans p95.6/YN52 polypeptide.
  • the host cell is a prokaryotic cell and in other embodiments the host cell is a eukaryotic cell.
  • the eukaryotic cell is a yeast cell, an insect cell, a mammalian cell or a nematode cell, which in certain further embodiments is a C. elegans cell.
  • the host cell lacks an endogenous C. elegans p95.6/YN52 homolog polypeptide, and in some embodiments expression of at least one gene encoding an endogenous C. elegans p95.6/YN52 homolog is substantially impaired.
  • the recombinant expression construct described above is a recombinant viral expression construct.
  • It is another aspect of the invention to provide a recombinant expression construct comprising at least one promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence encoding a C. elegans p95.6/YN52 polypeptide wherein the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with a polypeptide product of the second nucleic acid sequence.
  • the expression construct is a recombinant viral expression construct.
  • the polypeptide product of the second nucleic acid sequence is an enzyme.
  • the fusion protein localizes to membranes, which in certain further embodiments are mitochondrial membranes.
  • the expression construct further comprises at least one additional nucleic acid sequence that regulates transcription.
  • the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with at least one product of a second nucleic acid sequence encoding a polypeptide cleavable by a protease, the C. elegans p95.6/YN52 polypeptide being separable from the fusion protein by cleavage with the protease.
  • the invention also provides an isolated host cell comprising a recombinant expression construct comprising at least one promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence encoding a C. elegans p95.6/YN52 polypeptide wherein the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with a polypeptide product of the second nucleic acid sequence.
  • the host cell is a prokaryotic cell and in certain other embodiments the host cell is a eukaryotic cell, which may in certain further embodiments be a yeast cell, an insect cell, a mammalian cell or a nematode cell, which in certain still further embodiments is a C. elegans cell.
  • the host cell lacks an endogenous C. elegans p95.6/YN52 homolog polypeptide.
  • expression in the host cell of at least one gene encoding an endogenous C. elegans p95.6/YN52 homolog is substantially impaired.
  • the present invention provides a method of producing a recombinant C. elegans p95.6/YN52 polypeptide, comprising culturing a host cell comprising a recombinant expression construct comprising at least one regulated promoter operably linked to a first nucleic acid encoding a C. elegans p95.6/YN52 polypeptide. It is another aspect of the invention to provide a method of producing a recombinant a C.
  • elegans p95.6/YN52 polypeptide comprising culturing a host cell comprising a recombinant expression construct comprising at least one promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence encoding a C. elegans p95.6/YN52 polypeptide wherein the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with a polypeptide product of the second nucleic acid sequence.
  • the invention provides a method of producing a recombinant C.
  • the invention provides a method of producing a recombinant C.
  • elegans p95.6/YN52 polypeptide comprising culturing a host cell infected with a recombinant viral expression construct comprising at least one promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, the first nucleic acid sequence encoding a C. elegans p95.6/YN52 polypeptide wherein the C. elegans p95.6/YN52 polypeptide is expressed as a fusion protein with a polypeptide product of the second nucleic acid sequence. It is another aspect of the invention to provide a C. elegans p95.6/YN52 polypeptide produced by any of the methods just described.
  • the invention provides, in another aspect, an isolated C. elegans p95.6/YN52 polypeptide.
  • the C. elegans p95.6/YN52 polypeptide is a recombinant p95.6/YN52 polypeptide or a variant or fragment thereof.
  • the invention provides an isolated C. elegans p95.6/YN52 fusion protein comprising a C. elegans p95.6/YN52 polypeptide fused to at least one additional polypeptide sequence.
  • the one additional polypeptide sequence is an enzyme sequence or a variant or fragment thereof.
  • the fusion protein localizes to membranes, which in certain further embodiments are mitochondrial membranes.
  • an isolated C. elegans p95.6/YN52 fusion protein comprising a C. elegans p95.6/YN52 polypeptide fused to at least one additional polypeptide sequence cleavable by a protease, the C. elegans p95.6/YN52 polypeptide being separable from the fusion protein by cleavage with the protease.
  • the additional polypeptide sequence is a polypeptide having affinity for a ligand.
  • the present invention provides a method for determining the presence of a C. elegans p95.6/YN52 polypeptide in a biological sample comprising contacting a biological sample suspected of containing a C. elegans p95.6/YN52 polypeptide with a p95.6/YN52 ligand under conditions and for a time sufficient to allow binding of the p95.6/YN52 ligand to a C. elegans p95.6/YN52 polypeptide; and detecting the binding of the p95.6/YN52 ligand to a C. elegans p95.6/YN52 polypeptide, and therefrom determining the presence of a C.
  • the p95.6/YN52 ligand is an antibody, which in certain embodiments may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, a chimeric antibody or a functional antibody fragment.
  • the functional antibody fragment is a F(ab)', a F(ab') 2 and a F(V). It is another aspect of the invention to provide a method for identifying an agent that binds to a C. elegans p95.6/YN52 polypeptide, comprising contacting a candidate agent with a biological sample containing at least one recombinant C.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising contacting a biological sample comprising a recombinant C.
  • elegans p95.6/YN52 polypeptide with a detectable p95.6/YN52 ligand in the absence and presence of a candidate agent; and comparing binding of the detectable p95.6/YN52 ligand to the recombinant C. elegans p95.6/YN52 polypeptide in the absence of the agent to binding of the detectable p95.6/YN52 ligand to the recombinant C. elegans p95.6/YN52 polypeptide in the presence of the agent, and therefrom identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • the detectable p95.6/YN52 ligand is an antibody, which in certain further embodiments may be a polyclonal antibody, a monoclonal antibody, a single chain antibody, a chimeric antibody or a functional antibody fragment.
  • the functional antibody fragment is a F(ab'), a F(ab') 2 or a F(V).
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a biological sample containing mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C elegans p95.6/YN52 polypeptide.
  • the invention provides a method for identifying an agent that interacts with a C.
  • elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a first biological sample comprising mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent to a reference level of mitochondrial activity in a second biological sample comprising mitochondria which lack a recombinant C. elegans p95.6/YN52 polypeptide, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • the present invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a biological sample comprising a cell which contains mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • the cell is a permeabilized cell.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a first biological sample comprising a cell which contains mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent to a reference level of mitochondrial activity in a second biological sample comprising a cell which contains mitochondria that lack a recombinant C.
  • the cell is a permeabilized cell.
  • the mitochondrial activity is intracellular calcium level ora cellular response to an apoptogen.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a biological sample comprising at least one isolated mitochondrion, wherein the mitochondrion comprises a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a first biological sample comprising at least one isolated mitochondrion, wherein the mitochondrion comprises a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent to a reference level of mitochondrial activity in a second biological sample comprising one or more isolated mitochondria that lack a recombinant C.
  • the invention provides a pharmaceutical composition comprising an agent that interacts with a C. elegans p95.6/YN52 polypeptide, wherein the agent is identified according to any of the just described methods for identifying an agent that interacts with a C. elegans p95.6/Y 52 polypeptide.
  • the invention relates to a method of treatment comprising administering to a subject such a pharmaceutical composition.
  • the invention provides an isolated polypeptide having the amino acid sequence of SEQ ID NO:2, and in another aspect the invention provides an isolated polypeptide having the amino acid sequence of SEQ ID NO:20.
  • the invention provides an isolated nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:2, and in another aspect the invention provides an isolated nucleic acid encoding the polypeptide having the amino acid sequence of SEQ ID NO:20.
  • the nucleic acid is an RNA molecule and in certain still further embodiments the RNA molecule has the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid is an RNA molecule and in certain still further embodiments the RNA molecule has the nucleotide sequence of SEQ ID NO: 19. In certain other embodiments the nucleic acid is a DNA molecule, which in certain further embodiments has the nucleotide sequence SEQ ID NO:l . In certain other embodiments the nucleic acid is a DNA molecule, which in certain further embodiments has the nucleotide sequence SEQ ID NO: 19. In another embodiment the invention provides an expression vector comprising an isolated nucleic acid encoding the polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:20, operably linked to a promoter and a translation initiation sequence. In another embodiment the invention provides an antisense vector comprising the reverse complement of an isolated nucleic acid encoding the polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:20 operably linked to a promoter.
  • the invention provides an isolated nucleic acid encoding the polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:20, wherein the genetic code is the universal genetic code comprising the following codon usages: Ala is encoded by GCT, GCU, GCC, GCA, or GCG; Arg is encoded by CGT, CGU, CGC, CGA, CGG, AGA, or AGG; Asn is encoded by AAT, AAU, or AAC; Asp is encoded by GAT, GAU, or GAC; Cys is encoded by TGT, UGT, TGU, UGU, TGC, or UGC; Gin is encoded by CAA or CAG; Glu is encoded by GAA or GAG; Gly is encoded by GGT, GGU, GGA, GGG, or GGC; His is encoded by CAT, CAU, or CAC; Ile is encoded by ATT, AUT, ATU, AUU, ATC, AUC, ATA,
  • the invention provides an isolated nucleic acid encoding the polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:20, wherein the genetic code is the yeast mitochondrial genetic code comprising the following codon usages: Ala is encoded by GCT, GCU, GCC, GCA, or GCG; Arg is encoded by CGT, CGU, CGC, CGA, CGG, AGA, or AGG; Asn is encoded by AAT, AAU, or AAC; Asp is encoded by GAT, GAU, or GAC; Cys is encoded by TGT, UGT, TGU, UGU, TGC, or UGC; Gin is encoded by CAA or CAG; Glu is encoded by GAA or GAG; Gly is encoded by GGT, GGU, GGA, GGG, or GGC; His is encoded by CAT, CAU, or CAC; Ile is encoded by ATT, AUT, ATU, AUU, ATC, AUC,
  • the invention provides an isolated nucleic acid encoding the polypeptide having the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:20, wherein the genetic code is the mammalian mitochondrial genetic code comprising the following codon usages: Ala is encoded by GCT, GCU, GCC, GCA, or GCG; Arg is encoded by CGT, CGU, CGC, CGA, or CGG; Asn is encoded by AAT, AAU, or AAC; Asp is encoded by GAT, GAU, or GAC; Cys is encoded by TGT, UGT, TGU, UGU, TGC, or UGC; Gin is encoded by CAA or CAG; Glu is encoded by GAA or GAG; Gly is encoded by GGT, GGU, GGA, GGG, or GGC; His is encoded by CAT, CAU, or CAC; Ile is encoded by ATT, AUT, ATU, AUU, ATC, AUC, ATA, or
  • the present invention utilizes protein motifs, short (from 3 to about 300 amino acids in length) peptide sequences that define a portion (domain) of the protein having or directing a specific function such as, e.g., the reactive site of an enzyme, structural elements ( ⁇ -helix, ⁇ -sheet, etc.), or a binding site for a ligand or regulator of the protein. If a consensus sequence for every' type of peptide sequence motif were available, it would be possible (in theory, at least) to identify such motifs in polypeptide sequences in a protein of unknown function and thereby predict a limited number of potential functions for the protein.
  • a candidate sequence is a nucleotide or peptide sequence having features consistent with a nucleic acid or protein involved in a cellular or biochemical activity of interest, where the nucleic acid or protein has been postulated to exist, or has even been identified, but the sequence of which has not been determined.
  • the present invention provides a method of computer-assisted cloning of a coding sequence of interest, comprising the steps of: identifying a cellular or biochemical function of interest; using algorithms of embodiments of the present invention to identify one or more candidate sequences in a database; optionally, if the candidate sequence is a polypeptide sequence, reverse translating the polypeptide sequence to obtain a nucleotide sequence; isolating candidate nucleic acids having the candidate sequences using methods such as, e.g., the polymerase chain reaction (PCR); assaying the candidate nucleic acids, and/or the candidate proteins encoded thereby, for their ability to influence the cellular or biochemical activity of interest, wherein the coding sequence of interest is a candidate nucleic acid having the ability to influence the cellular or biochemical activity of interest.
  • PCR polymerase chain reaction
  • aspects of the invention include a computer-implemented method for extracting information from biological sequence databases.
  • the method includes effectively three steps: (i) receiving a motif that defines at least one polypeptide sequence of interest, (ii) searching a database of biological sequence databased on a query formulated from the motif to obtain a set of polypeptide sequence information from the database, and (iii) filtering the set of polypeptide sequence information to obtain a filtered set of polypeptide sequence information.
  • the defined polypeptide sequence is believed to exist within at least one database of biological sequence information, and the database includes biological sequence data for several sub-cellular molecular components of several biological organisms.
  • the filtered set of polypeptide sequence information includes: (a) polypeptide sequence information associated with at least one biological organism, (b) polypeptide sequence information associated with at least one desired sub-cellular molecular component, or (c) polypeptide sequence information associated with at least one protein domain, or (d) combinations of (a), (b) and/or (c).
  • the filtered set of polypeptide sequence information may also be provided as a report to a user.
  • Figure 1 is a block diagram of a computing system suitable for employing aspects of the invention to analyze data in, and extract data from, biological sequence databases.
  • Figure 2 is a flowchart depicting a method of extracting or targeting desired sequences from a biological database.
  • Figure 3 is a display screen showing results of a text search for sequences identified with calcium using the Pfam database and associated algorithm.
  • Figure 4 is a display screen showing motif generation using a MAST database and associated algorithm.
  • Figure 5 is a display screen showing a table or spreadsheet of sequences from which yeast sequences have been filtered.
  • Figure 6 is a display screen showing additional data associated with one line of the table of Figure 5.
  • Figure 7 is a display screen showing a dialogue box for querying a motif database.
  • Figure 8 is a display screen showing an example of output produced from a query using the display screen of Figure 7.
  • Figure 9 is a flowchart for targeting one protein in the GenBank biological database using IQ and EFHAND motifs.
  • Figure 10 is a display screen showing sample output of a sequence alignment algorithm.
  • Figure 1 1 is a display screen showing a table of shared motifs employed under one step of Figure 9.
  • Figure 12 shows a DNA sequence identified using the computing system [SEQ ID NO:l] that contains nucleotides encoding p95.6/YN52 from C. elegans with restriction endonuclease sites, and the deduced p95.6/YN52 amino acid sequence [SEQ ID NO:2].
  • Figure 13 shows a cDNA sequence [SEQ ID NO:3] that contains nucleotides encoding human PP7 (PPEF-1) serine/threonine phosphatase, and the deduced PP7 (PPEF-1) amino acid sequence [SEQ ID NO:4].
  • Figure 14 shows northern blot hybridization using P-labeled PCR products generated with the primer pairs (i) Unil for [SEQ ID NO:5] and Unil crev [SEQ ID NO:6] and (ii) Uni efor [SEQ ID NO:7] and Unilrev [SEQ ID NO:8] as probes.
  • Blot contained human polyA+ RNA from various tissue types as follows: Lane 1, heart; lane 2, brain; land 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas.
  • Figure 15 shows a cloned DNA sequence [SEQ ID NO: 19] that contains nucleotides encoding p95.6 from C. elegans.
  • Figure 16 shows the deduced p95.6 amino acid sequence [SEQ ID NO:20] encoded by a cloned DNA sequence [SEQ ID NO: 19].
  • the present invention is directed generally toward extracting from biological sequence databases, including gene and protein sequence databases, sequence information related to polypeptides of interest, and the use of such polypeptides as molecular targets in screening assays.
  • the invention is also directed generally to polynucleotide sequences that encode such polypeptides, for example, nucleic acid sequences refened to herein as "coding sequences of interest" (CSI).
  • CSI coding sequences of interest
  • the invention thereby provides compositions and methods related to specific CSI genes and gene products and related ligands, including compositions and methods for producing CSI polypeptide products and using such products in diagnostic, drug screening and therapeutic contexts.
  • CSI encode polypeptides that, ter alia, localize to mitochondria and are capable of calcium binding, calcium transport and/or other regulation of or by calcium.
  • Certain of such prefened embodiments relate to candidate mitochondrial calcium uniporters such as the C.
  • elegans p95.6/YN52 protein described herein and its homologs relate to a human serine/threonine phosphatase having a calcium binding EF-hand domain such as PP7 (PPEF-1) and its homologs, also described below.
  • PPEF-1 calcium binding EF-hand domain
  • a sequence database is searched and identifying numbers such as accession numbers are retrieved. Protein or polypeptide sequences associated with the accession numbers are retrieved and desired sequences filtered therefrom. For example, all yeast sequences are eliminated from the obtained protein sequences. An algorithm is performed on the resulting sequences to identify those sequences associated with a particular subcellular domain, such as a mitochondrial domain. Final filtering can include extracting only full length sequences and/or sequences for specific species or biological organisms. The resulting, final filtered data, is provided in a tabular format or input to a database for later query. Each of the above steps is described in more detail below.
  • nucleic acid coding sequences of interest may include nucleic acid molecules and may also include expression constructs comprising CSI.
