WO2004019880A2 - Proteines interagissant avec aw755252 et leur utilisation - Google Patents

Proteines interagissant avec aw755252 et leur utilisation Download PDF

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WO2004019880A2
WO2004019880A2 PCT/US2003/026997 US0326997W WO2004019880A2 WO 2004019880 A2 WO2004019880 A2 WO 2004019880A2 US 0326997 W US0326997 W US 0326997W WO 2004019880 A2 WO2004019880 A2 WO 2004019880A2
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protein
sequence
amino acid
set forth
acid sequence
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WO2004019880A3 (fr
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Chuck Hensel
Takeshi Sakamoto
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TADEKA CHEMICAL INDUSTRIES Ltd
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TADEKA CHEMICAL INDUSTRIES Ltd
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention generally relates to protein-protein interactions, particularly to protein complexes formed by protein-protein interactions and methods of use thereof.
  • phenotypic effects resulting from the disruption of expression of a particular gene can shed some light on the functions of the gene.
  • the techniques involved are complex and the time required for a phenotype to appear can be long, especially in animals.
  • disruption of a particular gene may not cause any detectable phenotypic effect.
  • test protein Because most proteins function through their interactions with other proteins, if a test protein interacts with a known protein, one can reasonably predict that the test protein is associated with the functions of the known protein, e.g., in the same cellular structure or same cellular process as the known protein. Thus, interaction partners can provide an immediate and reliable understanding towards the functions of the interacting proteins. By identifying interacting proteins, a better understanding of disease pathways and the cellular processes that result in diseases may be achieved, and important regulators and potential drug targets in disease pathways can be
  • a popular approach useful in large-scale screening is the phage display method, in which filamentous bacteriophage particles are made by recombinant DNA technologies to express a peptide or protein of interest fused to a capsid or coat protein of the bacteriophage.
  • a whole library of peptides or proteins of interest can be expressed and a bait protein can be used to screening the library to identify peptides or proteins capable of binding to the bait protein.
  • a bait protein can be used to screening the library to identify peptides or proteins capable of binding to the bait protein.
  • the phage display method only identifies those proteins capable of interacting in an in vitro environment, while the coimmunoprecipitation and copurification methods are not amenable to high throughput screening.
  • the yeast two-hybrid system is a genetic method that overcomes certain shortcomings of the above approaches.
  • the yeast two-hybrid system has proven to be a powerful method for the discovery of specific protein interactions in vivo. See generally, Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, NY, 1997.
  • the yeast two-hybrid technique is based on the fact that the DNA-binding domain and the transcriptional activation domain of a transcriptional activator contained in different fusion proteins can still activate gene transcription when they are brought into proximity to each other. In a yeast two-hybrid system, two fusion proteins are expressed in yeast cells.
  • One has a DNA-binding domain of a transcriptional activator fused to a test protein.
  • the other includes a transcriptional activating domain of the transcriptional activator fused to another test protein. If the two test proteins interact with each other in vivo, the two domains of the transcriptional activator are brought together reconstituting the transcriptional activator and activating a reporter gene controlled by the transcriptional activator. See, e.g., U.S. Patent No. 5,283,173.
  • yeast cells are eukaryotic cells.
  • the interactions between mammalian proteins detected in the yeast two-hybrid system typically are bona fide interactions that occur in mammalian cells under physiological conditions.
  • numerous mammalian protein-protein interactions have been identified using the yeast two-hybrid system.
  • the identified proteins have contributed significantly to the understanding of many signal transduction pathways and other biological processes.
  • the yeast two-hybrid system has been successfully employed in identifying a large number of novel mammalian cell cycle regulators that are important in complex cell cycle regulations.
  • the inventor of the present invention has discovered using the yeast two-hybrid system that AW755252 specifically interacts with GROUPl .
  • the specific interactions between these proteins and AW755252 suggest that AW755252 and the AW755252-interacting proteins may be involved in the same biological processes.
  • the interactions between such AW755252-interacting proteins and AW755252 may lead to the formation of protein complexes both in vitro and in vivo, which contain AW755252 and one or more of the AW755252-interacting proteins.
  • the protein complexes formed under physiological conditions may mediate the functions and biological activities of AW755252 and GROUPl proteins. For example, they are believed to be involved in cardiac function.
  • the AW755252-mteracting proteins and the protein complexes are potential drug targets for the development of drugs useful in treating or preventing diseases and disorders associated with the AW755252-containing protein complexes or a protein member thereof, or with abnormalcardiac function.
  • isolated protein complexes comprising AW755252 and one or more AW755252-interacting proteins selected from the group consisting of GROUPl .
  • homologues, derivatives, and fragments of AW755252 and of the AW755252-interacting proteins may also be used in forming protein complexes.
  • fragments of AW755252 and the AW755252-interacting proteins corresponding to the protein domains responsible for the interaction between AW755252 and the AW755252-mteracting proteins are used in forming a protein complex of the present invention.
  • a protein complex is provided from a hybrid protein, which comprises AW755252 or a homologue, derivative, or fragment thereof covalently linked, directly or through a linker, to a AW755252-interacting protein selected from the group consisting of GROUPl or a homologue, derivative, or fragment thereof.
  • the protem complexes can be prepared by isolation or purification from tissues and cells or produced by recombinant expression of their protein members.
  • the protein complexes can be incorporated into a protein microchip or microarray, which are useful in large-scale high throughput screening assays involving the protein complexes.
  • antibodies are provided which are immunoreactive with a protein complex of the present invention.
  • an antibody is selectively immunoreactive with a protein complex of the present invention.
  • a bifunctional antibody is provided which has two different antigen binding sites, each being specific to a different interacting protein member in a protein complex of the present invention.
  • the antibodies of the present invention can take various forms including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, antibody fragments such as Fv fragments, single-chain Fv fragments (scFv), Fab' fragments, and F(ab')2 fragments.
  • the antibodies are partially or fully humanized antibodies.
  • the antibodies of the present invention can be readily prepared using procedures generally known in the art.
  • recombinant libraries such as phage display libraries and ribosome display libraries may be used to screen for antibodies with desirable specificities.
  • various mutagenesis techniques such as site-directed mutagenesis and PCR diversification may be used in combination with the screening assays.
  • the present invention also provides detection methods for determining whether there is any aberration in a patient with respect to a protem complex having AW755252 and one or more AW755252-interacting protein selected from the group consisting of GROUPl .
  • the method comprises detecting an aberrant level of the protein complexes of the present invention.
  • the levels of one or more interacting protein members (at protein or cDNA or RNA level) of a protein complex of the present invention are measured.
  • the cellular localization, or tissue or organ distribution of a protein complex of the present invention is determined to detect any aberrant localization or distribution of the protein complex.
  • mutations in one or more interacting protein members of a protein complex of the present invention can be detected.
  • kits may be used for conducting the detection methods of the present invention.
  • the kit contains reagents useful in any of the above-described embodiments of the detection methods, including, e.g., antibodies specific to a protein complex of the present invention or interacting members thereof, and oligonucleotides selectively hybridizable to the cDNAs or mRNAs encoding one or more interacting protein members of a protein complex.
  • the detection methods may be useful in diagnosing a disease or disorder such as ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris, staging the disease or disorder, and identifying a predisposition to the disease or disorder.
  • the present invention also provides screening methods for selecting modulators of a protein complex formed between AW755252 or a homologue, derivative or fragment thereof and an AW755252-mteracting protein selected from the group consisting of GROUPl or a homologue, derivative, or fragment thereof. Screen methods are also provided for selecting modulators of an
  • AW755252-interacting protein selected from the group consisting of GROUPl .
  • the compounds identified in the screening methods of the present invention can be used in modulating the functions or activities of AW755252, the AW755252-interacting proteins, or the protein complexes of the present invention. They may also be effective in modulating the cellular functions involving AW755252, AW755252-interacting proteins or AW755252-containing protein complexes, and in preventing or ameliorating diseases or disorders such as ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris.
  • diseases or disorders such as ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris.
  • test compounds may be screened in an in vitro binding assay to identify compounds capable of binding a protein complex of the present invention or AW755252 or a AW755252-interacting protein identified in accordance with the present invention or a homologue, derivative or fragment thereof.
  • in vitro dissociation assays may also be employed to select compounds capable of dissociating the protein complexes identified in accordance with the present invention.
  • An in vitro screening assay may also be used to identify compounds that trigger or initiate the formation of, or stabilize, a protein complex of the present invention.
  • methods are provided for modulating the functions and activities of a AW755252-containing protein complex of the present invention, or interacting protein members thereof.
  • the methods may be used in treating or preventing diseases and disorders such as ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris.
  • the methods comprise reducing the protein complex level and/or inhibiting the functional activities of the protein complex.
  • the level and/or activity of AW755252 or one of the AW755252-mteracting proteins may be inhibited.
  • the method for modulating the functions and activities of a AW755252-containing protein complex of the present invention or interacting protein members thereof comprise increasing the protein complex level and/or activating the functional activities of the protein complex.
  • the level and/or activity of one of the AW755252-interactmg proteins or AW755252 may be increased.
  • a particular AW755252-containing protein complex, AW755252 or a AW755252-interacting protein of the present invention may be administered directly to a patient.
  • exogenous genes encoding one or more protein members of an AW755252-containing protein complex may be introduced into a patient by gene therapy techniques.
  • a patient needing treatment or prevention may also be administered with compounds identified in a screening assay of the present invention capable of triggering or initiating, enhancing or stabilizing protein-protein interactions between AW755252 or a homologue, derivative or fragment thereof and a AW755252-interactmg protein selected from the group consisting of GROUPl, or a homologue, derivative or fragment thereof.
  • GROUPl used herein means AW755252-interactmg proteins including mFHL2, mPN34854, mPRPHl, mTCTEX-1, ACTN2, mACTN4 and mMRJ, which have been identified using yeast two-hybrid system in the present invention.
  • PROTEIN2 used herein means any one of proteins in GROUPl .
  • polypeptide polypeptide
  • protein protein
  • peptide polypeptide
  • peptide polypeptide
  • protein protein
  • peptide polypeptide
  • peptide also encompass various modified forms thereof, including but not limited to glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, etc.
  • interacting means that two protein domains or complete proteins exhibit sufficient physical affinity to each other so as to bring the two "interacting" protein domains or proteins physically close to each other.
  • An extreme case of interaction is the formation of a chemical bond that results in continual and stable proximity of the two domains.
  • Interactions that are based solely on physical affinities, although usually more dynamic than chemically bonded interactions, can be equally effective in co-localizing two proteins. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, Nander-waals force, ionic force, covalent linkages, and combinations thereof.
  • the state of proximity between the interacting domains or entities may be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities.
  • an "interaction" is exhibited by the binding between the interacting domains or entities. Examples of interactions include specific interactions between antigen and antibody, ligand and receptor, enzyme and substrate, and the like.
  • an “interaction" between two protein domains or complete proteins can be determined by a number of methods. For example, an interaction can be determined by functional assays such as the two-hybrid systems. Protein-protein interactions can also be determined by various biochemical approaches based on the affinity binding between the two interacting partners. Such biochemical methods generally known in the art include, but are not limited to, protein affinity chromatography, affinity blotting, immunoprecipitation, and the like. The binding constant for two interacting proteins, which reflects the strength or quality of the interaction, can also be determined using methods known in the art. See Phizicky and Fields, Microbiol. Rev., 59:94-123 (1995).
  • protein complex means a composite unit that is a combination of two or more proteins formed by interaction between the proteins.
  • a "protein complex” is formed by the binding of two or more proteins together through specific non-covalent binding affinities.
  • covalent bonds may also be present between the interacting partners.
  • the two interacting partners can be covalently crosslinked so that the protein complex becomes more stable.
  • Isolated refers to that altered by the hand of a human being from its natural state, i.e., it has been altered outside of its natural environment or removed from its original environment, or both. "Isolated” can refer to, without limitation, isolated host cells, isolated polynucleotides or isolated polypeptides.
  • a polynucleotide or a polypeptide naturally present in a living organism is not isolated, but the same polynucleotide or polypeptide substantially separated from the coexisting materials of its natural state is isolated.
  • a polynucleotide or a polynucleotide encoding a polypeptide, which polynucleotide is introduced into a cell (e.g., a bacterial cell) or an organism by transformation, genetic manipulation or by any other recombinant method is isolated even if it is still present in the cell or organism, which cell or organism may be naturally occurring.
  • nucleic acids which include gene sequences
  • isolated nucleic acid when used in reference to nucleic acids (which include gene sequences) of this invention is intended to mean that a nucleic acid molecule is present in a form other than found in nature in its original environment with respect to its association with other molecules.
  • an "isolated nucleic acid” as used herein means a nucleic acid molecule having only a portion of the nucleic acid sequence in the chromosome but not one or more other portions present on the same chromosome.
  • an isolated gene typically includes no more than 50 kb, preferably no more than 25 kb, more preferably no more than 10 kb naturally occurring nucleic acid sequence which immediately flanks the gene in the naturally existing chromosome or genomic DNA.
  • an "isolated nucleic acid" as used herein is distinct from a clone in a conventional library such as genomic DNA library and cDNA library in that the clones in a library is still in admixture with almost all the other nucleic acids in a chromosome or a cell.
  • An isolated nucleic acid can be in a vector.
  • An isolated nucleic acid can also be part of a composition so long as the composition is substantially different from the nucleic acid's original natural environment.
  • an isolated nucleic acid can be in a semi-purified state, i.e., in a composition having certain natural cellular components, while it is substantially separated from other naturally occurring nucleic acids and can be readily detected and/or assayed by standard molecular biology techniques.
  • an "isolated nucleic acid" is separated from at least 50%, more preferably at least 75%, most preferably at least 90% of other naturally occurring nucleic acids.
  • isolated nucleic acid embraces "purified nucleic acid” which means a specified nucleic acid is in a substantially homogenous preparation of nucleic acid substantially free of other cellular components, other nucleic acids, viral materials, or culture medium, or chemical precursors or by-products associated with chemical reactions for chemical synthesis of nucleic acids.
  • a “purified nucleic acid” can be obtained by standard nucleic acid purification methods.
  • the specified nucleic acid molecule constitutes at least 75%, preferably at least 85, and more preferably at least 95 percent of the total nucleic acids in the preparation.
  • purified nucleic acid also means nucleic acids prepared from a recombinant host cell (in which the nucleic acids have been recornbinantly amplified and/or expressed) or chemically synthesized nucleic acids.
  • isolated nucleic acid also encompasses "recombinant nucleic acid” which is used herein to mean a hybrid nucleic acid produced by recombinant DNA technology having the specified nucleic acid molecule covalently linked to one or more nucleic acid molecules that are not the nucleic acids naturally flanking the specified nucleic acid.
  • nucleic acid molecules flanking the specified nucleic acid are no more than 50 kb, preferably no more than 25 kb.
  • isolated polypeptide as used herein means a polypeptide molecule is present in a form other than found in nature in its original environment with respect to its association with other molecules. Typically, an “isolated polypeptide” is separated from at least 50%, more preferably at least 75%, most preferably at least
  • isolated polypeptide encompasses a "purified polypeptide" which is used herein to mean a specified polypeptide is in a substantially homogenous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or when the polypeptide is chemically synthesized, chemical precursors or by-products associated with the chemical synthesis.
  • the specified polypeptide molecule constitutes at least 75%>, preferably at least 85, and more preferably at least 95 percent of the total polypeptide in the preparation.
  • a "purified polypeptide” can be obtained from natural or recombinant host cells by standard purification techniques, or by chemically synthesis.
  • isolated polypeptide also encompasses a "recombinant polypeptide” which is used herein to mean a hybrid polypeptide produced by recombinant DNA technology or chemical synthesis having a specified polypeptide molecule covalently linked to one or more polypeptide molecules which do not naturally flank the specified polypeptide.
  • An “isolated protein complex” may also be a "purified protein complex", that is, a substantially purified form in a substantially homogenous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or when the protein components in the protein complex are chemically synthesized, chemical precursors or by-products associated with the chemical synthesis.
  • a “purified protein complex” typically means a preparation containing preferably at least 15%, more preferably at least 85%, and most preferably at least 95%) a particular protein complex.
  • a “purified protein complex” may be obtained from natural or recombinant host cells or other body samples by standard purification techniques, or by chemical synthesis.
  • hybrid protein means a non-naturally occurring protein having a specified polypeptide molecule covalently linked to one or more polypeptide molecules which do not naturally link to the specified polypeptide.
  • a “hybrid protein” may be two naturally occurring proteins or fragments thereof linked together by a covalent linkage.
  • a “hybrid protein” may also be a protein formed by covalently linking two artificial polypeptides together. Typically but not necessarily, the two or more polypeptide molecules are linked or "fused” together by a peptide bond forming a single non-branched polypeptide chain.
  • antibody encompasses both monoclonal and polyclonal antibodies that fall within any antibody classes, e.g., IgG, IgM, IgA, or derivatives thereof.
  • antibody also includes antibody fragments including, but not limited to, Fab, F(ab') 2 , and conjugates of such fragments, and single-chain antibodies comprising an antigen recognition epitope.
  • antibody also means humanized antibodies, including partially or fully humanized antibodies. An antibody may be obtained from an animal, or from a hybridoma cell line producing a monoclonal antibody, or obtained from cells or libraries recornbinantly expressing a gene encoding a particular antibody.
  • selective immunoreactive means that an antibody is reactive thus binds to a specific protein or protein complex, but not other similar proteins or fragments or components thereof.
  • compound encompasses all types of organic or inorganic molecules, including but not limited proteins, peptides, polysaccharides, lipids, nucleic acids, small organic molecules, inorganic compounds, and derivatives thereof.
  • small molecule refers to acids (for example acetic acid, salicylic acid, ascorbic acid) bases, formamide, amino acids and their derivatives (for example protoheme, cytochrome heme) inorganic molecules (for example phosphoric acid), acetycholine, sugars, prosthetic groups, cofactors and inhibitors (for example, Flavin adenine dinucleotide, riboflavin, NAD, NDP + , NADPH, folic acid, methotrexate) aspirin, palmitic acid, caffeine, beta-mercaptoethanol, urea, minerals or vitamins.
  • acids for example acetic acid, salicylic acid, ascorbic acid
  • formamide amino acids and their derivatives (for example protoheme, cytochrome heme) inorganic molecules (for example phosphoric acid), acetycholine, sugars, prosthetic groups, cofactors and inhibitors (for example, Flavin adenine dinucleotide, riboflavin,
  • sequences for some or all of the interacting proteins in this disclosure are not novel and are available in public databases such as GenBank. See, Tables 1 and 3 for The GenBank Accession Nos.
  • the start and end numbers of the bait and prey fragments indicated in Tables 1-3 are based on the sequences of the corresponding full-length proteins known to one skilled in the art or the corresponding novel proteins of the present invention. These protem sequences are provided in the Figures presented herein.
  • the cDNA encoding AW755252 was isolated from a cardiac muscle cDNA expression library by Incyte Genomics, Inc. (GenBank accession number AW755252). Northern blot analysis demonstrates detectable AW755252 mRNA only in heart and skeletal muscle (Fig.1 ).
  • the amino acid sequence of AW755252 (SEQ ID NO: 1-5 in Table2 and SEQ ID NO: 10 in Figure 2) was deduced from the nucleotide sequence in GenBank accession number AW755252 in which only the nucleotide sequence information is available.
  • the amino acid sequence of AW755252 (SEQ ID NO: 10 in Figure 2) corresponds to that of human 187-2B protein (c.f, SEQ ID NO: 34 of
  • PCT/JP03/08666 shown in PCT/JP03/08666 filed on July 8, 2003.
  • Preferred example of the fragments of AW755252 includes a fragment consisting of amino acids 245 to 527 (of 527 total amino acids) of AW755252, which corresponds to that of human 187-2A protem (c.f, SEQ ID NO: 33 of PCT/JP03/08666) shown in PCT/JP03/08666 filed on July 8, 2003.
  • Structural analysis suggests the presence of a LIM domain (amino acids 1 to 49) and no other significant features. LIM domains are zinc-binding domains that are thought to mediate protein-protein interactions (Freyd et al., Nature.
  • AW755252 interacts with mFHL2.
  • a bait comprising amino acids 240 to 398 (of 527 total amino acids) of
  • AW755252 selected a single clone from a mouse embryo activation domain library encoding amino acids 100 to 235 (of 279 total amino acids) of mFHL2 (NM_010212).
  • the prey fragment of mFHL2 comprises amino acids 100 to 235
  • the sequence having a truncation of up to 99 amino acids at N-terminus and/or up to 44 (which is obtained by subtracting 235 from 279, the total amino acids number of mFHL2) amino acids at the C-terminus of the mFHL2 sequence set forth in Figure 3 does not render it unable to interact with AW755252.
  • mFHL2 also is known as skeletal muscle LIM-protein 3, SLIM 3, LIM-domain protein DRAL, four and a half LIM domains protein 2, and FHL-2.
  • the cDNA encoding mFHL2 was isolated based on its similarity to other LIM-domain containing proteins (Morgan and Madgwick, Biochem Biophys Res Commun. 1999 255(2):245-50).
  • the mRNA encoding mFHL2 is expressed primarily in heart tissue in mice (Morgan and Madgwick, Biochem Biophys Res Commun. 1999 255(2):245-50) similar to the pattern of expression of human FHL2 (DRAL) (NM_001450) (Chan et al, Gene. 1998 210(2):345-50).
  • LIM domains in addition to mediating protein-protein interactions, also are found in proteins that have a role in the nucleus, suggesting a possible nucleic acid binding function (Sanchez-Garcia and Rabbitts, Trends Genet. 1994 10(9):315-20.).
  • mFHL2 is a member of a family of proteins, the four and one half LIM domain family that, as its name implies, contains four and one half LIM domains as the only definable feature.
  • These proteins initially were thought to be involved primarily in cytoskeletal roles in the cytoplasm (Li et al, Cell Motil Cytoskeleton. 2001 48(l):ll-23). However, they were shown more recently to play a role in the nucleus as co-activators of transcription for the androgen receptor (Muller et al, EMBOJ. 2000 19(3):359-69, Muller et al, EMBOJ.
  • AW755252 interacts with a novel protein mPN34854 similar to KIAA0657.
  • a bait comprising amino acids 397 to 527 (of 527 total amino acids) of AW755252 selected a single clone from a mouse embryo activation domain library- spanning amino acids 1 to 486 (of 486 total amino acids) of a novel protein similar to KIAA0657.
  • the fragments of the interacting bait and prey should contain the minimal binding domain of each protein. Since the bait fragment of AW755252 comprises amino acids 397 to 527, the sequence having a truncation of up to 396 amino acids at
  • N-terminus of the AW755252 sequence set forth in Figure 2 does not render it unable to interact with mPN34854.
  • mRNA encoding the presumed human ortholog of KIAA0657 (AB014577) is expressed at highest levels in heart tissue, with lower levels in all other tissues examined (Ishikawa et al, DNA Res. 1998 5(3): 169-76.).
  • Structural analysis of mPN34854 reveals the presence of several immunoglobulin-like domains (5 to 71, 96 to 162, 187 to 253, 278 to 344 and 370 to 436). No other structural features are evident. Since mPN34854 is only a partial sequence, other structural features that give a better idea of the protein's function may be present.
  • AW755252 also is expressed highly in heart tissue, the interaction between AW755252 and mPN34854 may represent an interaction that is important for proper heart function.
  • AW755252 interacts with mPRPHl.
  • Two overlapping baits of AW755252 comprising amino acids 1 to 155 and 1 to 196 respectively, (of 527 total amino acids) each selected a single clone from a mouse embryo activation domain library spanning amino acids 215 to 438 (of 507 total amino acids) of mPRPHl (NM_013639).
  • the fragments of the interacting bait and prey should contain the minimal binding domain of each protein. Since the minimal bait fragment of AW755252 spans amino acids 1 to 155, the sequence having a truncation of up to 372 (which is obtained by subtracting 155 from 527, the total amino acids number of AW755252) amino acids at the C-terminus of the AW755252 sequence set forth in Figure 2 does not render it unable to interact with mPRPHl .
  • Peripherin mRNA is expressed in primarily in neuronal cells and in cell lines derived from neuroblastoma tumors (Landon et al, EMBOJ. 19898(6):1719-26, Goldstein et al, JNeurosciRes. 1991 30(1):92-104.).
  • Structural analysis of mPRPHl reveals the presence of an intermediate filament domain (amino acids 100 to 442), consistent with the function of mPRPHl as a neuronal intermediate filament protein.
  • two overlapping regions of potential coiled coil structure are present. No other structural features are evident.