  • expression of CSI provides CSI product (CSP) polypeptides such as C. elegans p95.6/YN52 and human PP7 that are useful for multiple purposes, including but not limited to polypeptides useful as molecular targets in screening assays for natural and synthetic small molecules that interact with CSP, including CSP binding ligands (e.g., p95.6/YN52 ligands).
  • CSP CSI product
  • the invention provides compositions and methods for producing recombinant CSP polypeptides that employ regulated promoters, and in certain of these and other aspects the invention provides compositions and methods for producing recombinant CSP polypeptides that are CSP fusion proteins, such as candidate calcium uniporter fusion proteins containing p95.6/YN52 polypeptide sequences.
  • the design of such expression systems includes the use of a host cell that lacks endogenous CSP or in which endogenous CSP gene expression is substantially impaired, as provided herein.
  • the present invention thus also pertains in part to methods for producing and isolating recombinant CSP polypeptides (e.g., p95.6/YN52 or PP7 (PPEF-1) polypeptides) that may then be used in various binding assays and screening assays and the like.
  • CSP polypeptides e.g., p95.6/YN52 or PP7 (PPEF-1) polypeptides
  • the present invention provides assays (including high throughput assays) for identifying agents that bind to recombinant CSP.
  • the present invention further relates in part to novel CSP ligands, the synthesis, selection and characterization of which would heretofore have not been possible given the need for expressed recombinant CSP polypeptides to use in binding assays.
  • the invention also pertains to agents that interact with CSP, including agents that enhance or impair any CSP functions known to the art, including but not limited to those described herein, and to incorporation of
  • the present invention also relates in part to the production of CSP polypeptides by using a recombinant expression vector having a regulatory nucleic acid operably linked to a CSI, for example, a sequence encoding a candidate calcium uniporter such as C. elegans p95.6/YN52 that has been identified using a computer system as described herein.
  • the invention provides compositions and methods for producing recombinant CSP polypeptides through the use of a regulated promoter; the invention also provides recombinant CSP polypeptides that are CSP polypeptide fusion proteins.
  • the invention also pertains to compositions and methods to identify the presence of CSP polypeptides and to isolate recombinant CSP polypeptides, and in addition to screening assays for compounds that interact with CSP polypeptides.
  • the present invention further relates to nucleic acids which hybridize to CSI as provided herein, as incorporated by reference or as will be readily apparent to those familiar with the art, if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences.
  • the present invention particularly relates to nucleic acids which hybridize under stringent conditions to the CSI refened to herein.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the nucleic acids which hybridize to CSI refened to herein, or their complements encode polypeptides which retain substantially the same biological function or activity as the CSP polypeptides encoded by the CSI.
  • hybridize under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules. Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed.
  • high stringency 0.1 x SSPE or SSC, 0.1% SDS, 65°C
  • medium stringency 0.2 x SSPE or SSC, 0.1% SDS, 50°C
  • low stringency 1.0 x SSPE or SSC, 0.1% SDS, 50°C.
  • the present invention is directed generally to the identification of a CSI encoding a CSP that is a divalent cation transporter polypeptide, and which in particularly prefened embodiments is a mitochondrial divalent cation transporter polypeptide.
  • Some divalent cations are recognized as intracellular messengers that are maintained at low cytoplasmic levels. For example, as noted above, excess levels of cytoplasmic Ca may lead to either necrotic or apoptotic forms of cell death in certain cells.
  • the concentration of free Ca 2+ in the cell cytoplasm e.g., ⁇ 10 nM is normally about a thousand fold lower than the
  • membrane transport protein may be used interchangeably with "transport protein,” “transporter polypeptide,” or “transporter.”
  • the methods of the present invention are directed to identifying a nucleic acid molecule encoding a divalent cation transporter that mediates cation transfer across the inner mitochondrial membrane, and in certain prefened embodiments the methods of the present invention are directed to identifying the presence of a divalent cation transporter in a biological sample.
  • a divalent cation transporter as provided herein is, in prefened embodiments, the mitochondrial calcium uniporter described above.
  • the mitochondrial calcium uniporter is an electrogenic calcium transporter.
  • ion transporters may be classified on the basis of whether they facilitate transmembrane movement of an ion by a mechanism that is electroneutral, or by a mechanism that is electrogenic. Electroneutral transporters do not carry a net charge, and mediate transmembrane delivery of one ionic species by exchanging such ion for a charge equivalent (e.g., Ca 2+ is exchanged for 2H + ) such that no net charge movement across the membrane accompanies transport of the ion(s).
  • a charge equivalent e.g., Ca 2+ is exchanged for 2H +
  • Transport catalyzed by electroneutral ionophores can be influenced by transmembrane pH gradients.
  • Electrogenic transporters contain no ionizable functions and form complexes with the ionic species being transported that are consequently charged, such that transmembrane charge movement does accompany transport of the ion (see, e.g., Dobler, 1981, Classification of Ionophores, In Ionophores and Their Structures, John Wiley and Sons, New York, NY; Westley, 1982, Notation and Classification, In Poly ether Antibiotics: Naturally Occurring Acid Ionophores (Westley, J.W., editor), Marcel Dekker, New York, NY).
  • a transporter polypeptide is a mitochondrial divalent cation transport polypeptide, and in certain particularly prefened embodiments the mitochondrial divalent cation transport polypeptide is a component of a calcium uniporter or other electrogenic transporter.
  • divalent cations i.e., chemical or biochemical species having a net charge of +2
  • divalent cations that may be delivered across a biological membrane include, but need not be limited, to calcium, cobalt, barium, iron, members of the lanthanide series including lanthanum, lead, magnesium, manganese, zinc and strontium.
  • an interaction between the divalent cation and the expressed transporter polypeptide includes any contact, binding, or association that leads to the transport of divalent cation from one cell compartment (e.g., cytoplasm) to another (e.g., mitochondrion), as detected by a the signal generated by the divalent cation-sensitive indicator molecule.
  • one cell compartment e.g., cytoplasm
  • another e.g., mitochondrion
  • Generation of such a signal may arise, according to non-limiting theory, from any contact, binding, association or other interaction between the divalent cation and the divalent cation-sensitive indicator molecule that leads to a detectable change in fluoresence, phosphoresence, bioluminesence, or the like (depending on the indicator selected), relative to the absence of such interaction, as an indication of divalent cation transport from one cell compartment (e.g., cytoplasm) to another (e.g., mitochondrion).
  • one cell compartment e.g., cytoplasm
  • another e.g., mitochondrion
  • a “divalent cation-sensitive indicator molecule” may include any enzyme, metabolic protein, marker protein (including fluorescent proteins) or other naturally occuning or synthetic detectable marker that is capable of generating a detectable signal as an indication of the presence of a divalent cation, preferably in proportion to the amount, level or conentration of the divalent cation that is present.
  • the invention pertains in part to detecting a signal generated by a calcium indicator molecule in a mitochondrion as provided herein.
  • the calcium indicator molecule may be endogenous to (e.g., naturally occuning in) the mitochondrion or it may be exogenous, which includes at least one calcium indicator molecule that does not occur naturally in the mitochondrion but that has been loaded, administered, admixed, expressed (including expression as the product of a genetically engineered nucleic acid construct), targeted, contacted, exposed or otherwise artificially introduced into the mitochondrion, as long as the calcium indicator molecule is capable of generating a detectable signal that is proportional to the level of calcium.
  • the calcium indicator molecule is exogenous and the detectable signal is a fluorescent signal.
  • the divalent cation-sensitive (e.g., calcium) indicator molecule is a fluorescent indicator
  • the signal generated by the indicator molecule may be detected by exposing the sample to light having an appropriate wavelength to excite the indicator, and determining resultant fluorescence with a suitable instrument for detecting a fluorescent light emission at an appropriate wavelength.
  • the divalent cation-sensitive (e.g., calcium) indicator molecule may be a light emission molecule, for example a fluorescent, phosphorescent, or chemiluminescent molecule or the like, which emits a detectable signal in the form of light when excited by excitation light of an appropriate wavelength.
  • fluorescent refers to luminescence (emission of light) that is caused by the absorption of radiation at one wavelength (“excitation”), followed by nearly immediate re-radiation (“emission”), usually at a different wavelength, that ceases almost at once when the incident radiation stops.
  • fluorescence occurs as certain compounds, known as fluorophores, are taken from a ground state to a higher state of excitation by light energy; as the molecules return to their ground state, they emit light, typically at a different wavelength.
  • Phosphorescence in contrast, refers to luminescence that is caused by the absorption of radiation at one wavelength followed by a delayed re-radiation that occurs at a different wavelength and continues for a noticeable time after the incident radiation stops.
  • “Chemiluminescence” refers to luminescence resulting from a chemical reaction
  • bioluminescence refers to the emission of light from living organisms or cells, organelles or extracts derived therefrom.
  • a variety of calcium indicators are known in the art and may be suitable for generating a detectable intracellular signal, for example, a signal that is proportional to the level of calcium, including but not limited to fluorescent indicators such as fura-2 (McCormack et al., 1989 Biochim. Biophys. Acta 973:420); mag-fura-2; BTC (U.S. Patent No. 5,501,980); fluo-3, fluo-4, fluo-5F and fluo-5N (U.S. Patent No.
  • Calcium Green 5N is a particularly prefened calcium indicator molecule for use according to the present invention.
  • a person having ordinary skill in the art can select a suitable calcium indicator from those described above or from other calcium indicators, according to the teachings herein and based on known properties (e.g., solubility, stability, etc.) of such indicators.
  • a cell permeant or cell impermeant indicator is needed (e.g., whether a sample comprises a permeabilized cell), affinity of the indicator for calcium (e.g., dynamic working range of calcium concentrations within a sample as provided herein), subcellular localization of the indicator (which may in turn depend on other properties of the host cell, such as one or more expressed, transfected components) and/or fluorescence spectral properties such as a calcium-dependent fluorescence excitation shift, may all be factors in the selection of a suitable calcium indicator.
  • affinity of the indicator for calcium e.g., dynamic working range of calcium concentrations within a sample as provided herein
  • subcellular localization of the indicator which may in turn depend on other properties of the host cell, such as one or more expressed, transfected components
  • fluorescence spectral properties such as a calcium-dependent fluorescence excitation shift
  • a variety of instruments can be used in methods of the invention to excite a calcium indicator molecule as provided herein that is a fluorescent compound, and to detect the signal generated by the calcium indicator molecule that is proportional to the level of calcium, e.g., to measure the resulting emission therefrom. Selection of a suitable instrument, light source, filter set, etc.
  • the calcium indicator molecule may depend on factors known to those familiar with the art, such as (i) application of energy (i.e., light) at a wavelength that will excite the calcium indicator molecule, preferably at or near the optimum excitation wavelength of the indicator molecule ( ⁇ max( ex )); (ii) detection of energy (i.e., light) within the emission spectrum of the acceptor compound, preferably at or near the optimum emission wavelength of the indicator molecule ( ⁇ max( e )); ( i) the type of samples to be assayed; and (iv) the number and formatting of samples to be assayed in a given program, for example, a high throughput screening format.
  • energy i.e., light
  • detection of energy i.e., light
  • the type of samples to be assayed preferably at or near the optimum emission wavelength of the indicator molecule
  • the number and formatting of samples to be assayed in a given program for example, a high throughput screening format.
  • the spectra of energy being applied to, and the spectra of energy being emitted by the samples will determine, in general, what type of instrument will be used.
  • ⁇ (ex) should not be identical to ⁇ (em)
  • the minimal acceptable amount of difference between these two values will be influenced by, among other factors, the instrumentation being used. That is, as ⁇ (ex) approaches ⁇ (em), instruments capable of resolving closely-spaced wavelengths are required, and an assay wherein the difference between ⁇ (ex) and ⁇ (em) is less than about 3 to about 5 nm requires a high resolution instrument. Conversely, an assay wherein the difference between ⁇ (ex) and ⁇ (em) is greater than about 50 to about 75 nm requires an instrument having relatively medium to low resolution.
  • the type of energy being emitted by an excited fluorophore and measured in samples will determine, in general, what type of instrument will be used.
  • a fluorometer for instance, is a device that measures fluorescent energy and should therefore be part of the instrumentation.
  • a fluorometer may be anything from a relatively simple, manually operated instrument that accommodates only a few reaction vessels (e.g., sample tubes) at a time, to a somewhat more complex manually operated or robotic instrument that accommodates a larger number of samples in a format having a plurality of reaction vessels, such as a 96-well microplate (e.g., an fmaxTM fluorimetric plate reader, Molecular Devices Corp., Sunnyvale, CA; or a CytofluorTM fluorimetric plate reader, model #2350, Millipore Corp., Bedford, MA), or a complex robotic instrument (e.g., a FLIPRTM instrument; see infra) that accommodates a multitude of samples in a variety of formats such as 96-well microplates, 384-well microplates or other high throughput screening formats wherein, for example, detection of signals from a calcium indicator molecule in a plurality or reaction vessels may be automated.
  • a 96-well microplate e.g., an fmaxTM flu
  • 96-well or 384-well microplates may be suitable in instances where the cells of interest adhere to the microplate substrate, or to some material applied to the wells of the microplate (e.g., a natural or synthetic coating with which the wells have been treated, such as collagen, fibronectin, vitronectin, RGD peptide, poly-L-lysine, CelTakTM, or the like). Interfering fluorescence derived from certain common plastic multiwell plate materials, however, may result in a large artifactual background component at excitation wavelengths below about 400 nm.
  • an instrument capable of reading fluorescent signals in glass or polymeric tubes or tubing, or another suitable non-interfering vessel may be prefened.
  • assay reaction vessels should allow for the introduction of biological samples, candidate agents, a source of calcium cations, control reagents and optionally additional compounds that may influence cytosolic calcium levels, as well as the ability to detect the signal generated by the calcium indicator molecule at one or a plurality of appropriate points in time.
  • the number of samples to be assayed in a given program may influence the degree of automation that can be implemented by the instrument selected. For example, when high throughput (HTS) screening, (i.e., assaying a large number of samples in a relatively brief time period) is desired, robotic or semi-robotic instruments are prefened. Alternatively, samples may be processed manually, even where formats that accommodate large sample numbers (e.g., 96-well microplates) are used.
  • HTS high throughput
  • a calcium uniporter may also be tested in suitable cell-based and cell-free systems for assaying mitochondrial activities as provided herein and with which those skilled in the art will be familiar, including but not limited to: activation by ADP, inhibition by ATP, Mg 2+ , ruthenium red or its derivative Ru360 (Matlib et al., 1998 J. Biol. Chem. 273:10223; Emerson et al., 1993 J.
  • calcium uniporter activity may be determined as it relates to mitochondrial membrane potential, which under certain conditions may be altered (e.g., increased or decreased in a statistically significant manner) in response to calcium transport by a functional uniporter.
  • Methods for determining mitochondrial membrane potential are provided, for example, in U.S. application number 09/161,172.
  • mitochondrial membrane potential may be determined according to methods with which those skilled in the art will be readily familiar, including but not limited to detection and/or measurement of detectable compounds such as fluorescent indicators, optical probes and/or sensitive pH and ion-selective electrodes (See, e.g., Ernster et al., 1981 J. Cell Biol.
  • the fluorescent probes 2-,4-dimethylaminostyryl-N-methyl pyridinium (DASPMI) and tetramethylrhodamine esters (such as, e.g., tetramethylrhodamine methyl ester, TMRM; tetramethylrhodamine ethyl ester, TMRE) or related compounds (see, e.g., Haugland, 1996, supra) may be quantified following accumulation in mitochondria, a process that is dependent on, and proportional to, mitochondrial membrane potential (see, e.g., Murphy et al., 1998 in Mitochondria & Free Radicals in Neurodegenerative Diseases, Beal, Howell and
  • fluorescent detectable compounds that may be used in the invention include but are not limited to rhodamine 123, rhodamine B hexyl ester, DiOC 6 (3), JC-1 [5, 5', 6,6'- Tetrachloro-l,l',3,3'-Tetraethylbezimidazolcarbocyanine Iodide] (see Cossarizza, et al., 1993 Biochem. Biophys. Res. Comm. 197:40; Reers et al., 1995 Meth.
  • Mitochondrial membrane potential can also be measured by non- fluorescent means, for example by using TTP (tetraphenylphosphonium ion) and a TTP- sensitive electrode (Kamo et al., 1979 J. Membrane Biol. 49:105; Porter and Brand, 1995 Am. J. Physiol 2 ⁇ 59:R1213).
  • TTP tetraphenylphosphonium ion
  • TTP-sensitive electrode Kamo et al., 1979 J. Membrane Biol. 49:105; Porter and Brand, 1995 Am. J. Physiol 2 ⁇ 59:R1213).
  • TMRM is somewhat preferable to TMRE because, following efflux from mitochondria, TMRE yields slightly more residual signal in the endoplasmic reticulicum and cytoplasm than TMRM.