  • the interaction between AW755252 and mPRPHl may be a representative of an interaction between AW755252 and a protein expressed in heart or muscle tissue that is structurally similar to mPRPHl .
  • mPRPHl interacts with a protein structurally similar to AW755252 in neuronal tissue. This interaction may play important role in the regulation of cytoskeleton formation at cardiomyocyte.
  • AW755252 interacts with mTCTEX-1 (M25825)
  • Two overlapping baits comprising amino acids 245 to 527 and 397 to 527 respectively, each selected a single clone from a mouse cardiac cell activation domain library spanning amino acids 1 to 113 (of 113 total amino acids) of mTCTEX-1 (M25825).
  • the fragments of the interacting bait and prey should contain the minimal binding domain of each protein. Since the minimal bait fragment of AW755252 spans amino acids 397 to 527, the sequence having a truncation of up to 396 amino acids at
  • N-terminus of the AW755252 sequence set forth in Figure 2 does not render it unable to interact with mTCTEX-1.
  • mTCTEX-1 also is known as cytoplasmic dynein light chain and T-complex testis-specific protein 1.
  • the cDNA encoding mTCTEX-1 was isolated from a mouse testicular cell cDNA library using a subtractive hybridization strategy.
  • mTCTEX-1 mRNA is expressed most highly in mouse testis, with much lower levels in heart, liver, brain, spleen, and kidney (Lander et al, Cell. 1989 58(5):969-79.).
  • publicly available EST data suggest a much broader range of expression.
  • mTCTEX-1 Structural analysis of mTCTEX-1 reveals no significant features. mTCTEX-1 appears to function as a dynein light chain, and as such is involved in transport of rhodopsin in rod photoreceptor cells (Tai et al, Cell. 1999 97(7):877-87). This function is consistent with the more broad expression pattern observed in public EST data.
  • the interaction between AW755252 and a dynein light chain protem suggests a role for AW755252 in the cellular transport machinery or in other cellular mechanical processes. For example, the transportation from cytoplasm to nuclear, or from membrane to cytoplasm and so on.
  • a bait comprising amino acids 240 to 398 (of 527 total amino acids) of
  • AW755252 selected a single clone from a heart activation domain library spanning amino acids 514 to 894 (of 894 total amino acids) of ACTN2.
  • the interacting fragments of the bait and prey should contain the minimal binding domain of each protein. Since the bait fragment of AW755252 comprises amino acids 240 to 398, the sequence having a truncation of up to 239 amino acids at N-terminus and/or up to 129 (which is obtained by subtracting 398 from 527, the total amino acids number of AW755252) amino acids at the C-terminus of the AW755252 sequence set forth in Figure 2 does not render it unable to interact with ACTN2.
  • ACTN2 since the prey fragment of ACTN2 comprises amino acids 514 to 894, the sequence having a truncation of up to 513 amino acids at N-terminus of the ACTN2 sequence set forth in Figure 8 does not render it unable to interact with AW755252.
  • ACTN2 also is known as alpha-actinin 2, alpha actinin skeletal muscle isoform 2, and F-actin cross linking protein.
  • the cDNA encoding ACTN2 was isolated based on its similarity to other alpha-actinin sequences (Beggs et al., J. Biol. Chem. 267:9281-8 (1992)).
  • the region of ACTN2 shown here to interact with AW755252 includes one of the spectrin repeats and all of the EF hand domains.
  • ACTN2 is thought to play a role in myofibril organization, and clearly plays a role in mediating protein-protein interactions in both cardiac and skeletal muscle (reviewed in Nag and Lee, Tsitologiia 39:907-12 (1997), Learn, FEBS Lett. 460:391-4 (1999), Watkins et al., Microsc. Res. Tech. 48:131-41 (2000)).
  • decreases in ACTN2 have been observed in cases of cardiac hypertrophy and failure (reviewed in Hein et al., Cardiovasc. Res. 45:273-8 (2000)).
  • these observations suggest a role for AW755252 and ACTN2 in allowing proper myofibril formation or in maintaining proper myofibrillar structure.
  • AW755252 interacts with mACTN4.
  • a bait comprising amino acids 240 to 398 (of 527 total amino acids) of AW755252 selected two clones from a mouse embryo activation domain library comprising amino acids 550 to 912 and 651 to 912 (of 912 total amino acids) of mACTN4.
  • the interacting fragments of the bait and prey should contain the minimal binding domain of each protein.
  • the sequence having a truncation of up to 239 amino acids at N-terminus and/or up to 129 (which is obtained by subtracting 398 from 527, the total amino acids number of AW755252) amino acids at the C-terminus of the AW755252 sequence set forth in Figure 2 does not render it unable to interact with mACTN4.
  • the minimal prey fragment of mACTN4 which interacts with AW755252 spans amino acids 651 to 912
  • the sequence having a truncation of up to 650 amino acids at N-terminus of the mACTN4 sequence set forth in Figure 9 does not render it unable to interact with AW755252.
  • mACTN4 also is known as mouse alpha-actinin 4, non-muscle alpha-actinin 4, and F-actin cross linking protein.
  • the gene encoding mACTN4 was isolated based on its overlap with the gene encoding mCAPN12 on mouse chromosome 7 (Dear et al., Genomics 68:152-60 (2000)).
  • mRNA encoding mACTN4 is expressed in a broad variety of tissues and cell types. Structural analysis of mACTN4 reveals the presence of two CH (calponin homology) domains (amino acids 53 to 153 and 166 to 265), and four spectrin repeats (amino acids 297 to 403, 417 to 518, 532 to 639, and 653 to 752).
  • CH calcium-binding domains
  • the CH domains are actin binding domains found in numerous actin binding proteins.
  • the region of mACTN4 shown here to interact with AW755252 includes one full and part of a second spectrin repeat and all of the EF hand domains.
  • mACTN4 is thought to play a role, through interactions with the cytoskeleton, in cell migration and to be involved in later stages of tumorigenesis (Honda et al., J. Cell Biol. 140:1383-93 (1998), Nikolopoulos et al., Oncogene 19:380-6 (2000)).
  • mutations in the human ortholog of mACTN4 have been shown to be causal for autosomal dominant focal and segmental glomerulosclerosis (FSGS) (Kaplan et al., Nat. Genet. 24:251-6 (2000)).
  • FSGS autosomal dominant focal and segmental glomerulosclerosis
  • AW755252 interacts with mMRJ.
  • the prey fragment of mMRJ which interacts with AW755252 comprises amino acids 32 to 176
  • the sequence having a truncation of up to 31 amino acids at N-terminus and/or up to 66 (which is obtained by subtracting 176 from 242, the total amino acids number of mMRJ) amino acids at the C-terminus of the mMRJ sequence set forth in Figure 10 does not render it unable to interact with AW755252.
  • mMRJ also is known as DnaJ homolog subfamily B member 6, heat shock protein J2, HSJ-2, and mDj4.
  • mMRJ The gene encoding mMRJ was isolated in a gene trap screen for genes involved in placental development (Hunter et al., Development 126:1247-58 ( 1999)). mMRJ mRNA is expressed at all stages of embryonic development, and is particularly high in trophoblast cells of the placenta. In addition, the gene trap procedure itself disrupted the mMRJ gene, resulting in a mouse line disrupted in placental development at about embryonic day 8.5 (Hunter et al., Development 126:1247-58 (1999)). This suggests a role for mMRJ in development, in particular in the placenta.
  • the present invention provides protem complexes formed between AW755252 and one or more AW755252-interacting proteins selected from the group consisting of GROUPl.
  • the present invention also provides a protein complex formed from the interaction between a homologue, derivative or fragment of AW755252 and one or more of the AW755252-interacting proteins in accordance with the present invention.
  • the present invention further encompasses a protein complex having AW755252 and a homologue, derivative or fragment of one or more of the AW755252-interacting proteins in accordance with the present invention.
  • protein fragments consisting of the amino acid sequence of the identified interaction domains or homologues or derivatives thereof can be used in forming the protein complexes of the present invention.
  • a hybrid protein containing such an interaction domain may also be used as an interacting partner in the protein complex of the present invention.
  • Homologues may be the counterpart proteins of other species including animals, plants, yeast, bacteria, and the like. Homologues may also be selected by, e.g., mutagenesis in AW755252 and its interacting partners. Homologues may be identified by site-specific mutagenesis in combination with assay systems for detecting protein-protein interactions, e.g., the yeast two-hybrid system described below.
  • Homology as used herein may refer to its precise meaning in biology of having a common evolutionary origin (such as mouse and human AW755252 proteins) and/or to structural resemblances. Structural resemblance is expressed in terms of identity or similarities. Identity or similarity as known in the art, is a relationship between two or more polypeptide sequences (or two or more polynucleotide sequences, as the case may be) as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polypeptide sequences or polynucleotide sequences, as determined by the match between strings of such sequences from the amino end to the carboxyl end or 5' to 3' end for polynucleotides.
  • the alterations may occur at the NH2- or COOH-terminal positions of the reference sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the alterations may occur at the 5' or 3'-terminal positions of the reference sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference polynucleotide sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations (Aa) for a given % identity is determined by first multiplying (x) the total number of amino acids (Ta) in the reference sequence by a number (n) which is obtained by dividing the percent identity by 100 (for example 0.80 for 80%, 0.90 for 90% 0.92 for 92%, 0.95 for 95%, 0.97 for 97% and so on) and then subtracting that product from said total number of amino acids (Ta) in the reference sequence. After this calculation, any non-integer value may be rounded off to the nearest integer to obtain the approximate number with out decimal values. For purposes of clarity, only the first decimal number is rounded off, to approximate the number of amino acid alterations to an integer to obtain a polypeptide of a given % identity.
  • the value of 26.35 is rounded down to 26, i.e., up to 26 amino acid alterations are needed over the entire length of the 527 amino acids of SEQ ID NO: 10 to obtain a polypeptide that is at least 95%o identical to the reference sequence of SEQ ID NO: 10.
  • derivative refers to a derivative or modified form of a protein.
  • modified forms include glycosylated forms, phosphorylated forms, myristylated forms, ribosylated forms, and the like.
  • Derivatives also include hybrid or fusion proteins containing one of the above native interacting proteins or a homologue or fragment thereof.
  • derivatives also encompass artificial proteins having substituted non-naturally occurring amino acids, e.g., D-amino acids.
  • a fragment of a polypeptide according to the present invention is also a variant polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any specific polypeptide disclosed herein.
  • Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ ID NOs:l-12. Further preferred are fragments characterized by structural or functional attributes such as fragments having alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet forming regions, beta-turn and beta-turn forming regions, coiled-coil and coiled-coil forming regions and other known in the art.
  • fragments include an isolated polypeptide comprising an amino acid sequence having 1 or more or at least 15, 20, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous amino acids truncated or deleted from the either amino- or carboxy-terminus of the amino acid sequences of SEQ ID NO: 1-12 disclosed herein.
  • fragments are those fragments that mediate activities of or retain properties for protem interactions including those with a similar activity/property or an improved activity/property, or with a decreased undesirable activity or property.
  • two or more interacting partners are directly fused together, or covalently linked together through a peptide linker, forming a hybrid protem having a single unbranched polypeptide chain.
  • the protein complex may be formed by "intramolecular" interactions between two portions of the hybrid protein.
  • one or both of the fused or linked interacting partners in this protein complex may be a native protein or a homologue, derivative or fragment of a native protein.
  • a variant polypeptide is a polypeptide that differs from a reference polypeptide but retains its essential properties (e.g., retains ability to interact with other protein(s) of the present protein-protein interaction).
  • a variant of AW755252 can have a sequence consisting of the amino acids identical to that set forth in SEQ ID NO: 10 (a reference polypeptide in this case) except that, over the entire length corresponding to the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of the variant can have one or more conservative amino acid substitutions, whereby an amino acid residue is replaced by another with like properties.
  • Typical conservative amino acid substitutions are among Ala, Val, Leu and He; among Thr and Ser; among the acidic residues Glu and Asp; among Asn and Gin; and among basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • a variant of AW755252 can also have a sequence consisting of the amino acids identical to that set forth in SEQ ID NO: 10, the reference polypeptide, except that, over the entire length corresponding to the amino acid sequence of SEQ ID NO: 10, the amino acid sequence of the variant can have one or more non-conservative amino acid substitutions, deletions or insertions at such positions of the amino acid sequence which do not alter its essential properties, such as for example, its interacting ability or activity with other polypeptides.
  • a variant and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. Substitutions, additions, deletions are also sometimes referred to as mutations.