  • membrane potential may be additionally or alternatively calculated from indirect measurements of mitochondrial permeability to detectable charged solutes, using matrix volume and/or pyridine nucleotide redox determination combined with spectrophotometric or fluorimetric quantification. Measurement of membrane potential dependent substrate exchange- diffusion across the inner mitochondrial membrane may also provide an indirect measurement of membrane potential. (See, e.g., Quinn, 1976, The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Maryland, pp. 200-217 and references cited therein.)
  • the invention is not intended to be limited to these examples of functionally testing a nucleic acid expression construct expressing calcium uniporter polypeptide activity, and may include other methodologies for determining calcium binding, calcium transport or other calcium regulatory activities.
  • the present invention will in certain embodiments be extremely useful by providing compositions and methods according to which regulators of the activity of a highly important physiological mediator, intracellular calcium, may be identified.
  • the nucleic acids of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • a CSI which encodes a CSP polypeptide (e.g., p95.6/YN52) for use according to the invention may be identical to the coding sequence known in the art and identified in a particular database as described herein, or may be a different coding sequence, which, as a result of the redundancy or degeneracy of the genetic code, encodes the same CSP polypeptide.
  • the nucleic acids which encode CSP polypeptides may include, but are not limited to: only the coding sequence for the CSP polypeptide (e.g., a CSI); the coding sequence for the CSP polypeptide and additional coding sequence; the coding sequence for the CSP polypeptide (and optionally additional coding sequence) and non- coding sequence, such as introns or non-coding sequences 5' and/or 3' of the coding sequence for the CSP polypeptide, which for example may further include but need not be limited to one or more regulatory nucleic acid sequences that may be a regulated or regulatable promoter, enhancer, other transcription regulatory sequence, repressor binding sequence, translation regulatory sequence or any other regulatory nucleic acid sequence.
  • nucleic acid encoding a CSP polypeptide encompasses a nucleic acid which includes only coding sequence for the polypeptide as well as a nucleic acid which includes additional coding and/or non-coding sequence(s).
  • the present invention further relates to variants of the herein described nucleic acids (including CSI) which encode for fragments, analogs and derivatives of a CSP polypeptide.
  • the variants of the nucleic acids encoding CSPs may be naturally occuning allelic variants of the nucleic acids or non-naturally occuning variants.
  • an allelic variant is an alternate form of a nucleic acid sequence which may have at least one of a substitution, a deletion or an addition of one or more nucleotides, any of which does not substantially alter the function of the encoded CSP polypeptide.
  • the present invention includes nucleic acids encoding the same C.
  • TRP elegans transient receptor potential
  • Variants and derivatives of CSP identified using a database search query as described herein may be obtained by mutations of an appropriate related CSI.
  • Alterations of the native amino acid sequence may be accomplished by any of a number of conventional methods. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.
  • oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion.
  • Exemplary methods of making such alterations are disclosed by Walder et al. (Gene 2:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 52:488, 1985); Kunkel et al. (Methods in Enzymol 154:367, 1987); and U.S. Patent Nos. 4,518,584 and 4,737,462.
  • Equivalent DNA constructs that comprise "silent" mutations (i.e., DNA changes that do not result in amino acid changes in the encoded polypeptide, including mutations that are silent due to the redundancy of the genetic code), or that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences not needed for biological activity are also encompassed by the invention.
  • sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of inconect intramolecular disulfide bridges upon renaturation.
  • EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein.
  • KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg- Arg, Arg-Lys, and Lys- Arg pairs to eliminate the occunence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and prefened approach to inactivating KEX2 sites.
  • nucleic acid preparations containing a CSI identified according to a database search as described herein may be obtained according to any of a number of well known methodologies in the art.
  • the CSI may be present in a readily available form such as a DNA plasmid or vector or the like. Suitable sources for many CSIs will be present in the database listing where the CSI is identified.
  • the CSI may be synthesized with a DNA synthesizer using established DNA synthesis chemistry.
  • a polypeptide sequence of interest or an appropriate related CSI e.g., a polynucleotide sequence that encodes the polypeptide sequence of interest
  • an appropriate related CSI e.g., a polynucleotide sequence that encodes the polypeptide sequence of interest
  • the person having ordinary skill in the art can readily obtain the CSI nucleic acid by designing suitable oligonucleotide primers that specifically hybridize to the CSI.
  • Molecular cloning of the CSI is then performed by hybridizing the primers to CSI present in a nucleic acid preparation appropriately selected according to information provided in the record retrieved from the search database, for example, one or more of the source organism (or a related organism), the source cell or tissue type, an induced or activated state of the source cell or the like.
  • oligonucleotide primer hybridization precedes nucleic acid amplification that is then performed to provide suitable quantities of CSI for various uses, including the construction of vectors, host cells, fusion proteins and expression systems as provided herein and known in the art. Details regarding molecular cloning procedures may be found, for example, in Ausubel et al. (1998 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA) and in Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY).
  • the present invention further relates to CSP polypeptides identified according to biological sequence database searching using a computer system as described herein, and in particular to methods for producing recombinant CSP polypeptides by culturing host cells containing CSP expression constructs, and to isolated recombinant CSP polypeptides, including, for example, the C. elegans TRP- like protein refened to herein as p95.6/YN52 (Fig. 12, SEQ ID NO:2 or GenBank Ace. No. P34586) or the human PP7 (PPEF-1) serine/threonine phosphatase (Fig. 13, SEQ ID NO:4), as well as fragments, analogs and derivatives of such polypeptides.
  • the polypeptides and nucleic acids of the present invention are preferably provided in an isolated form, and in certain prefened embodiments are purified to homogeneity.
  • fragment when refening to particular CSP polypeptides or fusion proteins refers to any CSP polypeptide or fusion protein that retains essentially the same biological function or activity as the CSP polypeptide identified according to the database searching methods described herein.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active CSP polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide or a synthetic polypeptide, and is preferably a recombinant polypeptide.
  • a fragment, derivative or analog of a CSP polypeptide or fusion protein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the CSP polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which additional amino acids are fused to the CSP polypeptide, including amino acids that are employed for purification of the CSP polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • the polypeptides of the present invention include CSP polypeptides and fusion proteins having amino acid sequences that are identical or similar to sequences known in the art.
  • the C. elegans p95.6/YN52 TRP-like protein of Figure 12 [SEQ ID NO:2] is contemplated for use according to the instant invention, as are polypeptides including homologous CSP polypeptides from other species such as human CSP polypeptides having at least 70% similarity (preferably a 70% identity) to the polypeptide of Figure 12 [SEQ ID NO:2] and more preferably 90% similarity (more preferably a 90% identity) to the polypeptide of Figure 12 [SEQ ID NO:2] and still more preferably a 95% similarity (still more preferably a 95% identity) to the polypeptide of Figure 12 [SEQ ID NO:2] and to portions of such polypeptides, wherein such portions of a CSP polypeptide generally contain at least 30 amino acids and more preferably at least 50 amino
  • fragments or portions of the polypeptides of the present invention may be employed for producing the conesponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
  • Fragments or portions of the nucleic acids of the present invention may be used to synthesize full-length nucleic acids of the present invention.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occuning).
  • a naturally occuning nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acids could be part of a vector and/or such nucleic acids or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • gene refers to the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer” as well as intervening sequences (introns) between individual coding segments (exons).
  • FIG. 1 and the following discussion provide a brief, general description of a suitable computing environment in which the invention can be implemented.
  • embodiments of the invention will be described in the general context of computer-executable instructions, such as program modules or macros being executed by a personal computer.
  • Those skilled in the relevant art will appreciate that the invention can be practiced with other computer system configurations, including hand-held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, mini computers, mainframe computers, and the like.
  • the invention can be practiced in distributed computing environments where tasks or modules are performed by remote processing devices, which are linked through a communications network.
  • program modules may be located in both local and remote memory storage devices.
  • a conventional personal computer 100 includes a processing unit 102, a system memory 104 and a system bus 106 that couples various system components including the system memory to the processing unit.
  • the processing unit 102 may be any logic processing unit, such as one or more central processing units (CPUs), digital signal processors (DSPs), application-specific integrated circuits (ASIC), etc.
  • CPUs central processing units
  • DSPs digital signal processors
  • ASIC application-specific integrated circuits
  • the system bus 106 can employ any known bus structures or architectures, including a memory bus with memory controller, a peripheral bus, and a local bus.
  • the system memory 104 includes read-only memory (“ROM”) 108 and random access memory (“RAM”) 110.
  • ROM read-only memory
  • RAM random access memory
  • a basic input/output system (BIOS) 112 which can form part of the ROM 108, contains basic routines that help transfer information between elements within the personal computer 100, such as during start-up.
  • the personal computer 100 also includes a hard disk drive 114 for reading from and writing to a hard disk (not shown), and an optical disk drive 1 16 and a magnetic disk drive 118 for reading from and writing to removable optical disks 120 and magnetic disks 122, respectively.
  • the optical disk 120 can be a CD-ROM, while the magnetic disk 122 can be a magnetic floppy disk.
  • the hard disk drive 114, optical disk drive 1 16 and magnetic disk drive 118 communicate with the processing unit 102 via the bus 106.
  • the hard disk drive 1 14, optical disk drive 116 and magnetic disk drive 118 may include interfaces or controllers (not shown) coupled between such drives and the bus 106, as is known by those skilled in the art.
  • the drives 114, 116 and 1 18, and their associated computer-readable media provide nonvolatile storage of computer readable instructions, data structures, program modules and other data for the personal computer 100.
  • the depicted personal computer 100 employs a hard disk, optical disk 120 and magnetic disk 122, those skilled in the relevant art will appreciate that other types of computer-readable media that can store data accessible by a computer may be employed, such as magnetic cassettes, flash memory cards, digital video disks ("DVD”), Bernoulli cartridges, RAMs, ROMs, smart cards, etc.
  • Program modules can be stored in the system memory 104, such as an operating system 124, one or more application programs 126, other programs or modules 128 and program data 130.
  • the system memory 104 may also include a web browser 131 for permitting the personal computer 100 to access and exchange data with web sites in the World Wide Web of the Internet, as described below.
  • the application programs 126 can include spreadsheet applications, such as Excel ® by Microsoft Corporation. While shown in Figure 1 as being stored in the system memory 104, the operating system 124, application programs 126, other modules 128, program data 130 and web browser 138 can be stored on the hard disk of the hard disk drive 114, the optical disk 120 of the optical disk drive 116 and/or the magnetic disk 122 of the magnetic disk drive 118.
  • a user can enter commands and information into the personal computer 100 through input devices such as a keyboard 132 and a pointing device such as a mouse 134.
  • Other input devices can include a microphone, joystick, game pad, scanner, etc.
  • These and other input devices are connected to the processing unit 102 through an interface 136 such as a serial port interface that couples to the bus 106, although other interfaces such as a parallel port, game port or universal serial bus (“USB”) can be used.
  • a monitor 138 or other display device is coupled to the bus 106 via a video interface 140, such as a video adapter.
  • the personal computer 100 can include other output devices, such as speakers, printers, etc.
  • the personal computer 100 can operate in a networked environment using logical connections to one or more remote computers, such as a remote computer 150.
  • the remote computer 150 can be another personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above for the personal computer 100.
  • the remote computer 150 includes a memory storage device such as a disk drive 152 shown in Figure 1.
  • the remote computer 150 is logically connected to the personal computer 100 under any known method of permitting computers to communicate, such as through a local area network (“LAN”) 154 or a wide area network (“WAN”) or Internet 156.
  • LAN local area network
  • WAN wide area network
  • Internet 156 Such networking environments are well known in offices, enterprise-wide computer networks, intranets and the Internet.
  • the personal computer 100 When used in a LAN networking environment, the personal computer 100 is connected to the LAN 154 through an adapter or network interface 158 (coupled to the bus 106). When used in a WAN networking environment, the personal computer 100 often includes a modem 160 or other device for establishing communications over the WAN/Internet 156.
  • the modem 160 is shown in Figure 1 as coupled between the interface 136 and the WAN/Internet 156.
  • program modules, application programs, or data, or portions thereof can be stored in the remote computer 150, such as in the disk drive 152.
  • the disk drive 152 represents an external database that may be accessed and searched based on queries formulated by the personal computer 100.
  • Various external databases, typically accessed via the Internet are described below.
  • a routine 200 extracts desired sequences from a database. Unless described otherwise herein, the steps depicted in Figure 2 are well known or described in detail in citations provided herein. Those skilled in the relevant art can create source code (such as in Perl), microcode or program logic anays or firmware based on the flowchart of Figure 2 and the detailed description provided herein.
  • the routine 200 can be stored in the system memory 104 and/or nonvolatile memory such as the magnetic disk 122.
  • the routine 200 begins by receiving a motif that effectively defines a search query for a database. The motif effectively defines a search for proteins having a functionality of interest or functional domain.
  • a motif defining a protein family or polypeptide of interest may be selected on the basis of the presence or absence of a functional domain responsible for association of the protein with a cell membrane.
  • proteins capable of such associations with membranes may include extrinsic membrane proteins, integral membrane proteins, secretory proteins and the like. Integral membrane proteins may further include membrane proteins having surfaces exposed to neither the cytosolic nor the extracellular (or intravesicular) milieu, to only one of the cytosolic or the extracellular (or intravesicular) milieu or, in the case of transmembrane proteins, to both the cytosolic and the extracellular (or intravesicular) milieu.
  • a cell membrane as used herein may be any cellular membrane, and typically refers to membranes that are in contact with cytosolic components, including intracellular membrane bounded compartments such as mitochondrial inner and outer membranes as described above, and also intracellular vesicles, endoplasmic reticulum ("ER”)-Golgi constituents, other organelles and the like, as well as the plasma membrane.
  • cytosolic components including intracellular membrane bounded compartments such as mitochondrial inner and outer membranes as described above, and also intracellular vesicles, endoplasmic reticulum ("ER”)-Golgi constituents, other organelles and the like, as well as the plasma membrane.
  • a protein or polypeptide of interest may stably or transiently associate with or localize to an inner or outer mitochondrial membrane, the intermembrane space or the mitochondrial matrix.
  • a protein of interest may contain polypeptide sequences that direct the protein to be retained in the cytosol, to reside in the lumen of the endoplasmic reticulum (ER), to be secreted from a cell via the classical ER-Golgi secretory pathway, to be inco ⁇ orated into the plasma membrane, to associate with a specific cytoplasmic component including the cytoplasmic domain of a transmembrane cell surface receptor or to be directed to a particular subcellular location by any of a variety of known intracellular protein sorting mechanisms with which those skilled in the art will be familiar. Accordingly, these and related embodiments are encompassed by the instant compositions and methods directed to identifying a polypeptide of interest that localizes to a predefined intracellular, membrane or extracellular localization.
  • the motif may be either a "pattern” or a "profile.”
  • a pattern is a short consensus sequence that is preferably identical universally with target sequences, but often includes a stretch having most but not all bases in the sequence conserved.
  • a pattern may be a sequence of nucleotide or amino acid values, with N number of wildcard values inserted therein, where each N represents one intervening nucleotide or amino acid value. Patterns applicable to the routine 200 may be obtained from the PROSITE database and associated algorithms or tools (http://www.expasy.ch/prosite/).
  • the motif takes the form of a profile, such as a hidden Markov model (HMM).
  • HMM hidden Markov model
  • Profile methods for building position- specific scoring models from multiple sequence alignments have existed, but often such scoring systems were ad hoc. See, e.g., S. Eddy, Profile Hidden Markov Models, Bioinformatics 74:755-763 (1998).
  • HMMs provide improved scoring systems because, in part, they rely on a class of probabilistic models that are generally applicable to time series or linear sequences. HMMs, previously used for recognizing words in digitized sequences of human speech, have recently been employed in computational biology and profile models.
  • An HMM is a generalization of a finite state machine or model in which each state in the model conesponds to a single residue. Probability distributions at each state represent the probabilities of point mutations; transition probabilities among states allow for insertions, deletions and, for some model topologies, repeated or shuffled domains. Thus, HMM profiles can tolerate N number of additional nucleotide or amino acid values, as well as the deletion of one or more values in the motif. An HMM is effectively a numerical matrix to which a database can be compared.
  • HMM profiles and motifs may obtain HMM profiles and motifs from databases and associated tools, such as Pfam (E. Sonnhammer et al., Pfam: Multiple Sequence Alignments and HMM-Profiles of Protein Domains, Nucl. Acids Res. 2r5:320-322 (1998)), and PROSITE, A. Bairoch et al., The PROSITE Database, Its Status in 1997, Nucl. Acids Res. 25:217-221 (1997).
  • the Pfam database is a database of multiple alignments of protein domains or conserved protein regions.
  • the Pfam database represents evolutionarily conserved structures that have implications for the function of a given protein.
  • the PROSITE database for example, is a database of protein families (i.e., groups of proteins having similar sequences) and domains (i.e., regions of proteins having similar sequences that may be found as portions of otherwise unrelated proteins).