  • variants are closely similar overall and, in many regions, identical. Particularly preferred are variants in which 5-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acids are substituted, deleted or added in any combination for every 100 amino acids.
  • a variant may be induced or naturally occurring such as an allelic variant.
  • Variants may be created by mutagenesis or by direct synthesis or other methods known to skilled workers in this art.
  • the protein complexes of the present invention can also be in a modified form.
  • an antibody selectively immunoreactive with the protein complex can be bound to the protein complex.
  • a non-antibody modulator capable of enhancing the interaction between the interacting partners in the protein complex may be included.
  • the protein members in the protein complex may be cross-linked for purposes of stabilization.
  • Various crosslinking methods may be used.
  • a bifunctional reagent in the form of R-S-S-R' may be used in which the R and R' groups can react with certain amino acid side chains in the protein complex forming covalent linkages.
  • crosslinking agents include, e.g., Denny- Jaffee reagent, a heterbifunctional photoactivable moiety cleavable through an azo linkage (See, Denny et al, Proc. Natl. Acad. Sci. USA, 81:5286-5290 (1984)), and
  • 125I- ⁇ S-[N-(3-iodo-4-azidosalicyl)cysteaminyl]-2-thiopyridine ⁇ a cysteine-specific photocrosslinking reagent (see Chen et al, Science, 265:90-92 (1994)).
  • the above-described protein complexes may further include any additional components, e.g., other proteins, nucleic acids, lipid molecules, monosaccharides or polysaccharides, ions, etc.
  • the protein complex of the present invention can be prepared by a variety of methods. Specifically, a protein complex can be isolated directly from an animal tissue sample, preferably a human tissue sample containing the protein complex. Alternatively, a protein complex can be purified from host cells that recornbinantly express the members of the protein complex. As will be apparent to a skilled artisan, a protein complex can be prepared from a tissue sample or recombinant host cell by coimmunoprecipitation using an antibody immunoreactive with an interacting protein partner, or preferably an antibody selectively immunoreactive with the protein complex as will be discussed in detail below.
  • the antibodies can be monoclonal or polyclonal.
  • Coimmunoprecipitation is a commonly used method in the art for isolating or detecting bound proteins. In this procedure, generally a serum sample or tissue or cell lysate is admixed with a suitable antibody. The protein complex bound to the antibody is precipitated and washed. The bound protein complexes are then eluted.
  • immunoaffmity chromatography and immunobloting techniques may also be used in isolating the protein complexes from native tissue samples or recombinant host cells using an antibody immunoreactive with an interacting protein partner, or preferably an antibody selectively immunoreactive with the protein complex.
  • the antibody may be covalently or non-covalently coupled to a matrix such as Sepharose in, e.g., a column.
  • the tissue sample or cell lysate from the recombinant cells can then be contacted with the antibody on the matrix.
  • the column is then washed with a low-salt solution to wash off the unbound components.
  • the protein complexes that are retained in the column can be then eluted from the column using a high-salt solution, a competitive antigen of the antibody, a chaotropic solvent, or sodium dodecyl sulfate (SDS), or the like.
  • a high-salt solution a competitive antigen of the antibody
  • a chaotropic solvent or sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • crude proteins samples from a tissue sample or recombinant host cell lysate can be fractionated on a polyacrylamide gel electrophoresis (PAGE) and then transferred to, e.g., a nitrocellulose membrane.
  • PAGE polyacrylamide gel electrophoresis
  • the location of the protein complex on the membrane may be identified using a specific antibody, and the protein complex is subsequently isolated.
  • individual interacting protein partners may be isolated or purified independently from tissue samples or recombinant host cells using similar methods as described above. The individual interacting protein partners are then contacted with each other under conditions conducive to the interaction therebetween thus forming a protein complex of the present invention. It is noted that different protein-protein interactions may require different conditions. As a starting point, for example, a buffer having 20 mM Tris-HCl, pH 7.0 and 500 mM NaCl may be used. Several different parameters may be varied, including temperature, pH, salt concentration, reducing agent, and the like. Some minor degree of experimentation may be required to determine the optimum incubation condition, this being well within the capability of one skilled in the art once apprised of the present disclosure.
  • the protem complex of the present invention may be prepared from tissue samples or recombinant host cells or other suitable sources by protein affinity chromatography or affinity blotting. That is, one of the interacting protein partners is used to isolate the other interacting protein partner(s) by binding affinity thus forming protein complexes.
  • an interacting protem partner prepared by purification from tissue samples or by recombinant expression or chemical synthesis may be bound covalently or non-covalently to a matrix such as Sepharose in, e.g., a chromatography column. The tissue sample or cell lysate from the recombinant cells can then be contacted with the bound protein on the matrix.
  • nucleic acid encoding an interacting protein member can be introduced into a suitable host cell.
  • nucleic acids encoding two or more interacting protein members should be introduced into the host cell.
  • the nucleic acids preferably in the form of DNA, are incorporated into a vector to form expression vectors capable of expressing the interacting protein member(s) once introduced into a host cell.
  • Many types of vectors can be used for the present invention.
  • the expression vectors may include a promoter operably linked to a DNA encoding an interacting protein member, an origin of DNA replication for the replication of the vectors in host cells.
  • the expression vectors also include a replication origin for the amplification of the vectors in, e.g., E. coli, and selection marker(s) for selecting and maintaining only those host cells harboring the expression vectors.
  • the expression vectors preferably also contain inducible elements, which function to control the transcription from the DNA encoding an interacting protein member.
  • Other regulatory sequences such as transcriptional enhancer sequences and translation regulation sequences (e.g., Shine-Dalgarno sequence) can also be operably included.
  • the expression vectors may also contain components that direct the expressed protein extracellularly or to a particular intracellular compartment.
  • Signal peptides, nuclear localization sequences, endoplasmic reticulum retention signals, mitochondrial localization sequences, myristoylation signals, palmitoylation signals, and transmembrane sequences are example of optional vector components that can determine the destination of expressed proteins.
  • the DNA fragments encoding the interacting protein members may be incorporated into a single vector or different vectors.
  • the thus constructed expression vectors can be introduced into the host cells by any techniques known in the art, e.g., by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, gene gun, and the like.
  • the expression of the interacting protein members may be transient or stable.
  • the expression vectors can be maintained in host cells in an extrachromosomal state, i.e., as self-replicating plasmids or viruses.
  • the expression vectors can be integrated into chromosomes of the host cells by conventional techniques such as selection of stable cell lines or site-specific recombination.
  • the vector construct can be designed to be suitable for expression in various host cells, including but not limited to bacteria, yeast cells, plant cells, insect cells, and mammalian and human cells. Methods for preparing expression vectors for expression in different host cells should be apparent to a skilled artisan.
  • the amino acid sequence of a native interacting protem member may be changed in predetermined manners by site-directed DNA mutagenesis to create or remove consensus sequences for, e g., phosphorylation by protein kinases, glycosylation, ribosylation, myristoylation, palmytoylation, and the like.
  • non-natural amino acids can be incorporated into an interacting protein member during the synthesis of the protem in recombinant host cells.
  • photoreactive lysine derivatives can be incorporated into an interacting protein member during translation by using a modified lysyl-tRNA. See, e.g.,
  • derivatives of the native interacting protein members of the present invention can also be prepared by chemically linking certain moieties to amino acid side chains of the native proteins.
  • the homologues and derivatives thus generated can be tested to determine whether they are capable of interacting with their intended interacting partners to form protein complexes. Testing can be conducted by e.g., the yeast two-hybrid system or other methods known in the art for detecting protein-protein interaction.
  • a hybrid protem as described above having AW755252 or a homologue, derivative, or fragment thereof covalently linked by a peptide bond or a peptide linker to a protein selected from the group consisting of GROUPl or a homologue, derivative, or fragment thereof, can be expressed recombinantly from a chimeric nucleic acid, e.g., a DNA or mRNA fragment encoding the fusion protem.
  • the present invention also provides a nucleic acid encoding the hybrid protein of the present invention.
  • an expression vector having incorporated therein a nucleic acid encoding the hybrid protein of the present invention is also provided. The methods for making such chimeric nucleic acids and expression vectors containing them should be apparent to skilled artisans apprised of the present disclosure.
  • the protein microarray of the present invention can be prepared in a number of methods known in the art.
  • An example of a suitable method is that disclosed in MacBeath and Schreiber, Science, 289:1760-1763 (2000).
  • glass microscope slides are treated with an aldehyde-containing silane reagent (SuperAldehyde Substrates purchased from TeleChem International, Cupertino, California). Nanoliter volumes of protein samples in a phosphate-buffered saline with 40% glycerol are then spotted onto the treated slides using a high-precision contact-printing robot.
  • BSA-NHS slides are fabricated by first attaching a molecular layer of BSA to the surface of glass slides and then activating the BSA with N,N'-disuccinimidyl carbonate.
  • a substrate or chip base is covered with one or more layers of thin organic film to eliminate any surface defects, insulate proteins from the base materials, and to ensure uniform protein array.
  • a plurality of protein-capturing agents e.g., antibodies, peptides, etc.
  • Proteins or protein complexes can then be bound to the capturing agents forming a protein microarray.
  • the protein microchips are kept in flow chambers with an aqueous solution.
  • the protein microarray of the present invention can also be made by the method disclosed in PCT Publication No. WO 99/36576 assigned to Packard Bioscience Company, which is incorporated herein by reference.
  • a three-dimensional hydrophilic polymer matrix i.e., a gel
  • the polymer matrix gel is capable of expanding or contracting and contains a coupling reagent that reacts with amine groups.
  • proteins and protein complexes can be contacted with the matrix gel in an expanded aqueous and porous state to allow reactions between the amine groups on the protein or protein complexes with the coupling reagents thus immobilizing the proteins and protein complexes on the substrate.
  • the gel is contracted to embed the attached proteins and protein complexes in the matrix gel.
  • proteins and protem complexes of the present invention can be incorporated into a commercially available protein microchip, e.g., the ProteinChip System from Ciphergen Biosystems Inc., Palo Alto, CA.
  • the ProteinChip System comprises metal chips having a treated surface, which interact with proteins.
  • a metal chip surface is coated with a silicon dioxide film.
  • the molecules of interest such as proteins and protein complexes can then be attached covalently to the chip surface via a silane coupling agent.
  • protem microchips of the present invention can also be prepared with other methods known in the art, e.g., those disclosed in U.S. Patent Nos. 6,087,102, 6,139,831, 6,087,103; PCT Publication Nos. WO 99/60156, WO 99/39210, WO 00/54046, WO 00/53625, WO 99/51773, WO 99/35289, WO 97/42507, WO 01/01142, WO 00/63694, WO 00/61806, WO 99/61148, WO 99/40434, all of which are incorporated herein by reference. 3. Antibodies
  • an antibody immunoreactive against a protem complex of the present invention is provided.
  • the antibody is selectively immunoreactive with a protein complex of the present invention.
  • the phrase "selectively immunoreactive with a protein complex" as used herein means that the immunoreactivity of the antibody of the present invention with the protein complex is substantially higher than that with the individual interacting members of the protein complex so that the binding of the antibody to the protein complex is readily distinguishable from the binding of the antibody to the individual interacting member proteins based on the strength of the binding affinities.
  • the binding constant differs by a magnitude of at least 2 fold, more preferably at least 5 fold, even more preferably at least 10 fold, and most preferably at least 100 fold.
  • the antibody is not substantially immunoreactive with the interacting protein members of the protein complex.
  • the antibody of the present invention can be readily prepared using procedures generally known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.
  • the protein complex against which the antibody to be generated will be immunoreactive is used as the antigen for the purpose of producing immune response in a host animal.
  • the protein complex used consists the native proteins.
  • the protein complex includes only the binding domains of AW755252 and one or more proteins selected from the group consisting of GROUP 1 , respectively. As a result, a greater portion of the total antibodies may be selectively immunoreactive with the protein complexes.
  • the binding domains can be selected from, e.g., those summarized in Table 1.
  • Suitable epitope-prediction computer programs include, e.g., Mac Vector from International Biotechnologies, Inc. and Protean from DNAStar.
  • a hybrid protein as described above in Section 2.1 is used as an antigen which has AW755252 or a homologues, derivative, or fragment thereof covalently linked by a peptide bond or a peptide linker to a protein selected from the group consisting of GROUPl or a homologue, derivative, or fragment thereof.
  • the hybrid protein consists of two interacting binding domains selected from Table 1, or homologues or derivatives thereof, covalently linked together by a peptide bond or a linker molecule.