  • the PROSITE database consists of biologically significant sites, patterns and profiles that help to reliably identify to which known protein family (if any) a new sequence belongs.
  • the PROSITE and Pfam databases are text word-searchable databases, as well as sequence-searchable databases for a given motif.
  • the user obtains a set of protein sequences containing a functional feature of interest, for example, proteins having functional polypeptide domains involved in binding, transport or other regulation of intracellular free calcium (i.e., Ca 2+ ), and then builds a hidden Markov model.
  • Figure 3 shows the results of a text search for the term "calcium" in description fields for all protein families in the Pfam database.
  • sequence refers to polypeptide sequences, unless otherwise noted.
  • the user or the routine 200, automatically performs multiple alignments of sequences in step 206 (http://bioweb.pasteur.fr/docs/main/doc/clustalw.).
  • the user may employ the known alignment algorithm CLUSTAL W.
  • CLUSTAL W Such alignment algorithms help identify a sequence containing a functional feature of interest, and from this sequence alignment a motif model such as an HMM may be created.
  • the user may employ MEME or MAST to generate a profile from multiple alignments of sequences, and subsequently search databases with the profile.
  • MAST or Motif Alignment and Search Tool is a tool for searching biological sequence databases for sequences that contain one or more of a group of known motifs.
  • MEME is a tool for developing motifs in groups of sequences.
  • MEME employs motifs having position-dependent letter (e.g. nucleotide value) frequency matrices that describe a set of similar sequences of equal length. In other words, a MEME-type motif is similar to a gapless profile.
  • a person of ordinary skill in the art may alternatively employ MEME to discover motifs (e.g., highly-conserved sequences) in groups of related polypeptide sequences, and then employ MAST to search sequence databases using the discovered motif.
  • MAST produces a "block" diagram showing non- contiguous alignment of portions in sequences previously identified under step 204 by, e.g. MEME.
  • Figure 4 shows the ordering and spacing of all non-overlapping motif occunences for sequences having high e-value scores.
  • GenBank accession numbers are listed in the "name” column, while expected scores of alignment are listed under the "expect" column.
  • the user Based on alignments produced in step 206, the user, or the routine 200 automatically, builds an HMM or other motif in step 208.
  • the HMM motif is built and then trained using training data.
  • the HMM is built from a family of aligned protein sequences that share a common functionality or identity.
  • a probabilistic mathematical model is generated therefrom which captures important statistical information about the degree of conservation at various positions in the multiple alignment.
  • HMM-type motifs are described above, other profile-type motifs can be employed.
  • profiles simpler than HMMs, may be employed under the PROSITE database.
  • profiles may be constructed under BLOCKS (Fred Hutchinson Cancer Research Center: http://www.fhcrc.org) or under PRINTS (http://www.biochem.ucl.ac.ak/lsm/dbrowser/prints/prints.html).
  • BLOCKS Red Hutchinson Cancer Research Center: http://www.fhcrc.org
  • PRINTS http://www.biochem.ucl.ac.ak/lsm/dbrowser/prints/prints.html.
  • a profile typically consists of a numerical series or matrix used as a query sequence for searching a sequence database to find new members of a desired protein family or domain.
  • the routine 200 searches a database of biological sequence on a query formulated from the motif. For example, the routine 200 searches the GenBank database using the HMM retrieved in step 202 or built in steps 204-208.
  • An example of an algorithm for querying a biological sequence database using HMMs is HMMER, which can be found at http://hmmer.wustl.edu.
  • Other available profile models include META-MEME, noted above, SAM, described in R. Hughey et al., Hidden Markov Articles for Sequence Analysis: Extension and Analysis of the Basic Method, Comptu. Applic. Biosci. 72:95-107 (1996), and PFTOOLS, described at P.
  • the user may request the routine 200 to extract species names associated with each accession number from a protein database (e.g., GenBank or SwissProt). For example, the user may request or extract sequences in the hit list associated with human sequences. This extracted data, based on a species limitation or filter, can then be output or otherwise provided in a report in step 224.
  • a protein database e.g., GenBank or SwissProt
  • step 216 hit lists produced from two HMM profiles can be compared to identify proteins found in both hit lists. For example, two different
  • HMM motifs can be used to search or query the GenBank database in step 210 and obtain two hit lists in step 212 therefrom. Then, in step 216, the routine 200 compares the two hit lists to identify proteins common in both hit lists, as described below.
  • the routine 200 converts the extracted accession numbers in the hit lists into their respective polypeptide sequences from the GenBank database.
  • the routine 200 employs the BLAST algorithm to retrieve such polypeptide sequences based on their conesponding accession numbers.
  • the routine 200 in step 218 employs an algorithm to estimate subcellular molecular components or localization of the retrieved sequences.
  • the routine 200 in step 218 employs the PSORT algorithm for each polypeptide sequence to determine which sequences are likely to be localized in mitochondria.
  • the PSORT algorithm is described, for example, in K. Nakai et al., Genomics 74:897-911 (1992), and K.
  • the PSORT algorithm applies a set of stored rules for biological sequence features of known protein sorting signals to the retrieved polypeptide sequences and extracts or identifies sets of subcellularly localized sequences.
  • PSORT calculates values of feature variables that reflect various characteristics of each input sequence, inte ⁇ rets the set of values obtained, and estimates a localization likelihood for each input sequence being sorted to each candidate site.
  • Localized sites include cytoskeleton, cytoplasm, nucleus, mitochondria, vesicles of a cellular secretory system, endoplasmic reticulum (ER), Golgi, vacuoles, plasma membrane, lysosomes, peroxisomes, and extracellular space including cell wall.
  • cytoskeleton cytoplasm, nucleus, mitochondria, vesicles of a cellular secretory system, endoplasmic reticulum (ER), Golgi, vacuoles, plasma membrane, lysosomes, peroxisomes, and extracellular space including cell wall.
  • PSORT II the successor to PSORT, employs the K-nearest neighbor algorithm, which is a method of pattern recognition in which K is a predefined parameter based on a training set of data. Further details on PSORT and PSORTII may be found, for example, at http://psort.nibb.ac.jp:8800/psort.
  • the PSORT algorithm receives as input a sequence, such as a full-length polypeptide or amino acid sequence containing all information for sorting.
  • a sequence such as a full-length polypeptide or amino acid sequence containing all information for sorting.
  • full-length amino acids sequences begin with Met (methionine).
  • any polypeptide sequences may be employed as input data to PSORT.
  • PSORT then analyzes each sequence to identify sorting signals that represent part of a protein or polypeptide sequence that indicates its origin or subcellular structure to which it couples. For example, in eukaryotes, proteins sorted through the vesicular pathway (bulk flow) usually have a signal sequence (also known as a "leader peptide") in the N-terminus, which is cleaved off after translocation through the ER membrane.
  • signal sequence also known as a "leader peptide”
  • PSORT employs numerous signal sequence recognition methods, which are described in more detail at http://psort.nibb. ac.jp:8800/psort/helpwww2.html.
  • PSORT produces a probability score based on N-terminal sequences. Possibly more importantly, PSORT may be used to accept unique training sets (e.g., human or animal sequences), and can be modified to recognize patterns (e.g., calcium-channel motifs).
  • unique training sets e.g., human or animal sequences
  • patterns e.g., calcium-channel motifs.
  • mitochondrial targeting sequences have no distinct consensus patterns, and sequence requirements for conect targeting appear to be rather unspecific, but usually are rich in basic amino acids (especially arginine), have a hydrophobic NH 2 end, and lack acidic amino acids at this end. Domains for mitochondrial targeting sequences are usually characterized by an N- terminal that is positively charged, amphiphilic and has an alpha-helix mo ⁇ hology.
  • the inventor has modified the original PSORT database and algorithm to include a total of 3,240 animal sequences in the underlying database, including 828 mitochondrial sequences and 2,412 non-mitochondrial sequences.
  • the new animal sequence database was constructed of proteins from the SwissProt database using only the following animal species: human, rat, mouse, rabbit, pig, hamster, cow, sheep, horse, chicken, Drosophila, C elegans, salmon, frog, monkey, and sea-hare (e.g., Aplysia). The subcellular localization of these sequences is described in the SwissProt database and this information was extracted by P.
  • the number of calculated parameters was reduced from 34 to 13 on the basis of multivariate discriminate analysis to determine which of the 34 parameters affected mitochondrial targeting to the greatest extent.
  • the original PSORT algorithm employed 1 ,080 sequences with the number of calculated parameters set at 34, resulting in a total of 36,720 calculations per query sequence.
  • the new PSORT algorithm improves localization predictions and reduces false positives over the original PSORT algorithm.
  • the original PSORT algorithm using the yeast database of 1,080 yeast sequences
  • 51% of the testing set of 165 mitochondrial sequences were predicted to have mitochondrial localization, and 8% of the set of 165 non-mitochondrial sequences gave the same prediction (false positives).
  • the modified PSORT algorithm using the animal database of 3,240 animal sequences
  • 78% of the testing set of 165 mitochondrial sequences were predicted to have mitochondrial localization, and 6% of the set of 165 non-mitochondrial sequences gave the same prediction (false positives).
  • MITOPROT Another subcellular localization routine for sequences that may be employed in step 218 is MITOPROT, which is described in M. Claros et al., Computational Method To Predict Mitochondrially-Imported Proteins and Their Targeting Sequences, Eur. J. Biochem., 247:779-786 (1996). MITOPROT provides statistical analysis of 47 different parameters under multivariate analysis and provides a probability score and identification of cleavage sites for sequences.
  • the routine 200 performs additional filtering to further isolate sequence functionality of interest from the biological sequence database. While the routine 200 in step 218 identifies sequences in the sequence database associated with a particular sub-cellular localization (e.g., mitochondrial sequences), the routine 200 in step 220 further filters the mitochondrial sequences to identify specific mitochondrial sequences. For example, the routine 200 in step 220 employs the BLAST algorithm to identify and exclude yeast sequences from the mitochondrial sequences obtained under step 218. The routine 200 queries a selected database of yeast sequences and employs BLAST to identify those sequences in the mitochondrial sequences obtained in step 218 that exhibit a high degree of homology with the yeast sequences. Such mitochondrial sequences exhibiting a high degree of homology with yeast sequences are eliminated under step 220.
  • a particular sub-cellular localization e.g., mitochondrial sequences
  • the routine 200 in step 220 further filters the mitochondrial sequences to identify specific mitochondrial sequences.
  • the routine 200 in step 220 employs the BLAST algorithm to identify and
  • step 220 Many other types of filtering can be performed in step 220. For example, sequences from unwanted sources can be eliminated, such as non-human sequences, if only human sequences are desired.
  • most polypeptide sequence information is stored in a database with fields or other data representing species data from which the sequences were obtained. Thus, a simple database query identifying all sequences associated with a given biological organism or species can be obtained.
  • routine 200 in step 220 can exclude sequences having an unwanted functionality.
  • a recuning sequence motif or theme known as a "zinc finger” may be undesirable.
  • the user in step 220 can instruct the routine 200 to exclude any polypeptides that included both a calcium-binding domain and a zinc-finger domain.
  • many techniques for identifying sequences having such a zinc finger functionality can be employed, such as simple text searching of descriptive fields associated with each polypeptide sequence in the database, or searching for actual sequence portions conesponding to zinc fingers.
  • routine 200 in step 220 can exclude sequences that are similar to a sequence in a particular organism, such as yeast (Saccharomyces cerevisiae).
  • a particular functionality is excluded, while in the first-described embodiment, all yeast sequences or sequences from a given organism are eliminated.
  • data produced under step 218 may include a sequence from a desired organism, and for a desired sub-cellular domain, but by another criteria, the sequence has properties the user wishes to eliminate from the search. For example, the user may know of a sequence of a mouse protein he or she is not interested in, so the user does not wish the protein's human homolog to be included.
  • routine 200 in step 220 employs the BLAST algorithm to compare all sequences produced under step 218 to the known sequence and eliminate any sequences having too low of a BLAST value (i.e., any sequences that are too similar to the undesired sequence).
  • the routine 200 employs an optional step to ensure that all sequences obtained from step 220 include full-length protein sequences.
  • the routine 200 may simply determine that each sequence obtained from step 220 has methionine (Met) at its N-terminal.
  • methionine Metal
  • Virtually all nuclear encoded proteins are encoded by regions of DNA that begin with the sequence ATG, or either ATG or ATA for mitochondrial DNA encoded genes, which encodes the amino acid methionine as the first amino acid, even though methionine may subsequently be removed as part of a naturally-occuning processing step (i.e., not all proteins have methionine as their first amino acid).
  • cDNA sequences are common in many prior art databases, including GenBank, because they are derived from reverse transcription of mRNA. cDNA sequences typically lack introns and other "junk” regions and typically include long stretches of continuous coding sequence in-frame. Genomic DNA sequences, also present in many prior art databases, include introns and other non-coding regions interspersed around coding sequences (e.g., open reading frames or "ORFs," exons). Genomic DNA sequences may include unique sequence stretches that help identify on what chromosome, and where on that chromosome, the gene resides. Importantly, by identifying partial sequences in a prior art database such as GenBank, the routine 200 allows the user to pursue determination of the full-length DNA sequence in the lab that encodes the desired protein or functionality of interest.
  • routine 200 in step 222 identifies any stop codons to determine the end of a sequence.
  • Stop codons i.e., TAA, TAG and TGA, (and AGA, AGG in mammalian mitochondria) signal the cellular protein assembly machinery to stop adding amino acids to a growing polypeptide during translation, as noted in Table 1 above.
  • the routine 200 in step 222 may thus extract or include only sequences having both start and stop codons that are "in frame".
  • DNA sequences are read in groups of 3-letter codons to deduce the amino acid sequence of an encoded polypeptide, such that, for example, a DNA region containing the sequence TAATATGTC might be read as the codons TAA, TAT and GTC.
  • start codon sequence ATG encoding initiating methionine must be read in-frame as a 3-letter codon to start a new protein, while in the above example, ATG would not be an in-frame codon while the stop codon TAA is in-frame.
  • the routine 200 in step 222 would identify the second codon ATG as being an in frame start codon.
  • step 224 the routine 200 receives the filtered sequence or sequences from step 220 and exports the sequences for formatting or otherwise provides a report of obtained sequence data.
  • the sequences obtained from step 222 are imported to a spreadsheet application.
  • the data provided as output under step 224 includes an identification number (ID), a species, a description of each identified sequence (including text descriptions), whether sequence begins with methionine, as well as additional information, such as PSORT primary and secondary sub-cellular localization prediction scores.
  • ID identification number
  • species a species
  • description of each identified sequence including text descriptions
  • additional information such as PSORT primary and secondary sub-cellular localization prediction scores.
  • the routine 200 not only filters desired sequences from the GenBank database, but adds additional data fields representing processing of such sequences under HMM (step 210), PSORT (step 218), BLAST (step 220), and so forth.
  • HMM HMM
  • PSORT step 218
  • BLAST step 220
  • the "MET START" column confirms that each sequence in the table of Figure 5 starts with methionine and is thus likely to be a full-length protein sequence, under step 222.
  • the R and D/E columns indicate, respectively, the number of arginine (R) and aspartic acid (D)/glutamic acid (E) amino acids in each sequence.
  • the “score” column is a weighted average of a number of parameters related to mitochondrial localization: number of R's, number of D/E's, hydropathic profile, and the like).
  • the columns “cleavage site”, “PI” (primary prediction) and “P2" (secondary prediction) are generated by the PSORT algorithm.
  • the "BLAST vs. Yeast” column lists e-values produced under step 220. Selecting one row in the spreadsheet of Figure 5, such as Row 502, provides a full view of the data associated with that sequence, as shown in Figure 6.
  • the table of Figure 6 shows the various parameters or fields for each sequence and its conesponding values.
  • the routine 200 preferably preserves all desired fields for sequences filtered from the desired database in step 210 (e.g., GenBank).
  • the filtered data may be exported or used in a variety of applications.
  • the data may be exported to a database and stored for future use or analysis with larger data sets.
  • the analyzed data may be imported to common gateway interface ("CGI") programs or scripts to permit the data to be exchanged or manipulated via the Internet 156.
  • CGI common gateway interface
  • the analyzed data may also be output to a printer for generating reports.
  • the data produced following step 222 can be input to an Oracle database, which can be searched to identify particular sequences of interest.
  • Figure 7 shows an example of a user interface for permitting a user to search the data resulting from step 222, such as the data shown in the spreadsheet of Figure 5.
  • the user interface provides several dialog boxes, or drop-down menus that allow the user to specify one of 6 motifs associated with functional polypeptide domains related to intracellular binding, transport or regulation of free calcium: EFHAND, IRK, TRP, Ion Channel, Ligand Channel and IQ.