  • the antibody of the present invention can be a polyclonal antibody to a protein complex of the present invention.
  • various animal hosts can be employed, including, e.g., mice, rats, rabbits, goats, guinea pigs, hamsters, etc.
  • a suitable antigen which is a protein complex of the present invention or a derivative thereof as described above can be administered directly to a host animal to illicit immune reactions. Alternatively, it can be administered together with a carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, and Tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • ovalbumin ovalbumin
  • Tetanus toxoid Tetanus toxoid
  • the antigen is conjugated to a carrier by a coupling agent such as carbodiimide, glutaraldeh de, and MBS.
  • a coupling agent such as carbodiimide, glutaraldeh de, and MBS.
  • Any conventional adjuvants may be used to boost the immune response of the host animal to the protein complex antigen.
  • Suitable adjuvants known in the art include but are not limited to Complete Freund's Adjuvant (which contains killed mycobacterial cells and mineral oil), incomplete Freund's
  • Adjuvant (which lacks the cellular components), aluminum salts, MF59 from Biocine, monophospholipid, synthetic trehalose dicorynomycolate (TDM) and cell wall skeleton (CWS) both from RIBI ImmunoChem Research Inc., Hamilton, MT, non-ionic surfactant vesicles (NISV) from Proteus International PLC, Cheshire, U.K., and saponins.
  • the antigen preparation can be administered to a host animal by subcutaneous, intramuscular, intravenous, intradermal, or intraperitoneal injection, or by injection into a lymphoid organ.
  • the antibodies of the present invention may also be monoclonal. Such monoclonal antibodies may be developed using any conventional techniques known in the art. For example, the popular hybridoma method disclosed in Kohler and others.
  • B-lymphocytes producing a polyclonal antibody against a protein complex of the present invention can be fused with myeloma cells to generate a library of hybridoma clones.
  • the hybridoma population is then screened for antigen binding specificity and also for immunoglobulin class (isotype). In this manner, pure hybridoma clones producing specific homogenous antibodies can be selected.
  • monoclonal antibodies which include but are not limited to the EBV hybridoma technique, the human N-cell hybridoma technique, and the trioma technique.
  • antibodies selectively immunoreactive with a protein complex of the present invention may also be recombinantly produced.
  • cDNAs prepared by PCR amplification from activated B-lymphocytes or hybridomas may be cloned into an expression vector to form a cDNA library, which is then introduced into a host cell for recombinant expression.
  • the cDNA encoding a specific desired protein may then be isolated from the library.
  • the isolated cDNA can be introduced into a suitable host cell for the expression of the protein.
  • recombinant techniques can be used to recombinantly produce specific native antibodies, hybrid antibodies capable of simultaneous reaction with more than one antigen, chimeric antibodies (e.g., the constant and variable regions are derived from different sources), univalent antibodies which comprise one heavy and light chain pair coupled with the Fc region of a third (heavy) chain, Fab proteins, and the like. See U.S. Patent No. 4,816,567; European Patent Publication No.
  • Antibody fragments such as Fv fragments, single-chain Fv fragments (scFv), Fab' fragments, and F(ab')2 fragments can also be recombinantly produced by methods disclosed in, e.g., U.S. Patent No.
  • the transgenic non-human host animal may be immunized with suitable antigens such as a protein complex of the present invention or one or more of the interacting protein members thereof to illicit specific immune response thus producing humanized antibodies.
  • suitable antigens such as a protein complex of the present invention or one or more of the interacting protein members thereof to illicit specific immune response thus producing humanized antibodies.
  • cell lines producing specific humanized antibodies can also be derived from the immunized transgenic non-human animals. For example, mature B-lymphocytes obtained from a transgenic animal producing humanized antibodies can be fused to myeloma cells and the resulting hybridoma clones may be selected for specific humanized antibodies with desired binding specificities. Alternatively, cDNAs may be extracted from mature B-lymphocytes and used in establishing a library which is subsequently screened for clones encoding humanized antibodies with desired binding specificities.
  • a bifunctional antibody which has two different antigen binding sites, each being specific to a different interacting protein member in a protein complex of the present invention.
  • the bifunctional antibody may be produced using a variety of methods known in the art. For example, two different monoclonal antibody-producing hybridomas can be fused together. One of the two hybridomas may produce a monoclonal antibody specific against an interacting protein member of a protein complex of the present invention, while the other hybridoma generates a monoclonal antibody immunoreactive with another interacting protein member of the protein complex. The thus formed new hybridoma produces different antibodies including a desired bifunctional antibody, i.e., an antibody immunoreactive with both of the interacting protein members.
  • the bifunctional antibody can be readily purified. See Milstein and Cuello, Nature, 305:537-540 (1983).
  • bifunctional antibodies can also be produced by recombinantly expressing light and heavy chain genes in a hybridoma that itself produces a monoclonal antibody. As a result, a mixture of antibodies including a bifunctional antibody is produced. See DeMonte et al, Proc. Natl. Acad. Set., USA, 87:2941-2945 (1990); Lenz and Weidle, Gene, 87:213-218 (1990).
  • bifunctional antibodies may also be used, which include those disclosed in Holliger et al, Proc. Nat 'I Acad. Sci. USA, 90:6444-6448 (1993); de Kruif et al, J. Biol. Chem., 271:7630-7634 (1996); Coloma and Morrison, Nat. Biotechnol, 15:159-163 (1997); Muller et al., FEBSLett, 422:259-264 (1998); and Muller et al., FEBSLett, 432:45-49 (1998), all of which are incorporated herein by reference.
  • Another aspect of the present invention relates to methods for detecting the protein complexes of the present invention, particularly for determining the level of a specific protem complex in a patient sample.
  • the level of a protein complex having AW755252 and one or more proteins selected from the group consisting of GROUPl in a cell, tissue, or organ of a patient is determined.
  • An aberrant level is thus detected.
  • the protein complex can be isolated or purified from a patient sample obtained from a cell, tissue, or organ of the patient and the amount thereof is determined.
  • the protein complex can be prepared from a cell, tissue or organ sample by coimmunoprecipitation using an antibody immunoreactive with an interacting protem member, a bifunctional antibody that is immunoreactive with two or more interacting protein members of the protein complex, or preferably an antibody selectively immunoreactive with the protein complex.
  • the level of the protein complex can be determined in a sample without separation, isolation or purification.
  • an antibody selectively immunoreactive with the specific protem complex is used in an immunoassay.
  • immunocytochemical methods can be used.
  • Other well known antibody-based techniques can also be used including, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA), fluorescent immunoassays, protein A immunoassays, and immunoenzymatic assays (IEMA). See e.g., U.S. Patent Nos. 4,376,110 and 4,486,530, both of which are incorporated herein by reference.
  • the level of an individual interacting protein member of a specific protein complex can be determined in a patient sample which can be used as a reasonably accurate indicator of the level of the protein complex in the sample.
  • antibodies against an individual interacting protein member of a specific complex can be used in any one of the methods described above.
  • the level of each of the interacting protein members of a protein complex is determined in a patient sample and the relative level of the protein complex is then deduced.
  • the relative protein complex level in a patient can also be determined by determining the level of the mRNA encoding an interacting protein member of the protein complex.
  • each interacting protein member's mRNA level in a patient sample is determined.
  • methods for determining mRNA level generally known in the art may all be used. Examples of such methods include, e.g., Northern blot assay, dot blot assay, PCR assay (preferably quantitative PCR assay), in situ hybridization assay, and the like.
  • the interactions between AW755252 and the proteins GROUPl suggest that these proteins and/or the protein complexes formed by such proteins may be involved in the same biological processes and disease pathways.
  • the interactions between AW755252 and GROUPl under physiological conditions may lead to the formation of protein complexes in vivo, which contain AW755252 and one or more of the AW755252-interacting proteins.
  • the protein complexes are expected to mediate the functions and biological activities of AW755252 and GROUPl .
  • the term "aberration" when used in the context of protein complexes of the present invention means any alterations of a protein complex including increased or decreased level of the protein complex in a particular cell or tissue or organ or the total body, altered localization of the protein complex in cellular compartments or in locations of a tissue or organ, changes in binding affinity of an interacting protein member of the protein complex, mutations in an interacting protein member or the gene encoding the protem, and the like.
  • the term “aberration” is used in a relative sense. That is, an aberration is relative to a normal individual or population of normal individuals.
  • the method of diagnosis is conducted by detecting, in a patient, the levels of one or more protein complexes of the present invention using any one of the methods described above, and determining whether the patient has an aberrant level of the protein complexes.
  • the diagnosis may also be based on the determination of the levels of one or more interacting protein members (at protem or cDNA or mRNA level) of a protein complex of the present invention.
  • An aberrant level of an interacting protein member may indicate a physiological disorder or a predisposition to a physiological disorder.
  • the method of diagnosis comprises determining, in a patient, the cellular localization, or tissue or organ distribution of a protein complex of the present invention and determining whether the patient has an aberrant localization or distribution of the protein complex.
  • immunocytochemical or immunohistochemical assays can be performed on a cell, tissue or organ sample from a patient using an antibody selectively immunoreactive with a protein complex of the present invention.
  • Antibodies immunoreactive with both an individual interacting protein member and a protein complex containing the protein member may also be used, in which case it is preferred that antibodies immunoreactive with other interacting protein members are also used in the assay.
  • nucleic acid probes may also be used in in situ hybridization assays to detect the localization or distribution of the mRNAs encoding the interacting protein members of a protein complex.
  • the mRNA encoding each interacting protein member of a protein complex is detected concurrently.
  • the method of diagnosis of the present invention comprises detecting any mutations in one or more interacting protein members of a protein complex of the present invention.
  • mutations include but are not limited to, e.g., deletions, insertions and rearrangements in the genes encoding the protein members, and nucleotide or amino acid substitutions and the like.
  • the kit can include one or more of the protein complexes of the present invention prepared or purified from a normal individual or an individual afflicted with a physiological disorder associated with an aberration in the protein complexes or an interacting protein member thereof.
  • the kit may further include one or more of the interacting protein members of the protein complexes of the present invention prepared or purified from a normal individual or an individual afflicted with a physiological disorder associated with an aberration in the protein complexes or an interacting protein member thereof.
  • Suitable oligonucleotide primers useful in the amplification of the genes or cDNAs for the interacting protein members may also be provided in the kit.
  • the kit includes a first oligonucleotide selectively hybridizable to the mRNA or cDNA encoding AW755252 and a second oligonucleotide selectively hybridizable to the mRNA or cDNA encoding a protein selected from the group consisting of GROUPl.
  • Additional oligos hybridizing to AW755252 and its interacting partners as identified in the present invention may also be included.
  • Such oligos may be used as PCR primers for, e.g., quantitative PCR amplification of mRNAs encoding AW755252 and an interacting partner thereof, or as hybridizing probes for detecting the mRNAs.
  • the oligonucleotides may have a length of from about 8 nucleotides to about 100 nucleotides, preferably from about 12 to about 50 nucleotides, and more preferably from about 15 to about 30 nucleotides.
  • the kit may also contain oligonucleotides that can be used as hybridization probes for detecting the cDNAs or mRNAs encoding the interacting protein members.
  • instructions for using the kit or reagents contained therein are also included in the kit.
  • the protem complexes of the present invention, AW755252 and AW755252-interacting proteins can also be used in screening assays to identify modulators of the protein complexes, AW755252, and/or the AW755252-interacting proteins.
  • homologues, derivatives and fragments of AW755252 and the AW755252-interacting proteins may also be used in such screening assays.
  • the term "modulator" encompasses any compounds that can cause any forms of alteration of the biological activities or functions of the proteins or protein complexes, including, e.g., enhancing or reducing their biological activities, increasing or decreasing their stability, altering their affinity or specificity to certain other biological molecules, etc.
  • a modulator also includes any compounds that simply bind AW755252, AW755252-interacting proteins, and/or the proteins complexes of the present invention.
  • a modulator can be a dissociator capable of interfering with or disrupting or dissociating protein-protein interaction between AW755252 or a homologue or derivative thereof and a protein selected from the group consisting of GROUPl or a homologue or derivative thereof.
  • a modulator can also be an enhancer or initiator that initiates or strengthens the interaction between the protein members of a protein complex of the present invention.