  • IRK inwardly-rectifying K channels
  • the fields included in output from a query can include: the number of arginines, the number of aspartic or glutamic acids, cleavage sites, HMM scores, best e-value of BLAST comparison with yeast and GenBank description fields.
  • a sample query defines the motif "TRP” and 5 PSORT prediction domain "MITOCHONDRIA” with values sorted by sub-cellular prediction value.
  • Figure 8 shows sample output resulting from such a query. As shown in Figure 8, the resulting output includes GenBank Accession Numbers ("ID”), as well as species prediction values ("PI”), localization ("PI TYPE”), together with the GenBank description ("GBDESC”).
  • Figure 10 shows an example of a multiple sequence or protein alignments performed under the routine 200.
  • sequences 2-10 are compared to a sequence of interest "GI
  • This C. elegans sequence represents a protein, p95.6/YN52, which contains both TRP and ion-transport motifs, is predicted to have a mitochondrial localization (under PSORT), contains a methionine at its amino terminus and shows no significant homology to any yeast proteins by BLAST analysis. It was therefore deemed to be a good candidate using the routine 200.
  • this sequence is aligned with 9 other known TRP proteins, there is high level of similarity in the central and C-terminal regions.
  • the C. elegans p95.6/YN52 sequence has a unique N-terminal end.
  • this sequence is likely to have the Ca 2+ channel functionality of the plasma-membrane localized TRP family, but may be localized in the mitochondria, due to its N-terminal sequence, which was found to have significant mitochondrial targeting potential.
  • a routine 700 begins under the step 202 by extracting HMM files for EFHAND and IQ models from the Pfam database.
  • Some transmembrane calcium channels contain calcium-binding EFHAND and calmodulin- binding IQ-domains.
  • HMM motifs directed to both EFHAND and IQ domains will likely identify transmembrane calcium channels in sequences within a sequence database such as GenBank.
  • FIG. 1 1 shows a spreadsheet table representing shared motifs between eight HMM motifs supplied to the GenBank database: DHP, ligand channel, ion ransport, IRK, EFHAND, IQ, ABC and TRP.
  • the table of Figure 11 confirms in the row labeled "# of hits vs. GenBank" that the EFHAND and IQ HMM motifs produce 1,209 and 352 hits, respectively.
  • step 216 the two hit lists produced in step 702 and 704 are compared to identify sequences contained in both lists. Under step 706, five such unique sequences or proteins contain both EFHAND and IQ HMM motifs. While the table of Figure 11 shows that 8 sequences share both motifs, 3 of the eight sequences are repeated.
  • step 2108 the 5 resulting sequences are analyzed using PSORT to predict sub-cellular localization. Thereafter, in step 220, all sequences having a high degree of homology with yeast protein sequences are filtered or eliminated. As shown in step 708, only one protein remains based on steps 218 and 220: PPEF-1 serine/threonine protein phosphatase. GenBank Ace. No.: 258641 1 ; Ref: Sherman et al., 1997 Proc. Nat. Acad. Sci. USA 94:11639; Wolf et al., 1999 Exp. Cell Res. 247:505- 513.
  • the invention provides CSP fusion proteins encoded by nucleic acids that have the conesponding CSI fused in frame to an additional coding sequence to provide for expression of a desired CSP polypeptide sequence, for example, a p95.6/YN52 or a PP7(PPEF-1) polypeptide sequence, fused to an additional functional or non-functional polypeptide sequence that permits, for example by way of illustration and not limitation, detection, isolation and/or purification of the CSP fusion protein.
  • the invention also contemplates polypeptides, expression constructs and host cells related to fusion proteins that localize to membranes, including p95.6/YH52 fusion proteins that localize to cellular membranes such as mitochondrial membranes.
  • Such CSP fusion proteins may permit detection, isolation and/or purification of the CSP fusion protein by protein-protein affinity, metal affinity or charge affinity-based polypeptide purification, or by specific protease cleavage of a fusion protein containing a fusion sequence that is cleavable by a protease such that the CSP polypeptide is separable from the fusion protein.
  • CSP fusion proteins may comprise polypeptide sequences added to a CSP to facilitate detection and isolation of CSP polypeptides.
  • Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., (1988 Bio/Technology 6:1204), the FLAG® epitope tagging system (Sigma-Aldrich, Inc., St. Louis, MO) or the XPRESSTM epitope tag (Invitrogen, Carlsbad, CA).
  • the affinity sequence may be a hexa-histidine tag as supplied, for example, by a pBAD/His (Invitrogen) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the affinity sequence may be a hemagglutinin (HA) tag when a eukaryotic host cell, for example a yeast cell or a mammalian cell line (e.g., COS-7 cells) is used.
  • the HA tag conesponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell 57:767).
  • CSP fusion proteins may further comprise immunoglobulin constant region polypeptides added to CSP to facilitate detection, isolation and/or localization of CSP.
  • the immunoglobulin constant region polypeptide preferably is fused to the C- terminus of a CSP polypeptide.
  • fusion proteins comprising heterologous polypeptides fused to a functional antibody fragment, which includes various portions of antibody-derived polypeptides that retain the ability to participate in binding interactions with a specific antigen (in the case of variable region containing fragments such as Fv, Fab, Fab' and F(ab') 2 fragments), or with a cognate receptor (e.g., Fc domain binding to Fc receptors) has been described, e.g., by Ashkenazi et al. (1991 Proc. Nat. Acad. Sci. USA 55. 10535) and Byrn et al. (1990 Nature 544:677).
  • CSP:Fc fusion proteins may be allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between Fc polypeptides, yielding dimeric CSP fusion proteins.
  • CSP fusion proteins having specific binding affinities for pre-selected antigens by virtue of fusion polypeptides comprising immunoglobulin V-region domains encoded by DNA sequences linked in-frame to sequences encoding CSP are also within the scope of the invention, including variants and fragments thereof as provided herein.
  • the nucleic acid of the present invention may also encode a fusion protein comprising a CSP polypeptide fused to other polypeptides having desirable affinity properties, for example an enzyme such as glutathione-S-transferase.
  • CSP fusion proteins may also comprise a CSP polypeptide fused to a Staphylococcus aureus protein A polypeptide; protein A encoding nucleic acids and their use in constructing fusion proteins having affinity for immunoglobulin constant regions are disclosed generally, for example, in U.S. Patent 5,100,788.
  • Other useful affinity polypeptides for construction of CSP fusion proteins may include streptavidin fusion proteins, as disclosed, for example, in WO 89/03422; U.S.
  • CSP polypeptide sequences may be fused to fusion polypeptide sequences that may be full length fusion polypeptides and that may alternatively be variants or fragments thereof.
  • CSP fusion proteins may comprise CSP polypeptides fused to green fluorescent protein (GFP) polypeptides, including mutant GFP polypeptides; such fusion proteins may be encoded by CSI fused in frame to nucleic acid sequences encoding the appropriate GFP polypeptide (see, e.g., Kendall et al., 1998 Trends in Biotechnology 76:216-224, and references cited therein).
  • GFP green fluorescent protein
  • green fluorescent protein encompasses the wildtype green fluorescent protein (wildtype GFP), as well as blue-shifted, cyan-shifted, red-shifted and yellow- shifted derivatives of wildtype GFP (designated, respectively, BFP, CFP, RFP and YFP; see, e.g, published PCT application WO 98/06737).
  • a CSP polypeptide including a fusion protein that comprises a CSP polypeptide sequence such as a p95.6/YN52 sequence, may preferentially localize to one or more membranes, which includes a biological membrane such as a cellular membrane described above.
  • such targeting of a CSP polypeptide of interest to a membrane may be to a cellular membrane, and may in certain further embodiments be to a mitochondrial membrane.
  • subcellular localization of a CSP polypeptide or fusion protein to a particular cellular membrane may arise as a result of a structural feature of the CSP or the fusion peptide or both, including primary structural features (i.e. amino acid sequences) identifiable using the modified PSORT algorithms provided by the present invention and described in greater detail above.
  • the invention is based in part on the unexpected observation that certain recombinant expression constructs as provided herein provide recombinant CSP polypeptides and fusion proteins capable of preferentially localizing to cell membranes.
  • the present invention provides a recombinant construct containing a CSI encoding a mitochondrially directed localizing sequence identifiable according to the computer database query steps as described herein, such as those that employ an improved PSORT algorithm for determining mitochondrial sequences.
  • the invention provides a fusion construct that includes a nucleic acid encoding a first polypeptide that is a CSP polypeptide, and that is expressed as a fusion protein with a second polypeptide sequence that targets the fusion protein to a desired subcellular localization such as a cellular membrane.
  • the second polypeptide sequence may be a CSP identified by using the database search steps described herein as directing the CSP to subcellularly localize in a particular cellular membrane.
  • the cell membrane is a prokaryotic cell membrane such as a bacterial cell membrane, for example an E. coli membrane.
  • the cell membrane is a eukaryotic cell membrane such as a yeast or a mammalian cell membrane, for example a membrane of any eukaryotic cell contemplated herein.
  • the present invention also relates to vectors and to constructs that include nucleic acids of the present invention, and in particular to "recombinant expression constructs" that include any nucleic acids encoding CSP polypeptides according to the invention as provided above, for example p95.6/YN52 or PP7 polypeptides.
  • the invention also relates to host cells which are genetically engineered with such vectors and/or constructs and to the production of CSP polypeptides and fusion proteins, or fragments or variants thereof, by recombinant techniques.
  • CSP proteins can be expressed in mammalian cells, yeast, bacteria, C. elegans or other cells under the control of appropriate promoters.
  • RNAs derived from the DNA constructs of the present invention can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989).
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • Such promoters can be derived from operons encoding -factor, acid phosphatase, heat shock proteins or glycolytic enzymes such as 3 -phosphogly cerate kinase (PGK) among others.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression constructs for bacterial use are constructed by inserting into an expression vector a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter.
  • the construct may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • commercial vectors include, for example, pKK223-3 (Pharmacia, Piscataway, NJ) and G ⁇ M1 (Promega Inc., Madison, WI). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter if it is a regulated promoter as provided herein, is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well know to those skilled in the art.
  • nucleic acids of the invention as provided herein may be included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing a CSP polypeptide.
  • expression vector constructs include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used for preparation of a recombinant expression construct as long as it is replicable and viable in the host.
  • the appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
  • Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc.
  • the DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis.
  • appropriate expression control sequences include LTR or SV40 promoter, the E. coli lac or trp, the phage lambda P promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P and t ⁇ .
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly prefened recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a CSP polypeptide is described herein.
  • the expression control sequence is a "regulated promoter", which may be a promoter as provided herein and may also be a repressor binding site, an activator binding site or any other regulatory sequence that controls expression of a nucleic acid sequence as provided herein.
  • the regulated promoter is a tightly regulated promoter that is specifically inducible and that permits little or no transcription of nucleic acid sequences under its control in the absence of an induction signal, as is known to those familiar with the art and described, for example, in Guzman et al. (1995 J. Bacteriol. 177:4121), Cana et al. (1993 EMBO J.
  • a regulated promoter is present that is inducible but that may not be tightly regulated.
  • a promoter is present in the recombinant expression construct of the invention that is not a regulated promoter; such a promoter may include, for example, a constitutive promoter such as an insect polyhedrin promoter or a yeast phosphoglycerate kinase promoter (see, e.g., Giraud et al., 1998 J. Mol. Biol. 281:409).
  • the expression construct also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the vector may be a viral vector such as a retroviral vector.
  • retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the viral vector includes one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ - actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters or promoters as described above.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2, ⁇ -AM, PA12, T19- 14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 7:5-14 (1990), which is inco ⁇ orated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and Ca PO 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the CSP polypeptides or fusion proteins. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the CSP polypeptide or fusion protein.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • host cells transduced by a recombinant viral construct directing the expression of CSP polypeptides or fusion proteins may produce viral particles containing expressed CSP polypeptides or fusion proteins that are derived from portions of a host cell membrane inco ⁇ orated by the viral particles during viral budding.
  • CSP encoding nucleic acid sequences are cloned into a baculovirus shuttle vector, which is then recombined with a baculovirus to generate a recombinant baculovirus expression construct that is used to infect, for example, Sf9 host cells, as described in Baculovirus Expression Protocols, Methods in Molecular Biology Vol. 39, CD.
  • CSP encoding nucleic acid sequences are cloned into an expression vector suitable for transformation of nematode cells, such as C elegans cells.
  • cosmids comprising a CSP-encoding nucleic acid can be micro-injected into N2 hermaphrodites to obtain transgenic C.
  • a CSI in a "reverse" orientation in an expression vector i.e., so that the antisense strand is transcribed, will yield an antisense vector expressing an antisense transcript that will specifically bind to and prevent the expression of endogenous CSI-comprising transcripts. It is also known in the art to prepare "knock-outs" (physical disruptions) of genes in C. elegans (see http://ko.cigenomics.bc.ca/elegans/).
  • the present invention relates to host cells and organisms containing the above described recombinant CSP expression, antisense or "knock-out" constructs, for example, p95.6/YN52 or PP7 constructs.
  • Host cells and organisms are genetically engineered (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention which may be, for example, a cloning vector, a shuttle vector or an expression construct.
  • the vector or construct may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding CSP polypeptides or CSP fusion proteins.
  • the culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell; or a nematode cell, or a lower eukaryotic cell, such as a yeast cell.
  • the host cell can also be a prokaryotic cell, such as a bacterial cell.
  • Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as E.
  • coli Streptomyces, Salmonella tvphimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera SJ9
  • animal cells such as CHO, COS or human cells, for example, 293 cells
  • adenoviruses plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo.
  • the host organism can be any organism that can be genetically manipulated to transiently or stably comprise the above described recombinant CSP expression, antisense or "knock-out" constructs.
  • Such organisms include, by way of example and not limitation, non-human animals including vertebrates such as rodents, non-human primates, sheep, dog, cow, amphibians, reptiles, etc.
  • Prefened non-human animals are selected from non-human mammalian species of animals, including, without limitation, animals from the rodent family such as rats and mice, most preferably mice (see, e.g., U.S. Patent Nos. 5,675,060 and 5,850,001).
  • Other genetically manipulated non-human animals that may be prepared include, without limitation, rabbits (U.S.
  • Patent No. 5,792,902 pigs
  • pigs U.S. Patent No. 5,573,933
  • bovine species U.S. Patent Nos. 5,633,076 and 5,741 ,957
  • ovine species such as goats and sheep
  • Cloned host animals, transgenic and otherwise, of the invention may also be prepared (for a review of mammalian cloning techniques, see Wolf et al, J. Assist. Reprod. Genet. 75:235-239, 1998).
  • Such cloned animals include, without limitation, ovine species such as sheep (Campbell et al., Nature 380:64-66, 1996; Wells et al., Biol. Reprod. 57:385-393, 1997) rodents such as mice (Wakayama et al., Nature 594:369-374, 1998) and non-human primates, such as rhesus monkeys (Meng et al., Biol Reprod. 57:454-459, 1997).
  • ovine species such as sheep (Campbell et al., Nature 380:64-66, 1996; Wells et al., Biol. Reprod. 57:385-393, 1997) rodents such as mice (Wakayama et al., Nature 594:369-374, 1998) and non-human primates, such as rhesus monkeys (Meng et al., Biol Reprod. 57:454-459, 1997).
  • C. elegans cells in vitro (see, e.g., Bloom, L., "Development of Techniques for Primary Culture of C. elegans Embryonic Neurons," Ph.D. thesis, Massachusetts Institute of Technology, 1993; see also http://www.dartmouth.edu/artsci/bio/ambros/ protocols/other/neuron culture l.html, _culture_2.html, and _culture_3.html), and these may be prefened host cells in certain embodiments.
  • expression or antisense constructs are introduced in C. elegans by the generation of transgenic animals, which in certain other embodiments may be prefened host organisms.
  • expression constructs can be microinjected into C. elegans oocytes (see, e.g., Koelle, M., "Microinjecting Worms," http://www.dartmouth.edu/ artsci/bio/ambros/protocols/MGH_protocols/koelle_prot/X0009_microinjection.l.html, 1994).
  • the expression or antisense constructs can be maintained as artificial chromosomes or integrated into the genome (see Koelle, M., "Integrating Extrachromosomal Anays into the C.
  • CSI encoding CSP such as p95.6/YN52
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-1 and COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 25: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences, for example as described herein regarding the preparation of CSP expression constructs.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, liposome-mediated transfection, transfection with naked DNA, biolistic particle-mediated transfection, DEAE-Dextran mediated transfection or electroporation (Davis et al., 1986 Basic Methods in Molecular Biology).
  • compositions of the invention may be desirable to prepare the compositions of the invention and to practice the methods of the invention under conditions where endogenous expression by a host cell of a coding sequence that is highly homologous to the CSI is compromised, in order to provide advantages associated with the expression of a desired CSP encoding construct.