  • the present invention provides screening methods for selecting modulators of AW755252, a AW755252-interacting protem selected from the group consisting of GROUPl, or a protein complex formed between AW755252 and one or more of the AW755252-interacting proteins. Screening methods are also provided for selecting modulators of AW755252 homologues, derivatives or fragments, or homologues, derivatives or fragments of a AW755252-interacting protem, or a protein complex formed between a AW755252 homologue, derivative or fragment and a homologue or derivative or fragment of a AW755252-interacting protein.
  • the modulators selected in accordance with the screen methods of the present invention can be effective in modulating the functions or activities of AW755252, a AW755252-interacting protein, or the protein complexes of the present invention.
  • compounds capable of binding to the protein complexes may be capable of modulating the functions of the protein complexes.
  • compounds that interfere with, weaken, dissociate or disrupt, or alternatively, initiate, facilitate or stabilize the protein-protein interaction between the interacting protem members of the protein complexes can also be effective in modulating the functions or activities of the protein complexes.
  • test compounds may be screened in the screening assays of the present invention to select modulators of AW755252, a AW755252-containing protein complex and/or a AW755252-interacting protein of the present invention.
  • selecting or “select” compounds it is intended to encompass both (a) choosing compounds from a group previously unknown to be modulators of AW755252, a AW755252-containing protein complex and/or a AW755252-interacting protein of the present invention, and (b) testing compounds that are known to be capable of binding, or modulating the functions and activities of, AW755252, a AW755252-containing protein complex and/or a AW755252-interacting protein of the present invention.
  • test compounds Both types of compounds are generally referred to herein as "test compounds.”
  • the test compounds may include, by way of example, proteins (e.g., antibodies, small peptides, artificial or natural proteins), nucleic acids, and derivatives, mimetics and analogs thereof, and small organic molecules having a molecular weight of no greater than 10,000 dalton, more preferably less than 5,000 dalton.
  • the test compounds are provided in library formats known in the art, e.g., in chemically synthesized libraries, recombinantly expressed libraries (e.g., phage display libraries), and in vitro translation-based libraries (e.g., ribosome display libraries).
  • the screening assays of the present invention can be used in the antibody production processes described in Section 3 to select antibodies with desirable specificities.
  • Various forms antibodies or derivatives thereof may be screened, including but not limited to, polyclonal antibodies, monoclonal antibodies, bifunctional antibodies, chimeric antibodies, single chain antibodies, antibody fragments such as Fv fragments, single-chain Fv fragments (scFv), Fab' fragments, and F(ab') 2 fragments, and various modified forms of antibodies such as catalytic antibodies, and antibodies conjugated to toxins or drugs, and the like.
  • the antibodies can be of any types such as IgG, IgE, IgA, or IgM. Humanized antibodies are particularly preferred.
  • the various antibodies and antibody fragments may be provided in libraries to allow large-scale high throughput screening.
  • expression libraries expressing antibodies or antibody fragments may be constructed by a method disclosed, e.g., in Huse et al, Science, 246:1275-1281 (1989), which is incorporated herein by reference.
  • Single-chain Fv (scFv) antibodies are of particular interest in diagnostic and therapeutic applications.
  • Methods for providing antibody libraries are also provided in U.S. Patent Nos. 6,096,551; 5,844,093; 5,837,460; 5,789,208; and 5,667,988, all of which are incorporated herein by reference.
  • Peptidic test compounds may be peptides having L-amino acids and/or D-amino acids, phosphopeptides, and other types of peptides.
  • the screened peptides can be of any size, but preferably have less than about 50 amino acids. Smaller peptides are easier to deliver into a patient's body.
  • Various forms of modified peptides may also be screened.
  • peptides can also be provided in, e.g., combinatorial libraries. See generally, Gallop et al, J. Med. Chem., 37: 1233-1251 (1994). Methods for making random peptide libraries are disclosed in, e.g., Devlin et al, Science, 249:404-406 (1990).
  • random-sequence peptide phage display libraries may be generated by cloning synthetic oligonucleotides into the gene III or gene VIII of an E. coli. filamentous phage.
  • the thus generated phage can propagate in E. coli. and express peptides encoded by the oligonucleotides as fusion proteins on the surface of the phage. Scott and Smith, Science, 249:368-390 (1990).
  • the "peptides on plasmids" method may also be used to form peptide libraries.
  • random peptides may be fused to the C-terminus of the E. coli. Lac repressor by recombinant technologies and expressed from a plasmid that also contains Lac repressor-binding sites. As a result, the peptide fusions bind to the same plasmid that encodes them.
  • Small organic or inorganic non-peptide non-micleotide compounds are preferred test compounds for the screening assays of the present invention. They too can be provided in a library format. See generally, Gordan et al J. Med. Chem., 37:1385-1401 (1994). For example, benzodiazepine libraries are provided in Bunin and Ellman, J. Am. Chem. Soc, 114:10997-10998 (1992), which is incorporated herein by reference. A method for constructing and screening peptoid libraries are disclosed in Simon et al, Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992).
  • 6,162,926 multiply-substituted fullerene derivatives); 6,093,798 (hydroxamic acid derivatives); 5,962,337 (combinatorial l,4-benzodiazepin-2, 5-dione library); 5,877,278 (Synthesis of N-substituted oligomers); 5,866,341 (compositions and methods for screening drug libraries); 5,792,821 (polymerizable cyclodextrin derivatives); 5,766,963 (hydroxypropylamine library); and 5,698,685 (morpholino-subunit combinatorial library), all of which are incorporated herein by reference.
  • oligonucleotides and peptide nucleic acids may also be screened to identify clinically useful compounds.
  • Combinatorial libraries of oligos are also known in the art. See Gold et al, J. Biol. Chem., 270:13581-13584 (1995).
  • test compounds may be screened in an in vitro assay to identify compounds capable of binding the protein complexes or interacting protein members thereof in accordance with the present invention.
  • a test compound is contacted with a protein complex or an interacting protein member thereof under conditions and for a time sufficient to allow specific interaction between the test compound and the target components to occur and thus binding of the compound to the target forming a complex. Subsequently, the binding event is detected.
  • Agonists as used herein are those compounds that enhance the desired activities or properties for protein interactions.
  • Antagonists are those compounds that interfere with or block the desired activities or properties for protein interactions.
  • Various screening techniques known in the art may be used in the present invention.
  • the protein complexes and the interacting protein members thereof may be prepared by any suitable methods, e.g., by recombinant expression and purification.
  • the protein complexes and/or interacting protein members thereof both are referred to as "target” hereinafter in this section) may be free in solution.
  • a test compound may be mixed with a target forming a liquid mixture.
  • the compound may be labeled with a detectable marker.
  • the binding complex having the compound and the target may be co-immunoprecipitated and washed.
  • the compound in the precipitated complex may be detected based on the marker on the compound.
  • the target is immobilized on a solid support or on a cell surface.
  • the target can be a ⁇ ayed into a protein microchip in a method described in Section 2.3.
  • a target may be immobilized directly onto a microchip substrate such as glass slides or onto a multi-well plates using non-neutralizing antibodies, i.e., antibodies capable of binding to the target but do not substantially affect its biological activities.
  • test compounds can be contacted with the immobilized target to allow binding to occur to form complexes under standard binding assay conditions.
  • Either the targets or test compounds are labeled with a detectable marker using well-known labeling techniques.
  • the test compounds can be immobilized on a solid support, e.g., forming a mciroarray of test compounds.
  • the target protein or protein complex is then contacted with the test compounds.
  • the target may be labeled with any suitable detection marker.
  • the target may be labeled with radioactive isotopes or fluorescence marker before binding reaction occurs.
  • antibodies that are immunoreactive with the target and are labeled with radioactive materials, fluorescence markers, enzymes, or labeled secondary ' anti-Ig antibodies may be used to detect any bound target thus identifying the binding compound.
  • One example of this embodiment is the protein probing method.
  • the target provided in accordance with the present invention is used as a probe to screen expression libraries of proteins or random peptides.
  • the expression libraries can be phage display libraries, in vitro translation-based libraries, or ordinary expression cDNA libraries.
  • the libraries may be immobilized on a solid support such as nitrocellulose filters. See e.g., Sikela and Hahn, Proc. Natl. Acad. Sci. USA, 84:3038-3042 (1987).
  • the probe may be labeled by a radioactive isotope or a fluorescence marker.
  • the probe can be biotmylated and detected with a streptavidin-alkaline phosphatase conjugate. More conveniently, the bound probe may be detected with an antibody.
  • a known ligand capable of binding to the target can be used in competitive binding assays. Complexes between the known ligand and the target can be formed and then contacted with test compounds. The ability of a test compound to interfere with the interaction between the target and the known ligand is measured.
  • One exemplary ligand is an antibody capable of specifically binding the target. Particularly, such an antibody is especially useful for identifying peptides that share one or more antigenic determinants of the target protein complex or interacting protein members thereof.
  • a protein complex used in the screening assay includes a hybrid protein as described in Section 2.1, which is formed by fusion of two interacting protein members or fragments or domains thereof.
  • the hybrid protein may also be designed such that it contains a detectable epitope tag fused thereto.
  • epitope tags include sequences derived from, e.g., influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc, lacZ, GST, and the like.
  • the assay can be conducted in similar manners as the binding assays described above.
  • the presence or absence of a particular protein complex can be detected by an antibody selectively immunoreactive with the protein complex.
  • immunoprecipitation assay can be conducted with the antibody. If the test compound disrupts the protein complex, then the amount of immunoprecipitated protein complex in this assay will be significantly less than that in a control assay in which the same protem complex is not contacted with the test compound.
  • two proteins the interaction between which is to be enhanced may be incubated together with a test compound. Thereafter, protein complex may be detected by the selectively immunoreactive antibody. The amount of protein complex may be compared to that formed in the absence of the test compound.
  • Various other detection methods may be suitable in the dissociation assay, as will be apparent to skilled artisan apprised of the present disclosure.
  • Test compounds can also be screened in any in vivo assays select modulators of the protein complexes or interacting protein members thereof in accordance with the present invention.
  • any in vivo assays known in the art useful in identifying compounds capable of strengthening or interfering with the stability of the protein complexes of the present invention may be used.
  • yeast two-hybrid systems or their analogous or derivative forms is used.
  • suitable two-hybrid systems known in the art include, but are not limited to, those disclosed in U.S. Patent Nos. 5,283,173; 5,525,490; 5,585,245; 5,637,463; 5,695,941; 5,733,726; 5,776,689; 5,885,779; 5,905,025; 6,037,136; 6,057,101; 6,114,111; and Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, NY, 1997, all of which are incorporated herein by reference.
  • two chimeric genes are prepared encoding two fusion proteins: one contains a transcription activation domain fused to an interacting protein member of a protein complex of the present invention or an interacting domain of the interacting protein member, while the other fusion protein includes a DNA binding domain fused to another interacting protein member of the protein complex or an interacting domain thereof.
  • the two interacting protein members or interacting domains thereof are referred to as “bait fusion protein” and "prey fusion protein,” respectively.
  • the chimeric genes encoding the fusion proteins are termed “bait chimeric gene” and "prey chimeric gene,” respectively.
  • a "bait vector” and a “prey vector” are provided for the expression of a bait chimeric gene and a prey chimeric gene, respectively.
  • vectors can be used in a transcription-based two-hybrid assay.
  • the bait and prey vectors may include a promoter operably linked to a chimeric gene for the transcription of the chimeric gene, an origin of DNA replication for the replication of the vectors in host cells and a replication origin for the amplification of the vectors in, e.g., E. coli, and selection marker(s) for selecting and maintaining only those host cells harboring the vectors.
  • the vectors preferably also contain inducible elements, which function to control the expression of a chimeric gene. Making the expression of the chimeric genes inducible and controllable is especially important in the event that the fusion proteins or components thereof are toxic to the host cells.
  • Termination sequences such as the bovine growth hormone, SV40, lacZ and AcMNPV polyhedral polyadenylation signals may also be operably linked to a chimeric gene.
  • An epitope tag coding sequence for detection and/or purification of the fusion proteins can also be incorporated into the expression vectors. Examples of useful epitope tags include, but are not limited to, influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc, lacZ, GST, and the like.
  • Proteins with polyhistidine tags can be easily detected and/or purified with Ni affinity columns, while specific antibodies to many epitope tags are generally commercially available.
  • the vectors can be introduced into the host cells by any techniques known in the art, e.g., by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, gene gun, and the like.
  • the bait and prey vectors can be maintained in host cells in an extrachromosomal state, i.e., as self-replicating plasmids or viruses.