  • detection of particular CSP encoding nucleic acid sequences or CSP polypeptides that are highly similar to those encoded by the host cell genome may be facilitated by inhibiting host cell CSI expression.
  • functional activity of an exogenously introduced recombinant CSP polypeptide e.g., an expressed C.
  • elegans p95.6/YN52 or human PP7 polypeptide that results from transfection is to be determined in a host cell or in a biological sample derived therefrom, it may also be advantageous to inhibit endogenous host cell expression of the related CSI encoding a CSP homolog (e.g., a calcium regulatory protein or serine/threonine phosphatase).
  • a CSP homolog e.g., a calcium regulatory protein or serine/threonine phosphatase.
  • host cells may lack at least one endogenous CSP homolog, and in certain prefened embodiments the host cells may lack any endogenous CSP homologs.
  • expression in host cells of at least one gene encoding an endogenous CSP homolog is substantially impaired.
  • Substantial impairment of endogenous CSP homolog expression may be achieved by any of a variety of methods that are well known in the art for blocking specific gene expression, including site-specific or site-directed mutagenesis as described above, antisense inhibition of gene expression, ribozyme mediated inhibition of gene expression and generation of mitochondrial DNA depleted (p° ) cells.
  • Antisense oligonucleotides are oligonucleotides that bind in a sequence- specific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that has complementary sequences, antisense oligonucleotides prevent translation of the mRNA, or hasten the degradation of the RNA, or otherwise interfere with one or more biological functions of RNA (see, e.g., U.S. Patent No. 5,168,053 to Altman et al.; U.S. Patent No. 5,190,931 to Inouye, U.S. Patent No. 5,135,917 to Burch; U.S. Patent No. 5,087,617 to Smith and Clusel et al. (1993) Nucl.
  • Triplex molecules refer to single DNA strands that bind duplex DNA forming a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Patent No. 5,176,996 to Hogan et al., which describes methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
  • Antisense oligonucleotides are typically designed to resist degradation by endogenous nucleolytic enzymes by using such linkages as: phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages (see, e.g., Agrwal et al., Tetrahedron Lett 25:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 95:6657-6665 (1971); Stec et al., Tetrahedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. Acids Res.
  • particularly useful antisense nucleotides and triplex molecules are molecules that are complementary to or bind the sense strand of DNA or mRNA that encodes a CSP homolog polypeptide or a protein mediating any other process related to expression of particular endogenous CSP homolog genes, such that inhibition of translation of mRNA encoding the CSP homolog polypeptide is effected.
  • a ribozyme is an RNA molecule that specifically cleaves RNA substrates, such as mRNA, resulting in specific inhibition or interference with cellular gene expression. There are at least five known classes of ribozymes involved in the cleavage and/or ligation of RNA chains.
  • Ribozymes can be targeted to any RNA transcript and can catalytically cleave such transcripts (see, e.g., U.S. Patent No. 5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,1 16,742 and 5,093,246 to Cech et al.).
  • any such CSP homolog mRNA-specific ribozyme, or a nucleic acid encoding such a ribozyme may be delivered to a host cell to effect inhibition of CSP homolog gene expression.
  • Ribozymes, and the like may therefore be delivered to the host cells by DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, such that upon introduction into the nucleus, the ribozyme will be directly transcribed.
  • a eukaryotic promoter such as a eukaryotic viral promoter
  • expression of a gene encoding an endogenous CSP homolog is substantially impaired by any of the above methods for inhibiting gene expression when cells are substantially but not necessarily completely depleted of functional DNA or functional mRNA encoding the endogenous CSP homolog, or of the relevant CSP homolog polypeptide.
  • CSP homolog expression is substantially impaired when cells are preferably at least 50% depleted of DNA or mRNA encoding the endogenous CSP homolog (as measured using high stringency hybridization as described above) or at least 50% depleted of CSP homolog polypeptide (as measured, e.g., by Western immunoblo ); and more preferably at least 75% depleted of endogenous CSP homolog DNA, mRNA or polypeptide.
  • CSP homolog expression is substantially impaired when cells are depleted of >90% of their endogenous CSP homolog DNA, mRNA, or polypeptide.
  • a CSI encodes a CSP that is a mitochondrial molecular component
  • expression of a gene encoding an endogenous CSP homolog may be substantially impaired through the use of mitochondrial DNA depleted p° cells, which are incapable of mitochondrial replication and so may not continue to express functional CSP homolog polypeptides.
  • Methods for producing p° cells are known and can be found, for example in U.S. Patent No. 5,888,498, which is hereby inco ⁇ orated by reference.
  • C. elegans p95.6/YN52 (GenBank Ace. No. P34586) or human serine/ threonine phosphatase (GenBank Ace. Nos. AAC05825, AAB82795) polypeptides or fusion proteins derived from CSI or CSP identified by database searching using a Computer System as described above, may be useful in intact host cells; in intact organelles such as mitochondria, cell membranes, intracellular vesicles other cellular organelles; or in disrupted cell preparations including but not limited to cell homogenates or lysates, submitochondrial particles, uni- and multilamellar membrane vesicles or other preparations.
  • expressed recombinant CSP polypeptides or fusion proteins can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
  • a "biological sample containing mitochondria” may comprise any tissue or cell preparation in which intact mitochondria capable of maintaining a membrane potential when supplied with one or more oxidizable substrates such as glucose, glutamate, malate or galactose are or are thought to be present.
  • Mitochondrial membrane potential may be determined according to methods with which those skilled in the art will be readily familiar, including but not limited to detection and/or measurement of detectable compounds such as fluorescent indicators, optical probes and/or sensitive pH and ion-selective electrodes (See, e.g., Ernster et al., 1981 J.
  • a detectable compound e.g., DASPMI [2-,4-dimethylaminostyryl-N- methylpyridinium], TMRM [tetramethylrhodamine methyl ester], etc.
  • a biological sample containing mitochondria may, for example, be derived from a normal (i.e., healthy) individual or from an individual having a disease associated with altered mitochondrial function.
  • Biological samples containing mitochondria may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source.
  • the subject or biological source may be a biological organism such as a human or non-human animal, a plant, a unicellular organism or a multicellular organism.
  • the subject or biological source may also be a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like.
  • a “biological sample” may comprise any tissue or cell preparation as just described for a biological sample containing mitochondria, but does not require the presence of intact mitochondria.
  • a “biological sample” may comprise any tissue or cell preparation and a "biological sample containing at least one recombinant CSP polypeptide” comprises any tissue or cell preparation in which an expressed recombinant CSP polypeptide or fusion protein as provided herein, for example, a p95.6/YN52 polypeptide or a PP7 polypeptide, is thought to be present.
  • a biological sample may, for example, be derived from a recombinant cell line or from a transgenic animal.
  • Biological samples containing recombinant CSP may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source.
  • the subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like.
  • Certain aspects of the present invention as it relates to identification and use in screening assays of particular CSPs that are mitochondrial molecular components that bind, transport or otherwise regulate intracellular calcium and that are selected according to criteria described herein (e.g., calcium-binding domain, subcellular localization to mitochondria, absence of related CSI from yeast cDNA libraries, absence of dihydropyridine-binding domain, etc.), for example C. elegans p95.6/YN52 polypeptides as described herein, involve the relationship between mitochondrial ⁇ and intracellular calcium homeostasis. As noted above, fluctuations in mitochondrial calcium are normal occurrences that are part of intracellular calcium homeostasis.
  • Mitochondrial calcium levels may also reflect transient high cytosolic calcium concentrations, which, in combination with reduced ATP or other conditions associated with mitochondrial pathology can yield mitochondrial permeability transition (MPT, see, e.g., Gunter et al., 1998 Biochim. Biophys. Acta 1366:5; Rottenberg et al., 1990, Biochim. Biophys. Acta 1016:87). Under unstimulated conditions in a normal cell, the extramitochondrial (cytosolic) level of Ca + is greater than that present within mitochondria.
  • mitochondrial or cytosolic calcium levels may vary from the above ranges and may range from, e.g., about 1 nM to about 500 mM, more typically from about 10 nM to about 100 ⁇ M and usually from about 20 nM to about 1 ⁇ M, where "about” indicates ⁇ 10%.
  • a variety of calcium indicators are known in the art including but not limited to fura-2 (McCormack et al., 1989 Biochim. Biophys. Acta 973:420); mag-fura-2; BTC (U.S. Patent No. 5,501,980); fluo-3, fluo-4 and fluo-5N (U.S. Patent No. 5,049,673); Calcium Green 5N; benzothiaza-1 ; and benzothiaza-2 and others, which are available from Molecular Probes, Eugene, OR.
  • Ca + influx into mitochondria appears to be largely dependent, and may be completely dependent, upon the negative transmembrane electrochemical potential ( ⁇ ) established by electron transfer, and such influx fails to occur in the absence of ⁇ even when an eight-fold Ca + concentration gradient is imposed (Kapus et al., 1991 FEBS Lett. 252:61).
  • mitochondria may release Ca 2+ via the calcium uniporter (described above) when the membrane potential is dissipated following Ca 2+ accumulation, as occurs, for example, with inhibitors or uncouplers of oxidative phosphorylation (i.e., uncouplers of the mitochondrial ETC from ADP phosphorylation) such as 2,4-dinitrophenol and carbonyl cyanide p-trifluoro- methoxyphenylhydrazone (FCCP).
  • oxidative phosphorylation i.e., uncouplers of the mitochondrial ETC from ADP phosphorylation
  • FCCP carbonyl cyanide p-trifluoro- methoxyphenylhydrazone
  • 9+ compounds that induce increased intracellular concentrations of Ca include but are not limited to thapsigargin, carbachol and, in the case of cells having appropriate receptors, amino acid neurotransmitters such as glutamate or N-methyl-D-aspartic acid.
  • amino acid neurotransmitters such as glutamate or N-methyl-D-aspartic acid.
  • the particular cells that are exposed to a given compound such as glutamate require a receptor therefor, in order for the compound to influence intracellular Ca 2+ levels.
  • NT-2 teratocarcinoma cells express such glutamate receptors, whereas SH-SY5Y neuroblastoma cells do not.
  • the choice of cell line in which it may be desirable to increase intracellular calcium levels will determine which compounds are most appropriate.
  • ionomycin in certain prefened embodiments of the invention related to determination of mitochondrial regulation of intracellular calcium, ionomycin (Toeplitz et al., 1979 J Amer. Chem. Soc. 707:3344) may be used as a calcium ionophore and Rhod-2 (Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, Oregon, pp. 504-526) may be a fluorescent indicator of intramitochondrial calcium content.
  • any appropriate compound that results in increased intracellular concentrations of Ca 2+ and any indicator of intracellular calcium levels that permits measuring mitochondrial regulation of calcium homeostasis in a biological sample may be used.
  • MPT may also be induced by compounds that bind one or more mitochondrial molecular components.
  • Such compounds include, but are not limited to, atractyloside and bongkrekic acid. Methods of determining appropriate amounts of such compounds to induce MPT are known in the art (see, e.g., Beutner et al., 1998 Biochim. Biophys. Acta 1368:1; Obatomi and Bach, 1996 Toxicol Lett 59:155; Green and Reed, 1998 Science 257:1309; Kroemer et al., 1998 Annu. Rev. Physiol. 60:619; and references cited therein).
  • an altered mitochondrial state is induced by exposing a biological sample to compositions refened to as "apoptogens" that induce programmed cell death, or "apoptosis".
  • apoptogens are known to those familiar with the art (see, e.g., Green et al., 1998 Science 281 :1309 and references cited therein) and may include by way of illustration and not limitation: tumor necrosis factor-alpha (TNF- ⁇ ); Fas ligand; glutamate; N-methyl-D-aspartate (NMD A); interleukin-3 (IL-3); herbimycin A (Mancini et al., 1997 J. Cell. Biol.
  • isolation of a mitochondrial molecular component with which an agent identified according to the methods of the invention interacts refers to physical separation of such a complex from its biological source, and may be accomplished by any of a number of well known techniques including but not limited to those described herein, and in the cited references.
  • a compound that "binds a mitochondrial component” can be any discrete molecule, agent compound, composition of matter or the like that may, but need not, directly bind to a mitochondrial molecular component, and may in the alternative bind indirectly to a mitochondrial molecular component by interacting with one or more additional components that bind to a mitochondrial molecular component.
  • any CSP polypeptide including recombinant CSP polypeptides and fusion proteins may be a mitochondrial molecular component and/or a mitochondrial pore component
  • any CSP ligand or agent that binds to an CSP polypeptide may be a compound that binds a mitochondrial component and/or an agent that affects mitochondrial pore activity.
  • mitochondria are comprised of "mitochondrial molecular components", which may be a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof, a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or another biological molecule that is a constituent of a mitochondrion.
  • Mitochondrial molecular components includes but is not limited to "mitochondrial pore components".
  • a "mitochondrial pore component” is any mitochondrial molecular component that regulates the selective permeability characteristic of mitochondrial membranes as described above, including those that bind calcium, transport calcium or are otherwise involved in the maintenance of calcium or other ion levels on either side of the mitochondrial membrane. Mitochondrial pore components also include mitochondrial molecular components responsible for establishing ⁇ m and those that are functionally altered during MPT.
  • Isolation and, optionally, identification and/or characterization of the mitochondrial pore component or components with which an agent that affects mitochondrial pore activity interacts may also be desirable and are within the scope of the invention.
  • a mitochondrial activity such as mitochondrial permeability properties, for example, mitochondrial binding, transport or regulation of calcium as provided herein and in U.S. application serial numbers 09/161,172 and 09/338,122 or, for example, MPT according to the methods provided herein and in U.S.A.N.
  • Techniques for isolating a mitochondrial molecular component such as a CSP polypeptide or fusion protein may include any biological and/or biochemical methods useful for separating the complex from its biological source, and subsequent characterization may be performed according to standard biochemical and molecular biology procedures. Those familiar with the art will be able to select an appropriate method depending on the biological starting material and other factors.
  • Such methods may include, but need not be limited to, radiolabeling or otherwise detectably labeling cellular and mitochondrial components in a biological sample, cell fractionation, density sedimentation, differential extraction, salt precipitation, ultrafiltration, gel filtration, ion-exchange chromatography, partition chromatography, hydrophobic chromatography, electrophoresis, affinity techniques or any other suitable separation method that can be adapted for use with the agent with which the mitochondrial pore component interacts.
  • Antibodies to partially purified components may be developed according to methods known in the art and may be used to detect and/or to isolate such components.
  • Antibodies specific for a CSP protein or polypeptide of interest are prepared according to a variety of methods known in the art. In general, such antibodies may be polyclonal, monoclonal or monospecific antibodies.
  • Primary antibodies of the invention bind specifically to a particular protein or polypeptide of interest and are thus used in assays to detect and quantify such proteins and polypeptides.
  • a primary antibody of the invention is detectably labeled or is specifically recognized and monitored by a detectably labeled secondary antibody or a combination of a secondary antibody and a tertiary molecule (which may also be an antibody) that is detectably labeled.
  • the primary antibody of the invention provides a means by which a protein or polypeptide of interest is specifically bound and subsequently detected.
  • One prefened assay format is the Enzyme-Linked Immunosorbent Assay (ELISA) format.
  • a nucleic acid of interest may encode a known CSP protein or a portion thereof, or a polypeptide sequence that is homologous to a known protein.
  • antisera to the known protein, or the known protein itself may be commercially available.
  • the known or recombinantly-produced protein can be used to immunize a mammal of choice (e.g., a rabbit, mouse or rat) in order to produce antisera from which polyclonal antibodies can be prepared (see, e.g., Cooper and Paterson, Units 11.12 and 11.13 in Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al, eds., John Wiley & Sons, New York, New York, 1992, pages 11-37 to 11-41).
  • nucleic acid sequence of interest encodes a polypeptide sequence for which no complete protein (or homolog thereof) is known, or is too short to encode more than about 30 amino acids (i.e., the nucleic acid of interest is less than about 100 nucleotides in length), or encodes more than one polypeptide sequence of potential interest
  • candidate amino acid sequences can be used to synthesize one or more polypeptide molecules, each of which has a defined amino acid sequence.
  • Such synthetic polypeptides can then be used to immunize animals (e.g., rabbits) according to methods known in the art (Collawn and Paterson, Units 11.14 and 11.15 in Chapter 1 1 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al, eds., John Wiley & Sons, New York, New York, 1992, pages 1 1-42 to 11-46; Cooper and Paterson, Units 11.12 and 11.13 in Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al, eds., John Wiley & Sons, New York, New York, 1992, pages 11-37 to 1 1-41).
  • the resulting antisera may then be used to probe cells from which the nucleic acid of interest was isolated.
  • a positive response to a given antiserum indicates that the candidate reading frame from which the synthetic polypeptide used to raise the antiserum was derived is a reading frame used to encode at least one protein in the cell(s) so examined.
  • such an antiserum can be used to identify proteins of interest in the cells from which the nucleic acid of interest was isolated.