  • one or both vectors can be integrated into chromosomes of the host cells by conventional techniques such as selection of stable cell lines or site-specific recombination.
  • the in vivo assays of the present invention can be conducted in many different host cells, including but not limited to bacteria, yeast cells, plant cells, insect cells, and mammalian cells. A skilled artisan will recognize that the designs of the vectors can vary with the host cells used.
  • the assay is conducted in prokaryotic cells such as Escherichia coli, Salmonella, Klebsiella, Pseudomonas, Caulobacter, and Rhizobium.
  • Suitable origins of replication for the expression vectors useful in this embodiment of the present invention include, e.g., the ColEl, pSClOl, and Ml 3 origins of replication.
  • suitable promoters include, for example, the T7 promoter, the lacZ promoter, and the like.
  • inducible promoters are also useful in modulating the expression of the chimeric genes.
  • the lac operon from bacteriophage lambda plac5 is well known in the art and is inducible by the addition of IPTG to the growth medium.
  • Other known inducible promoters useful in a bacteria expression system include pL of bacteriophage lambda, the trp promoter, and hybrid promoters such as the tac promoter, and the like.
  • selection marker sequences for selecting and maintaining only those prokaryotic cells expressing the desirable fusion proteins should also be incorporated into the expression vectors.
  • Numerous selection markers including auxotrophic markers and antibiotic resistance markers are known in the art and can all be useful for purposes of this invention.
  • the bla gene which confers ampicillin resistance is the most commonly used selection marker in prokaryotic expression vectors.
  • Other suitable markers include genes that confer neomycin, kanamycin, or hygromycin resistance to the host cells.
  • vectors are commercially available from vendors such as Invitrogen Corp. of San Diego, Calif., Clontech Corp. of Palo Alto, Calif, BRL of Bethesda, Maryland, and Promega Corp. of Madison, Wisconsin.
  • pBR322 e.g., pBR322, pSPORT, pBluescriptllSK, pcDNAI, and pcDNAII all have a multiple cloning site into which the chimeric genes of the present invention can be conveniently inserted using conventional recombinant techniques.
  • the constructed expression vectors can be introduced into host cells by various transformation or transfection techniques generally known in the art.
  • mammalian cells are used as host cells for the expression of the fusion proteins and detection of protein-protein interactions.
  • mammalian cells can be used including normal tissue cells, stable cell lines, and transformed tumor cells.
  • mammalian cell lines such as CHO cells, Jurkat T cells, NIH 3T3 cells, HEK-293 cells, CV-1 cells, COS-1 cells, HeLa cells, VERO cells, MDCK cells, WI38 cells, and the like are used.
  • Mammalian expression vectors are well known in the art and many are commercially available.
  • Suitable inducible promoters include, for example, the tetracycline responsive element (TRE) (See Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), metallothionein IIA promoter, ecdysone-responsive promoter, and heat shock promoters.
  • TRE tetracycline responsive element
  • metallothionein IIA promoter promoter
  • ecdysone-responsive promoter ecdysone-responsive promoter
  • heat shock promoters e.g., the Epstein Barr origin of replication in the presence of the Epstein Barr nuclear antigen (see Sugden et al, Mol. Cell.
  • SV40 origin of replication in the presence of the SV40 T antigen (which is present in COS-1 and COS-7 cells) (see Margolskee et al, Mol. Cell. Biol, 8:2837 (1988)).
  • Suitable selection markers include, but are not limited to, genes conferring resistance to neomycin, hygromycin, zeocin, and the like.
  • Many commercially available mammalian expression vectors may be useful for the present invention, including, e.g., pCEP4, pcDNAI, pIND, pSecTag2, pVAXl, pcDNA3.1, and pBI-EGFP, and pDisplay.
  • the vectors can be introduced into mammalian cells using any known techniques such as calcium phosphate precipitation, lipofection, electroporation, and the like.
  • the bait vector and prey vector can be co-transformed into the same cell or, alternatively, introduced into two different cells which are subsequently fused together by cell fusion or other suitable techniques.
  • Viral expression vectors which permit introduction of recombinant genes into cells by viral infection, can also be used for the expression of the fusion proteins.
  • Viral expression vectors generally known in the art include viral vectors based on adenovirus, bovine papilloma virus, murine stem cell virus (MSCV), MFG virus, and retrovirus. See Sarver, et al, Mol. Cell. Biol, 1: 486 (1981); Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659 (1984); Mackett, et al, Proc Natl. Acad. Sci. USA, 79:7415-7419 (1982); Mackett, et al, J.
  • a chimeric gene according to the present invention can be operably linked to a suitable promoter.
  • the promoter-chimeric gene construct is then inserted into a non-essential region of the viral vector, typically a modified viral genome. This results in a viable recombinant virus capable of expressing the fusion protein encoded by the chimeric gene in infected host cells. Once in the host cell, the recombinant virus typically is integrated into the genome of the host cell. However, recombinant bovine papilloma viruses typically replicate and remain as extrachromosomal elements.
  • the detection assays of the present invention are conducted in plant cell systems.
  • Methods for expressing exogenous proteins in plant cells are well known in the art. See generally, Weissbach & Weissbach, Methods for
  • Recombinant virus expression vectors based on, e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV) can all be used.
  • recombinant plasmid expression vectors such as Ti plasmid vectors and Ri plasmid vectors are also useful.
  • the chimeric genes encoding the fusion proteins of the present invention can be conveniently cloned into the expression vectors and placed under control of a viral promoter such as the 35S
  • a plant promoter e.g., the promoter of the small subunit of RUBISCO and heat shock promoters (e.g., soybean hspl7.5-E or hspl7.3-B promoters).
  • the in vivo assay of the present invention can also be conducted in insect cells, e.g., Spodoptera frugiperda cells, using a baculovirus expression system.
  • Expression vectors and host cells useful in this system are well known in the art and are generally available from various commercial vendors.
  • the chimeric genes of the present invention can be conveniently cloned into a non-essential region (e.g., the polyhedrin gene) of an Autographa californica nuclear polyhedrosis virus (AcNPV) vector and placed under control of an AcNPV promoter (e.g., the polyhedrin promoter).
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • the non-occluded recombinant viruses thus generated can be used to infect host cells such as Spodoptera frugiperda cells in which the chimeric genes are expressed. See U.S. Patent No. 4,215,051.
  • the fusion proteins are expressed in a yeast expression system using yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, and Schizosaccharomyces pombe as host cells.
  • yeasts such as Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, and Schizosaccharomyces pombe as host cells.
  • the expression of recombinant proteins in yeasts is a well-developed field, and the techniques useful in this respect are disclosed in detail in The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Vols.
  • each of the two chimeric genes is included in a separate expression vector (bait vector and prey vector). Both vectors can be co-transformed into a single yeast host cell. As will be apparent to a skilled artisan, it is also possible to express both chimeric genes from a single vector.
  • the bait vector and prey vector are introduced into two haploid yeast cells of opposite mating types, e.g., a-type and ⁇ -type, respectively. The two haploid cells can be mated at a desired time to form a diploid cell expressing both chimeric genes.
  • suitable constitutive promoters include but are not limited to the yeast ADH1 , PGK1 , TEF2 , GPD1 , HIS3, and CYC1 promoters.
  • suitable inducible promoters include but are not limited to the yeast GAL1 (inducible by galactose), CUP1 (inducible by Cu ++ ), and FUS1 (inducible by pheromone) promoters; the AOX/MOX promoter from H. polymorpha and P. Pastoris (repressed by glucose or ethanol and induced by methanol); chimeric promoters such as those that contain LexA operators (inducible by LexA-containing transcription factors); and the like.
  • a transcriptional termination signal is operably linked to the chimeric genes in the vectors.
  • transcriptional termination signal sequences derived from, e.g., the CYC1 and ADH1 genes can be used.
  • auxotrophic markers including, but not limited to, URA3, HIS3, TRP1, LEU2, LYS2, ADE2, and the like.
  • the yeast host cells transformed with bait vector and/or prey vector are cultured in a medium lacking a particular nutrient.
  • selectable markers are not based on auxotrophies, but rather on resistance or sensitivity to an antibiotic or other xenobiotic.
  • markers include but are not limited to chloramphenicol acetyl transferase (CAT) gene, which confers resistance to chloramphenicol; CAN1 gene, which encodes an arginine permease and thereby renders cells sensitive to canavanine (see Sikorski et al, Meth. Enzymol, 194:302-318 (1991)); the bacterial kanamycin resistance gene (kan R ), which renders eucaryotic cells resistant to the aminoglycoside G418 (see Wach et al, Yeast, 10:1793-1808 (1994)); and CYH2 gene, which confers sensitivity to cycloheximide (see Sikorski et al, Meth.
  • CAT chloramphenicol acetyl transferase
  • CAN1 gene which encodes an arginine permease and thereby renders cells sensitive to canavanine
  • kan R bacterial kanamycin resistance gene
  • CYH2 gene which confers sensitivity
  • L40 strain which has the genotype MATa his3 delta200 trpl-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ;
  • Y190 strain which is available from Clontech, Palo Alto, California and has the genotype MATalpha gal4 gal80 his3delta200 trpl-901 ade2-101 ura3-52 leu2-3, 112 URA3::GALl-lacZ LYS2::GAL1-HIS3 cyl ; and
  • YRG-2 Strain which is available from Stratagene, La Jolla, California and has the genotype MATalpha ur ⁇ 3-52 his3-200 ⁇ de2-101 lys2-801 trpl-901 leu2-3, 112 g ⁇ l4-542 g ⁇ l80-538 LYS2::GAL1-HIS3 URA3::GALl/CYCl-l ⁇ cZ.
  • a transcription-based two-hybrid assay the interaction between a bait fusion protein and a prey fusion protein brings the DNA-binding domain and the transcription-activation domain into proximity forming a functional transcriptional factor, which acts on a specific promoter to drive the expression of a reporter protein.
  • the transcription activation domain and the DNA-binding domain may be selected from various known transcriptional activators, e.g., GAL4, GCN4, ARD1, the human estrogen receptor, E. coli LexA protein, herpes simplex virus VPl 6 (Triezenberg et ⁇ /.,
  • the reporter gene and the promoter driving its transcription typically are incorporated into a separate reporter vector.
  • the host cells are engineered to contain such a promoter-reporter gene sequence in their chromosomes.
  • the interaction or lack of interaction between two interacting protem members of a protein complex can be determined by detecting or measuring changes in the reporter in the assay system.
  • the reporters and selection markers can be of similar types and used in a similar manner in the present invention, the reporters and selection markers should be carefully selected in a particular detection assay such that they are distinguishable from each other and do not interfere with each other's function.
  • a reporter protein may be a fusion protein having an epitope tag fused to a protein.
  • epitope tags include sequences derived from, e.g., influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc, lacZ, GST, and the like. Antibodies specific to these epitope tags are generally commercially available.
  • the expressed reporter can be detected using an epitope-specific antibody in an immunoassay.
  • the reporter is selected such that it can be detected by a color-based assay.
  • reporter include, e.g., the lacZ protein (beta-galactosidase), the green fluorescent protein (GFP), which can be detected by fluorescence assay and sorted by flow-activated cell sorting (FACS) (See Cubitt et al, Trends Biochem. Sci., 20:448-455 (1995)), secreted alkaline phosphatase, horseradish peroxidase, the blue fluorescent protem (BFP), and luciferase photoproteins such as aequorin, obelin, mnemiopsin, and berovin (See U.S. Patent No. 6,087,476, which is incorporated herein by reference).
  • FACS flow-activated cell sorting
  • auxotrophic factor is used as a reporter in a host strain deficient in the auxotrophic factor.
  • suitable auxotrophic reporter genes include, but are not limited to, URA3, HIS3, TRP1, LEU2, LYS2, ADE2, and the like.
  • yeast cells containing a mutant URA3 gene can be used as host cells (Ura ⁇ phenotype). Such cells lack l7 43-encoded functional orotidine-5' -phosphate decarboxylase, an enzyme required by yeast cells for the biosynthesis of uracil. As a result, the cells are unable .to grow on a medium lacking uracil.
  • wild-type orotidine-5 '-phsphate decarboxylase catalyzes the conversion of a non-toxic compound 5-fiuoroorotic acid (5-FOA) to a toxic product, 5-fluorouracil.