  • monoclonal antibodies are often the prefened type of antibody for a variety of applications.
  • Methods for producing and preparing monoclonal antibodies are known in the art (see, e.g., Fuller et al., Units 11.4 to 11.1 1 in Chapter 1 1 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al, eds., John Wiley & Sons, New York, New York, 1992, pages 11-22 to 11-36).
  • Murine monoclonal antibodies may be "humanized” and used as therapeutic agents (see, e.g., G ⁇ ssow and Seemann, Methods in Enzymology 203:99- 121 , 1991 ; Vaughan et al, Nature Biotechnology 76:535-539, 1998).
  • Antibodies specific for CSP proteins and polypeptides of interest may be used to detect such proteins and polypeptides in a variety of assay formats. Such immunoassays may be useful in diagnostic, prognostic or pharmacogenetic methods of the invention, or in methods in which various cell types, tissues or organs are probed for the presence of a protein of interest. Monoclonal antibodies are generally preferred for such methods due to their high degree of specificity and homogeneity.
  • Affinity techniques may be particularly useful in the context of the present invention, and may include any method that exploits a specific binding interaction between a mitochondrial pore component and an agent identified according to the invention that interacts with the pore component.
  • an affinity binding technique for isolation of the pore component may be particularly useful.
  • affinity labeling methods for biological molecules in which a known ion channel-active agent, MPT-active agent or a novel CSP ligand as provided herein may be modified with a reactive moiety, are well known and can be readily adapted to the interaction between the agent and a pore component, for pu ⁇ oses of introducing into the pore component a detectable and/or recoverable labeling moiety.
  • a known ion channel-active agent, MPT-active agent or a novel CSP ligand as provided herein are well known and can be readily adapted to the interaction between the agent and a pore component, for pu ⁇ oses of introducing into the pore component a detectable and/or recoverable labeling moiety.
  • Characterization of mitochondrial pore component molecular species may be accomplished using physicochemical properties of the pore component such as spectrometric absorbance, molecular size and/or charge, solubility, peptide mapping, sequence analysis and the like. (See, e.g., Scopes, supra.) Additional separation steps for biomolecules may be optionally employed to further separate and identify molecular species that co-purify with mitochondrial pore components.
  • extracts of cultured cells and in particularly prefened embodiments extracts of biological tissues or organs may be sources of mitochondrial molecular components, including CSP polypeptides.
  • Prefened sources may include blood, brain, fibroblasts, myoblasts, liver cells or other cell types.
  • the invention further provides for the identification of agents (for example, CSP ligands such as p95.6/YN52 ligands or PP7 ligands) that bind to, or that interact with, such polypeptides and homologous polypeptides from other species, including agents that interact with a mitochondrial CSP polypeptide or its homolog in a manner that alters (e.g., increases or decreases) a mitochondrial activity.
  • agents for example, CSP ligands such as p95.6/YN52 ligands or PP7 ligands
  • agents for example, CSP ligands such as p95.6/YN52 ligands or PP7 ligands
  • agents for example, CSP ligands such as p95.6/YN52 ligands or PP7 ligands
  • agents for example, CSP ligands such as p95.6/YN52 ligands or PP7 ligands
  • Such compositions and methods
  • compositions and methods are useful as high throughput screening assays. Accordingly, it is another aspect of the present invention to provide compositions and methods for identifying agents that bind to or alter (e.g., increase or decrease) the activity of a C.
  • elegans p95.6/YN52 polypeptide which may include a mitochondrial calcium uniporter activity as described above or another ion (e.g., calcium) binding, transport or regulatory function; such agents are also refened to herein as p95.6/YN52 ligands.
  • the invention provides a method for determining the presence of a CSP polypeptide such as a C. elegans p95.6/YN52 polypeptide (or a PP7 polypeptide) in a biological sample, comprising contacting a biological sample suspected of containing a C. elegans p95.6/YN52 polypeptide (or a PP7 polypeptide) with a p95.6/YN52 ligand (or a PP7 ligand) under conditions and for a time sufficient to allow binding of the p95.6/YN52 ligand (or PP7 ligand) to the C. elegans p95.6/YN52 (or PP7) polypeptide, and detecting such binding.
  • a CSP polypeptide such as a C. elegans p95.6/YN52 polypeptide (or a PP7 polypeptide) in a biological sample
  • C elegans p95.6/YN52 ligands may include any novel C. elegans p95.6/YN52 ligands as provided herein.
  • PP7 ligands may include any novel PP7 ligands as provided herein.
  • Prefened CSP ligands include antibodies specific for the CSP (e.g., for p95.6 or for PP7), such as polyclonal, monoclonal, single chain or chimeric antibodies, or functional antibody fragments, as provided herein.
  • prefened embodiments include the use of any C. elegans p95.6/YN52 polypeptide or C. elegans p95.6/YN52 fusion protein as provided herein. Accordingly, the instant method for determining the presence of a C. elegans p95.6/YN52 polypeptide in a sample will be useful for monitoring expression of C. elegans p95.6/YN52 encoding constructs provided herein.
  • a C. elegans p95.6/YN52 fusion protein is used that is a GST fusion protein
  • the C. elegans p95.6/YN52 fusion protein is a FLAG®-tagged fusion protein.
  • a p95.6 fusion protein comprises a green fluorescent protein (GFP) domain or a related domain of a modified or mutated GFP (WO 98/06737) such as a yellow-shifted (YFP), a blue-shifted (BFP), a cyan-shifted (CFP) or a red-shifted fluorescent protein (RFP), which may be used, for example, to verify subcellular localization to mitochondria of an expressed p95.6 fusion protein encoded by a recombinant expression construct according to the invention.
  • GFP green fluorescent protein
  • WO 98/06737 a modified or mutated GFP
  • YFP yellow-shifted
  • BFP blue-shifted
  • CFP cyan-shifted
  • RFP red-shifted fluorescent protein
  • Determining the presence of a CSP that is suspected of having or contributing to calcium uniporter activity may also be conducted using functional assays for a calcium uniporter as known in the art and provided herein.
  • a CSI identified as provided herein is recombinantly expressed to provide a biological sample comprising a CSP that can be functionally characterized, for example, by determining calcium binding, calcium transport or other calcium regulatory activity in the sample or in material prepared therefrom.
  • calcium uniporter activity of a CSP such as p95.6/YN52 may be detected as a gain of function in a cell or organism known to lack such function (for instance, s. cerevisiae). Determination of a calcium uniporter role for a CSP such as p95.6 may also be made by detecting a specific loss of uniporter function, for example in cells that lack a p95.6/YN52 polypeptide and/or cells in which p95.6 expression is substantially impaired as provided herein, for instance, as a consequence of exposure to a p95.6-specific antisense reagent or ribozyme.
  • a calcium uniporter can also be tested in suitable cell-based and cell-free systems for assaying mitochondrial activities as provided herein and with which those skilled in the art will be familiar, including but not limited to: activation by ADP, inhibition by ATP, Mg 2+ , ruthenium red and its derivative Ru360 (Matlib et al., 1998 J. Biol. Chem. 273:10223; Emerson et al., 1993 J. Amer. Chem. Soc. 115:11799) and competitive inhibition by Sr 2+ , Mn 2+ and La 3+ .
  • calcium uniporter activity may be determined as it relates to mitochondrial membrane potential, which under certain conditions may be altered (e.g., increased or decreased) in response to calcium transport by a functional uniporter.
  • the invention is not intended to be limited to these examples of functionally testing a CSP polypeptide for calcium uniporter activity, and may include other methodologies for determining calcium binding, calcium transport or other calcium regulatory activities.
  • the invention provides a method for identifying an agent that binds to a C. elegans p95.6/YN52 polypeptide, comprising contacting a candidate agent with a biological sample containing at least one recombinant C. elegans p95.6/YN52 polypeptide under conditions and for a time sufficient to permit binding of the agent to the C. elegans p95.6/YN52 polypeptide and detecting such binding.
  • the biological sample may comprise a cell that may be a prokaryotic cell or a eukaryotic cell. In certain other prefened embodiments the cell may lack an endogenous homolog of the C.
  • elegans p95.6/YN52 polypeptide for example, due to a mutation in one or more endogenous p95.6/YN52-like polypeptide encoding genes.
  • expression by the cell of at least one gene encoding an endogenous C. elegans p95.6/YN52 homolog is substantially impaired, for example, through the use of p95.6/YN52 nucleic acid-specific ribozyme or antisense constructs as provided herein, or through the use of p° cells and/or cybrid cells, which are provided, for example, in U.S. Patent No. 5,888,498.
  • expression of a gene encoding an endogenous p95.6/YN52 homolog is substantially impaired by any of the above methods for inhibiting when cells are substantially but not necessarily completely depleted of functional DNA or functional mRNA encoding the endogenous p95.6/YN52 isoform, or of the relevant p95.6/YN52 polypeptide.
  • p95.6/YN52 isoform expression is substantially impaired when cells are preferably at least 50% depleted of DNA or mRNA encoding the endogenous p95.6/YN52 polypeptide (as measured using high stringency hybridization as described above) or depleted of p95.6 polypeptide (as measured by Western immunoblot); and more preferably at least 75% depleted of endogenous p95.6 DNA, mRNA or polypeptide.
  • p95.6 isoform expression is substantially impaired when cells are depleted of >90% of their endogenous p95.6 DNA, mRNA, or polypeptide.
  • this aspect of the invention it may be prefened to use intact cells or, alternatively, in certain other embodiments the use of permeabilized cells or isolated mitochondria may be prefened.
  • permeabilized cells for example by way of illustration and not limitation, through the use of surfactants, detergents, phospholipids, phospholipid binding proteins, enzymes, viral membrane fusion proteins and the like; through the use of osmotically active agents; by using chemical crosslinking agents; by physicochemical methods including electroporation and the like, or by other permeabilizing methodologies.
  • Detection of binding of an agent (or of a CSP ligand) to a CSP such as a p95.6/YN52 polypeptide may be by any of a variety of methods and will depend on the nature of the candidate agent (or ligand) being screened. For example, certain candidate agents (or ligands) are inherently detectable as a consequence of their physicochemical properties, such as will be apparent to those skilled in the art and including spectrophotometric, colorimetric, fluorimetric, solubility, hydrophobic, hydrophilic, electrostatic charge, molecular mass or other physicochemical properties.
  • certain candidate agents may be radioactively labeled with a readily detectable radionuclide, as is well known in the art.
  • the subject invention methods also contemplate competitive binding assays to identify agents (or ligands) that specifically bind to a CSP such as p95.6.
  • certain candidate agents may also be directly or indirectly detectable by protein affinity methodologies, for example, by their ability to interfere with binding of a C.
  • a candidate agent bound to a p95.6/YN52 polypeptide may be detected by any method known for the detection, identification or characterization of relevant molecules, including spectrophotometric, mass spectrometric, chromatographic, electrophoretic, calorimetric or any other suitable analytical technique.
  • the invention provides a method for identifying an agent that binds to or interacts with a C. elegans p95.6/YN52 polypeptide comprising contacting a biological sample comprising a recombinant C. elegans p95.6/YN52 polypeptide with a detectable p95.6/YN52 ligand in the absence and presence of a candidate agent; and comparing binding of the detectable p95.6/YN52 ligand to the recombinant C. elegans p95.6/YN52 polypeptide in the absence of the agent to binding of the detectable p95.6/YN52 ligand to the recombinant C.
  • the detectable p95.6/YN52 ligand is detected as described above, and in certain embodiments the detectable ligand is an antibody or a functional antibody fragment as provided herein.
  • Certain other embodiments of the invention are directed to screening assays using a biological sample containing mitochondria as provided herein. In one such embodiment the invention relates to a method for identifying an agent that interacts with a C.
  • elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a biological sample containing mitochondria wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent, wherein an altered (e.g., increased or decreased) level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • altered levels of mitochondrial activity pertain to readily detectable increases or decreases that may vary quantitatively depending on the particular mitochondrial activity being monitored and on the particular reagents, instrumentation and methodology selected.
  • an altered level of mitochondrial activity refers to a statistically significant increase or decrease.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide comprising detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a first biological sample comprising mitochondria, wherein the mitochondria comprise a recombinant C.
  • elegans p95.6/YN52 polypeptide comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent to a reference level of mitochondrial activity in a second biological sample comprising mitochondria which lack a recombinant C. elegans p95.6/YN52 polypeptide, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • a suitable reference level will be according to criteria with which those having ordinary skill in the art will be familiar, and may vary depending on the particular mitochondrial activity being assayed and upon the particular reporter molecule or other source of detectable signal that is employed as the reference. As provided herein and as will be known to those familiar with the art, for example, any number of suitable procedures for detecting a particular mitochondrial activity may be selected.
  • mitochondrial activity may be determined by detecting intracellular calcium levels, mitochondrial membrane potential, mitochondrial ATP production, oxygen consumption as a measure of aerobic respiration or mitochondrial ETC activity (e.g., specific enzymological assays of one or more of ETC Complexes I- V) using a variety of established methodologies (see, e.g., Miller et al., 1996 J. Neurochem. 67:1897 and references cited therein; Sheehan et al. 1997 J. Neurosci. 17:4612 and references cited therein). Accordingly, a person having ordinary skill in the art can select a suitable biological sample, reporter molecule and metric technique for use as the source of a detectable reference signal.
  • the invention provides a method for identifying an agent that interacts with a C. elegans p95.6/YN52 polypeptide that includes contacting a candidate agent with a cell, which in certain further embodiments may be a permeabilized cell as described above.
  • the method comprises detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a biological sample comprising a cell which contains mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • the method comprises detecting a level of at least one mitochondrial activity before and after contacting a candidate agent with a first biological sample comprising a cell which contains mitochondria, wherein the mitochondria comprise a recombinant C. elegans p95.6/YN52 polypeptide; and comparing the level of mitochondrial activity in the absence of the candidate agent and in the presence of the candidate agent to a reference level of mitochondrial activity in a second biological sample comprising, as described herein, a cell which contains mitochondria that lack a recombinant C. elegans p95.6/YN52 polypeptide, wherein an altered level of mitochondrial activity indicates the presence of an agent that interacts with a C. elegans p95.6/YN52 polypeptide.
  • mitochondrial activity may be measured as a level of intracellular calcium. As described in detail herein, mitochondria contribute significantly to regulation of intracellular Ca 2+ levels. In certain other embodiments of any of the above described methods that relate to a biological sample comprising a cell that contains mitochondria, mitochondrial activity may be measured as a cellular response to an apoptogen, which includes any detectable alteration in a cell that indicates involvement of a component of an apoptotic pathway.
  • apoptosis Various methods for detecting apoptosis are known in the art, including by way of non- limiting example, determination of: cell viability, cell membrane blebbing, chromosomal disintegration, plasma membrane phosphatidylserine translocation, mitochondrial release of cytochrome c, caspase activation or the use of any other criterion related to apoptosis (see, e.g., Green and Reed, Science 257:1309-1312, 1998; Green, Cell 94:695-698, 1998; Kroemer, Nature Medicine 5:614-620, 1997).
  • the invention provides competitive binding assays wherein novel C. elegans p95.6/YN52 ligands as provided herein are useful.
  • the invention need not be so limited and may be modified to employ known detectable molecules capable of interacting with a mitochondrial molecular component as provided herein.
  • Examples of known detectable molecules capable of interacting with a mitochondrial molecular component include suitably labeled ATP, ADP, atractyloside (ATR), carboxyatracyloside (CATR), palmitoyl-CoA, bongkrekic acid, thyroxin, eosin Y and erythrosin B or other molecules capable of interacting with a mitochondrial molecular component known in the art (see, e.g., Block et al., 1986 Meths. Enzymol 125:658).
  • ATR atractyloside
  • CAR carboxyatracyloside
  • elegans p95.6/YN52 a candidate mitochondrial calcium uniporter provided by the present invention, offer unexpected advantages heretofore unknown in the art for identifying mitochondrially active agents.
  • Prefened embodiments include the use of any C. elegans p95.6/YN52 polypeptide or p95.6/YN52 fusion protein or p95.6/YN52 analog, homolog or fragment, as provided herein.
  • a p95.6/YN52 fusion protein is used that is a GST fusion protein, and in other prefened embodiments the p95.6/YN52 fusion protein is a FLAG®-tagged fusion protein.
  • a candidate agent for use according to the present invention may be any composition of matter that is suspected of binding to or altering (e.g., increasing or decreasing) the activity of a particular CSP target such as a p95.6/YN52 (or a PP7) polypeptide.
  • the candidate agent is provided in soluble form.
  • a candidate agent may alter the activity of the CSP target directly (e.g., by physical contact with the target molecule) or indirectly (e.g., by interaction with one or more additional molecular components such as mitochondrial molecular components present in a sample, where such additional components alter target activity in response to contact with the agent).
  • candidate agents are provided as "libraries” or collections of compounds, compositions or molecules.
  • Such molecules typically include compounds known in the art as "small molecules” and having molecular weights less than 10 5 daltons, preferably less than IO 4 daltons and still more preferably less than 10 3 daltons.
  • members of a library of test compounds can be administered to a plurality of samples, each containing at least one CSP polypeptide as provided herein (e.g., a p95.6/YN52 polypeptide or homolog), and then assayed for their ability to bind to the CSP or to enhance or inhibit CSP activity.
  • candidate agents may be screened on a biological sample containing cells having mitochondria that comprise a recombinant p95.6/YN52 polypeptide, for their ability to alter a mitochondrial activity such as calcium transport, mitochondrial membrane potential, ETC activity or response to apoptogenic stimuli.
  • Compounds so identified as capable of influencing mitochondrial function are valuable for therapeutic and/or diagnostic pu ⁇ oses, since they permit treatment and/or detection of diseases associated with activity of a p95.6/YN52 polypeptide or homolog.
  • Such compounds are also valuable in research directed to molecular signaling mechanisms that involve p95.6/YN52, and to refinements in the discovery and development of future p95.6/YN52-specific compounds exhibiting greater specificity.
  • Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels.
  • various starting compounds may be prepared employing one or more of solid- phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions.
  • the resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694 and PCT/US91/04666) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. 5,798,035, U.S. 5,789,172, U.S. 5,751,629).
  • a diverse assortment of such libraries may be prepared according to established procedures, and tested using a CSP target such as a p95.6/YN52 polypeptide or homolog according to the present disclosure.
  • the C. elegans p95.6/YN52 ligand compounds are preferably part of a pharmaceutical composition when used in the methods of the present invention.
  • the pharmaceutical composition will include at least one of a pharmaceutically acceptable carrier, diluent or excipient, in addition to one or more C. elegans p95.6/YN52 ligands and, optionally, other components.
  • “Pharmaceutically acceptable earners” for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985).
  • sterile saline and phosphate-buffered saline at physiological pH may be used.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of -hydroxybenzoic acid may be added as preservatives. Id. at 1449.
  • antioxidants and suspending agents may be used. Id.
  • “Pharmaceutically acceptable salt” refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts).
  • the compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.
  • compositions that contain one or more C. elegans p95.6/YN52 substrates/ ligands compounds may be in any form which allows for the composition to be administered to a patient.
  • the composition may be in the form of a solid, liquid or gas (aerosol).
  • routes of administration include, without limitation, oral, topical, parenteral (e.g., sublingually or buccally), sublingual, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrameatal, intraurethral injection or infusion techniques.
  • compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.
  • an excipient and/or binder may be present.
  • examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose.
  • Coloring and/or flavoring agents may be present.
  • a coating shell may be employed.
  • the composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • prefened compositions contain, in addition to one or more C. elegans p95.6/YN52 ligand compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • a liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a prefened adjuvant.
  • An injectable pharmaceutical composition is preferably sterile.
  • a liquid composition intended for either parenteral or oral administration should contain an amount of C. elegans p95.6/YN52 ligand compound such that a suitable dosage will be obtained. Typically, this amount is at least 0.01 wt% of an C. elegans p95.6/YN52 ligand compound in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition.
  • Prefened oral compositions contain between about 4% and about 50% of C. elegans p95.6/YN52 ligand compound(s).
  • Prefened compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of active compound.
  • the pharmaceutical composition may be intended for topical administration, in which case the canier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the C. elegans p95.6/YN52 ligand compound of from about 0.1 to about 10% w/v (weight per unit volume).
  • the composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable noninitating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the C. elegans p95.6/YN52 ligand compound(s) may be administered through use of insert(s), bead(s), timed-release formulation(s), patch(es) or fast-release formulation(s).
  • the optimal dosage of the C. elegans p95.6/YN52 ligands compound(s) may depend on the weight and physical condition of the patient; on the severity and longevity of the physical condition being treated; on the particular form of the active ingredient, the manner of administration and the composition employed. It is to be understood that use of a C. elegans p95.6/YN52 ligand compound in a chemotherapy can involve such a compound being bound to an agent, for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound.
  • an agent for example, a monoclonal or polyclonal antibody, a protein or a liposome, which assist the delivery of said compound.
  • C. elegans p95.6/YN52 a mitochondrial calcium-binding protein
  • a calcium-binding domain (motif) HMM was created under steps 204-208 (or extracted from a previously created HMM in step 202). The HMM was used to query the GenBank database to obtain a hit list under steps 210 and 212.
  • the routine 200 retrieved conesponding sequences and employed PSORT to identify those sequences estimated to have mitochondrial localization.
  • the routine 200 eliminated sequences that, have similar homologs in yeast (S. cerevisiae).
  • routine 200 in step 220 eliminated sequences having a dihydropyridine-binding domain (motif), and the routine 200 checked for full-length protein sequences under step 222.
  • a dihydropyridine-binding domain motif
  • FIG. 12 an approximately 2.2 Mb genomic DNA sequence from chromosome III of the nematode C. elegans (Accession No. P34586) was identified in the GenBank database.
  • the DNA sequence (Fig. 12, SEQ ID NO:l) contained a coding region for p95.6/YN52, a polypeptide having 823 amino acids (Fig. 12, SEQ ID NO:2) and a predicted molecular weight of 95.6 kDa.
  • TRP transient receptor potential
  • a human serine/threonine phosphatase having a calcium- binding functional domain is identified using a computer system as described herein.
  • a 653 amino acid polypeptide having a predicted molecular weight of approximately 75 kDa was identified in the NCBI database (www.ncbi.nlm.nih.gov/), conesponding to a human protein known as PP7 (Huang et al., 1998 J. Biol. Chem. 273:1462) or as PPEF-1 (Sherman et al., 1997 Proc. Nat Acad. Sci. USA 94:11639). Analysis of the amino acid sequence for this polypeptide (GenBank Accession Nos.
  • AAC05825, AAB82795) reveals this protein to be a serine/threonine phosphatase possessing a characteristic phosphatase catalytic core domain that has high homology to the catalytic regions of other serine/threonine phosphatases PP1, PP2A, PP2B, PP4, PP5 and PP6.
  • the N- and C-terminal regions of PP7 are distinct from other known phosphatases, however, and the C-terminal domain includes multiple EF-hand motif calcium-binding sites.
  • the amino acid sequence of PP7 contains a mitochondrial localization sequence and also includes an N-terminal R2 cleavage site (Gavel et al., 1990 Prot. Engineering 4:33) between the amino acids at positions 22 and 23.
  • EXAMPLE 3 CLONING OF P95.6/YN52 FROM A C ELEGANS CDNA LIBRARY
  • This example describes molecular cloning of DNA sequences encoding the C. elegans p95.6/YN52 polypeptide identified in Example 1.
  • Two overlapping PCR products were generated, conesponding to nucleic acid sequences encoding the amino terminal portion and the carboxy terminal portion of the protein, respectively, using standard PCR protocols and the Stratagene (La Jolla, CA) C. elegans UniZAP XRTM cDNA Library as a template.
  • References to nucleotide position numbers use the initiation codon (ATG) encoding methionine at the 5' end of the coding sequence for p95.6/YN52 as nucleotides 1-3.
  • oligonucleotide primers used for amplifying the N-terminal fragment were:
  • SEQ ID NO:5 which, going from 5' to 3', contained a Sail restriction site (underlined), a Kozak consensus sequence and a start sequence encoding initiating methionine and conesponding to nucleotides 1-25 of C. elegans cDNA encoding p95.6/YN52 (double- underlined); and
  • the PCR cDNA fragments were digested with the following restriction enzymes according to standard procedures: N-terminal fragment (Sail and either Acll, BamHl, or BstXI) and C-terminal fragment (Bglll and either Acll, BamHI or BstXI, respectively).
  • N-terminal fragment Sail and either Acll, BamHl, or BstXI
  • C-terminal fragment Bglll and either Acll, BamHI or BstXI, respectively.
  • the N-terminal and C-terminal restriction fragments were ligated together into Xhol- and BamHI-digested pCDNA3.1 (Invitrogen, Carlsbad, CA), a mammalian expression vector with a CMV promoter and selectable markers for eukaryotic and bacterial cells (i.e., genes encoding neomycin and ampicillin resistance).
  • the ligation mixture was transformed into competent E. coli cells and plated under conditions selective for ampicillin resistant colonies. Plasmids were rescued from six bacterial colonies grown on the selection plates and screened for the recombinant cDNA by PCR amplification of plasmid DNA with either of the primer pairs (SEQ ID NOS:5 and 6; or SEQ ID NOS:7 and 8) used to generate the C-terminal and N-terminal fragments of the p95.6 gene, as described above in this Example.
  • the primer pairs SEQ ID NOS:5 and 6; or SEQ ID NOS:7 and 8
  • a point mutation in the N-terminal portion was detected at nucleotide position number 800 in only one of the six clones, relative to SEQ ID NO:l.
  • This mutation was determined to have arisen as an artifact of PCR amplification and was conected based on SEQ ID NO:l using a QuickChangeTM (Stratagene, La Jolla, CA) site-directed mutagenesis kit, according to the manufacturer's instructions. Sequence analysis of the six clones permitted the identification in each one of the p95.6 DNA sequence shown in Fig. 15 (SEQ ID NO: 19), which includes a continuous open reading frame encoding the amino acid sequence shown in Fig. 16 (SEQ ID NO:20).
  • SEQ ID NO: 19 Comparison of SEQ ID NO: 19 to SEQ ID NO:l revealed a 75 nucleotide in-frame deletion in SEQ ID NO: 19; i.e., SEQ ID NO: 19 was identical to SEQ ID NO:l except for the absence from SEQ ID NO: 19 of nucleotides 142-216 of SEQ ID NOT . Accordingly, a conesponding peptide of 25 amino acids (residues 48-72) that is present in SEQ ID NO:2 was absent from the translated p95.6 polypeptide product encoded by SEQ ID NO: 19, as shown in Figure 16 (SEQ ID NO:20).
  • Plasmids containing the C. elegans sequence of interest were used to transiently transfect mammalian COS-1 cells (ATCC, Manassas, VA) to assess intracellular localization of the recombinant protein by performing immunofluorescence analysis with an antibody against the FLAGTM epitope tag (Sigma, St. Louis, MO) according to the supplier's recommendations.
  • Such antibodies are commercially available and were obtained from BabCo (Berkley Antibody Company, Richmond, CA), and Zymed Laboratories Inc. (South San Francisco, CA).
  • the PCR fragments digested with restriction enzymes as described above are also ligated into a yeast expression vector, such as pYADH2 (Brunelli et al., Yeast 9:1299-1308, 1993), which has the inducible ADH2 (alcohol dehydrogenase II) promoter (glucose repressible and strongly transcribed on non-fermentable carbon sources, not inducible by galactose) and T ⁇ /Amp selectable markers.
  • the ligation mixture is transfected and screened as above. Plasmids containing the cDNA of interest are used to transfect yeast, which are selected on media without the amino acid tryptophan. Positive colonies are used in functional assays to assess calcium uniporter activity.
  • PCR-amplified C. elegans p95.6/YN52 encoding sequences are shown to hybridize to human polyA+ RNA from various tissue sources. Overlapping PCR products conesponding to the nucleic acid sequences encoding the amino and carboxy termini of the C.
  • elegans p95.6/YN52 polypeptide identified in Example 1 were generated for use as hybridization probes, as described in Example 3 using the primer pairs (i) Unil for [SEQ ID NO:5] and Unil crev [SEQ ID NO:6] and (ii) Uni efor [SEQ ID NO:7] and Unilrev [SEQ ID NO:8], except that primers were 32 P end-labeled as described in Ausubel et al. (1998 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA).
  • Probes were hybridized to a blot containing human polyA+ RNA derived from multiple human tissues (#7760-1, Clontech, Inc., Palo Alto, CA) in Express HybTM solution (Clontech) for 1 hour at 37°C. The blot was then washed for 15 minutes twice in 2X SSC, 0.1% SDS at room temperature and twice in the same buffer at 37°C, and then exposed to XomatTM autoradiographic film (Kodak, Rochester, NY) for 3 hours. The blot was next washed twice for 15 minutes in IX SSC, 0.1% SDS at 37°C and twice in the same buffer at 40°C, and again exposed to autoradiographic film.
  • Example 3 p95.6/YN52 cDNA was cloned following ligation of two distinct but overlapping PCR products, one encoding the N-terminal portion and the other encoding the C-terminal portion of the p95.6/YN52 polypeptide having the amino acid sequence of SEQ ID NO:20.
  • This example describes molecular cloning of a single cDNA [SEQ ID NO: 19] encoding full length p95.6/YN52 [SEQ ID NO:20]. With the C.
  • the resulting PCR product which lacks the putative TGA stop codon of p95.6/YN52, and which includes the FLAG epitope tag as a fusion peptide at the p95.6 C-terminus, is digested with Sail and Bglll and purified. The purified fragment is then ligated into Xhol- and BamHI-digested pCDNA3.1 (Invitrogen, Carlsbad, CA) to produce a construct with which competent E. coli cells are transformed. Selection, characterization and transfection are as described in Example 3.

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Abstract

La présente invention concerne des séquences de protéines mitochondriales spécifiques liant, transportant et/ou régulant les ions calcium intracellulaires. L'invention permet d'identifier ces séquences d'acides aminés et les séquences de nucléotides les codant dans des bases de données de séquences biologiques ne comportant aucune description ni de fonction ni de localisation sub-cellulaire. A cet effet, on a eu recours à un système informatique et à une technique d'interrogation de base de données tenant compte de multiples paramètres préférés. L'invention concerne également la production et l'utilisation de ces séquences dans des constructions d'expressions de recombinaison, des polypeptides, des cellules hôtes, des essais de recherche systématique de médicaments, ainsi que les compositions et procédés s'y rapportant. Pour l'identification de telles séquences, l'invention porte notamment sur un moteur automatisé d'extraction de motifs qui reçoit et crée un motif, tel qu'un modèle de Markov caché. Sur la base de ce modèle de Markov caché, on recherche dans une base de données de séquences telle que la GenBank des numéros d'identification tels que les numéros d'accession, et on les extrait. On extrait également les protéines et séquences de polypeptides associés aux numéros d'accession et on en filtre les séquences souhaitées d'après des paramètres spécifiques tels que l'espèce d'origine, ou la conformité avec un ou plusieurs algorithmes, notamment le PSORT ou le domaine de ciblage mitochondrial. Les données résultant du filtrage sont fournies sous forme de tableaux, ou sous forme d'entrée dans une base de données en vue d'interrogations ultérieures.
PCT/US2000/029787 1999-10-26 2000-10-26 Sequences geniques identifiees par recherche de motif de proteines dans une base de donnees Ceased WO2001030830A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002816A3 (fr) * 2000-06-29 2003-01-09 Mitokor Procedes d'identification de transporteurs de cations divalents mitochondriaux
WO2003019417A1 (fr) * 2001-08-29 2003-03-06 Bioinfomatix Inc. Systeme et procede d'analyse du proteome et gestion de donnees
EP1450845A4 (fr) * 2001-12-04 2005-07-06 Millennium Pharm Inc 15603, membre de la famille des canaux ioniques humains
CN108491692A (zh) * 2018-03-09 2018-09-04 中国科学院生态环境研究中心 一种构建抗生素抗性基因数据库的方法
CN116130004A (zh) * 2023-01-06 2023-05-16 成都侣康科技有限公司 一种抗菌肽的鉴定处理方法和系统

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CN111243679B (zh) * 2020-01-15 2023-03-31 重庆邮电大学 微生物群落物种多样性数据的存储检索方法

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NZ507063A (en) * 1998-04-02 2003-11-28 Rigel Pharmaceuticals Inc Self-dimerising peptides causing the formation of compact structures

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002816A3 (fr) * 2000-06-29 2003-01-09 Mitokor Procedes d'identification de transporteurs de cations divalents mitochondriaux
WO2003019417A1 (fr) * 2001-08-29 2003-03-06 Bioinfomatix Inc. Systeme et procede d'analyse du proteome et gestion de donnees
EP1450845A4 (fr) * 2001-12-04 2005-07-06 Millennium Pharm Inc 15603, membre de la famille des canaux ioniques humains
CN108491692A (zh) * 2018-03-09 2018-09-04 中国科学院生态环境研究中心 一种构建抗生素抗性基因数据库的方法
CN108491692B (zh) * 2018-03-09 2023-07-21 中国科学院生态环境研究中心 一种构建抗生素抗性基因数据库的方法
CN116130004A (zh) * 2023-01-06 2023-05-16 成都侣康科技有限公司 一种抗菌肽的鉴定处理方法和系统
CN116130004B (zh) * 2023-01-06 2024-05-24 成都侣康科技有限公司 一种抗菌肽的鉴定处理方法和系统

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