  • yeast cells containing a wild-type URA3 gene are sensitive to 5-FOA and cannot grow on a medium containing 5-FOA. Therefore, when the interaction between the interacting protein members in the fusion proteins results in the expression of active orotidine-5 '-phosphate decarboxylase, the Ura " (Foa R ) yeast cells will be able to grow on a uracil deficient medium (SC-Ura plates). However, such cells will not survive on a medium containing 5-FOA. Thus, protein-protein interactions can be detected based on cardiac function.
  • antibiotic resistance reporters can also be employed in a similar manner. In this respect, host cells sensitive to a particular antibiotics is used.
  • Antibiotics resistance reporters include, for example, chloramphenicol acetyl transferase (CAT) gene and the kan R gene, which confers resistance to G418 in eukaryotes and to kanamycin in prokaryotes.
  • CAT chloramphenicol acetyl transferase
  • the screening assay of the present invention is useful in identifying compounds capable of interfering with or disrupting or dissociating protein-protein interaction between AW755252 or a homologue or derivative thereof and a protein selected from the group consisting of GROUPl or a homologue or derivative thereof.
  • AW755252 and its interacting partners are believed to play a role in cardiac function, and thus are involved in ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris. It may be possible to ameliorate or alleviate the diseases or disorders in a patient by interfering with or dissociating normal interactions between AW755252 and one of GROUPl.
  • the disease or disorder may be treated or prevented by weakening or dissociating the interaction between AW755252 and the member in a patient.
  • the disease or disorder may be treated with a compound that weakens or interferes with the interaction between the mutant form of AW755252 and the member.
  • AW755252, a mutant form or a binding domain thereof, and a AW755252-interacting protein, or a mutant form or a binding domain thereof are used as test proteins expressed in the form of fusion proteins as described above for purposes of a two-hybrid assay.
  • the fusion proteins are expressed in a host cell and allowed to interact with each other in the presence of one or more test compounds.
  • a counterselectable marker is used as a reporter such that a detectable signal (e.g., appearance of color or fluorescence, or cell survival) is present only when the test compound is capable of interfering with the interaction between the two test proteins.
  • a detectable signal e.g., appearance of color or fluorescence, or cell survival
  • the reporters used in various "reverse two-hybrid systems" known in the art may be employed. Reverse two-hybrid systems are disclosed in, e.g., U.S. Patent Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al, Proc. Natl. Acad. Sci. USA, 93:10315-10320 (1996); and Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10321-10326 (1996), all of which are incorporated herein by reference.
  • Suitable counterselectable reporters useful in a yeast system include the URA3 gene (encoding orotidine-5 '-decarboxylase, which converts 5-fluroorotic acid (5-FOA) to the toxic metabolite 5-fluorouracil), the CANl gene (encoding arginine permease, which transports toxic arginine analog canavanine into yeast cells), the GAL1 gene (encoding galactokinase, which catalyzes the conversion of 2-deoxygalactose to toxic 2-deoxygalactose-l -phosphate), the LYS2 gene (encoding alpha-aminoadipate reductase, which renders yeast cells unable to grow on a medium containing alpha-aminoadipate as the sole nitrogen source), the MET15 gene (encoding O-acetylhomoserine sulfhydrylase, which confers on yeast cells sensitivity to methyl mercury), and the CYH2 gene (encoding L
  • any known cytotoxic agents including cytotoxic proteins such as the diphtheria toxin (DTA) catalytic domain can also be used as counterselectable reporters. See U.S. Patent No. 5,733,726. DTA causes the ADP-ribosylation of elongation factor-2 and thus inhibits protein synthesis and causes cell death. Other examples of cytotoxic agents include recin, Shiga toxin, and exotoxin A of Pseudomonas aeruginosa.
  • DTA diphtheria toxin
  • yeast cells containing a mutant URA3 gene can be used as host cells (Ura " Foa R phenotype) for the in vivo assay.
  • Such cells lack L7&4-?-encoded functional orotidine-5 '-phsphate decarboxylase, an enzyme required for the biosynthesis of uracil. As a result, the cells are unable to grow on media lacking uracil.
  • yeast cells cannot convert non-toxic 5-fluoroorotic acid (5-FOA) to a toxic product, 5-fluorouracil.
  • 5-FOA non-toxic 5-fluoroorotic acid
  • yeast cells are resistant to 5-FOA and can grow on a medium containing 5-FOA. Therefore, for example, to screen for a compound capable of disrupting interaction between AW755252 and PROTEIN2, AW755252 can be expressed as a fusion protein with a DNA-binding domain of a suitable transcription activator while PROTEIN2 is expressed as a fusion protein with a teanscription activation domain of a suitable transcription activator.
  • the reporter URA3 gene may be operably linked to a promoter specifically responsive to the association of the transcription activation domain and the DNA-binding domain.
  • an in vivo screening assay can be conducted in the presence of a test compound with the yeast cells being cultured on a medium containing uracil and 5-FOA. If the test compound does not disrupt the interaction between AW755252 and PROTEIN2, active URA3 gene product, i.e., orotidine-5 '-decarboxylase, which converts 5-FOA to toxic 5-fluorouracil, is expressed. As a result, the yeast cells cannot grow.
  • the screening assay of the present invention can be applied in a format appropriate for large-scale screening.
  • combinatorial technologies can be employed to construct combinatorial libraries of small organic molecules or small peptides. See generally, e.g., Kenan et al, Trends Biochem. S , 19:57-64 (1994); Gallop et al, J. Med. Chem., 37:1233-1251 (1994); Gordon et al, J. Med. Chem., 37:1385-1401 (1994); Ecker et al, Biotechnology, 13:351-360 (1995).
  • Such combinatorial libraries of compounds can be applied to the screening assay of the present invention to isolate specific modulators of particular protein-protein interactions.
  • the random peptides can be co-expressed with the fusion proteins of the present invention in host cells and assayed in vivo. See e.g., Yang et al, Nucl Acids Res., 23:1152-1156 (1995). Alternatively, they can be added to the culture medium for uptake by the host cells.
  • yeast mating is used in an in vivo screening assay.
  • haploid cells of alpha-mating type expressing one fusion protein as described above is mated with haploid cells of a-mating type expressing the other fusion protein.
  • the diploid cells are spread on a suitable medium to form a lawn.
  • Drops of test compounds can be deposited onto different areas of the lawn. After culturing the lawn for an appropriate period of time, drops containing a compound capable of modulating the interaction between the particular test proteins in the fusion proteins can be identified by stimulation or inhibition of growth in the vicinity of the drops.
  • the screening assays of the present invention for identifying compounds capable of modulating protein-protein interactions can also be fine-tuned by various techniques to adjust the thresholds or sensitivity of the positive and negative selections. Mutations can be introduced into the reporter proteins to adjust their activities.
  • the uptake of test compounds by the host cells can also be adjusted. For example, yeast high uptake mutants such as the ergo mutant steams can facilitate yeast uptake of the test compounds. See Gaber et al, Mol. Cell. Biol, 9:3447-3456 (1989).
  • the uptake of the selection compounds such as 5-FOA, 2-deoxygalactose, cycloheximide, alpha-aminoadipate, and the like can also be fine-tuned.
  • the screening assay of the present invention can also be used in identifying compounds that trigger or initiate, enhance or stabilize protein-protein interaction between AW755252 or a mutant thereof and a protein selected from the group consisting of GROUPl or a mutant thereof. For example, if a disease or disorder is associated with decreased expression of AW755252 and/or a member of selected from the group of GROUPl, then the disease or disorder may be treated or prevented by strengthening or stabilizing the interaction between AW755252 and the AW755252-interacting member in a patient.
  • a disease or disorder is associated with mutant forms of AW755252 and/or a AW755252-interacting protein that lead to weakened or abolished protein-protein interaction therebetween, then the disease or disorder may be treated with a compound that initiates or stabilizes the interaction between the mutant forms of AW755252 and/or the AW755252-interacting protein.
  • a screening assay can be performed in the same manner as described above, except that a positively selectable marker is used.
  • a positively selectable marker is used.
  • AW755252 or a mutant form or a binding domain thereof, and a protein selected from the group consisting of GROUPl, or a mutant form or a binding domain thereof are used as test proteins expressed in the form of fusion proteins as described above for purposes of a two-hybrid assay.
  • the fusion proteins are expressed in a host cell and allowed to interact with each other in the presence of one or more test compounds.
  • a gene encoding a positively selectable marker such as the lacZ protein may be used as a reporter gene such that when a test compound enables or enhances the interaction between AW755252, or a mutant form or a binding domain thereof, and a protein selected from the group consisting of GROUPl or a mutant form or a binding domain thereof, the lacZ protein, i.e., beta-galactosidase is expressed.
  • the lacZ protein i.e., beta-galactosidase is expressed.
  • the compound may be identified based on the appearance of a blue color when the host cells are cultured in a medium containing X-Gal.
  • control assay is performed in which the above screening assay is conducted in the absence of the test compound. The result is then compared with that obtained in the presence of the test compound.
  • each of the interacting pair can be expressed and purified.
  • the purified interacting protein pairs are then allowed to interact with each other in vitro under appropriate conditions.
  • the interacting protem complex can be stabilized by crosslinking or other techniques.
  • the interacting complex can be studied using various biophysics techniques including, e.g., X-ray crystallography, NMR, computer modeling, mass spectrometry, and the like.
  • structural information can also be obtained from protein complexes formed by interacting proteins and a compound that initiates or stabilizes the interaction of the proteins.
  • understanding of the interaction between the proteins of interest in the presence or absence of a modulator can also be derived from mutagenesis analysis using yeast two-hybrid system or other methods for detection protein-protein interaction.
  • various mutations can be introduced into the interacting proteins and the effect of the mutations on protein-protein interaction is examined by a suitable method such as the yeast two-hybrid system.
  • an identified peptide compound capable of modulating particular protein-protein interactions can also be analyzed by the alanine scanning technique and/or the two-hybrid assay to determine the domains or residues of the peptide important to its modulating effect on particular protein-protein interactions.
  • the peptide compound can be used as a lead molecule for rational design of small organic molecules or peptide mimetics. See Huber et al, Curr. Med. Chem., 1:13-34 (1994).
  • a structural model can be established by a modeling process that may incorporate data from NMR analysis, X-ray diffraction data, alanine scanning, spectroscopic techniques and the like.
  • Various techniques including computational analysis, similarity mapping and the like can all be used in this modeling process. See e.g., Perry et al, in OSAR: Quantitative Structure-Activity Relationships in Drug Design, pp.189-193, Alan R. Liss, Inc., 1989; Rotivinen et al, Ada Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al., Proc. R. Soc.
  • a template can be formed based on the established model.
  • Various compounds can then be designed by linking various chemical groups or moieties to the template.
  • Various moieties of the template can also be replaced.
  • the peptide or mimetics thereof can be cyclized, e.g., by linking the N-terminus and C-terminus together, to increase its stability.
  • AW755252 and the AW755252-interacting proteins suggest that these proteins and/or the protein complexes formed by such proteins may be involved in the same biological processes and disease pathways.
  • one may modulate such biological processes by modulating the functions and activities of AW755252, a AW755252-interacting protein, and a protein complex formed by the proteins.
  • modulating the functions or activities of AW755252, a AW755252-interacting protein, and a protein complex formed by the proteins means causing any forms of alteration of the properties, biological activities or functions of the proteins or protein complexes, including, e.g., increasing the levels of AW755252, a AW755252-interacting protein or a protein complex formed by the proteins, enhancing or reducing their biological activities, increasing or decreasing their stability, altering their affinity or specificity to certain other biological molecules, etc.
  • an AW755252-containing protein complex of the present invention or its members thereof may be involved in cardiac function.
  • assays such as those described in Section 4 may be used in determining the effect of an abe ⁇ ation in a particular AW755252-containing complex or an interacting member thereof on cardiac function.
  • the diagnostic methods as described in Section 4 can be used in diagnosing the disease or disorder.
  • various in vitro and in vivo assays may be employed to test the therapeutic or prophylactic efficacies of the various therapeutic approaches described in Sections 6.2 and 6.3 which are aimed to modulate the functions and activities of a particular
  • AW755252-containing complex of the present invention or an interacting member thereof. Similar assays can also be used to test whether the therapeutic approaches described in Sections 6.2 and 6.3 result in the modulation of cardiac function.
  • the cell model or transgenic animal model described in Section 7 may be employed in the in vitro and in vivo assays.
  • the method for modulating the function and activities of AW755252-containing protein complexes of the present invention or interacting members thereof may be employed to modulate cardiac function.
  • the methods may also be used in the treatment or prevention of ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris.
  • methods are provided for reducing in a patient the level and/or activity of a protein complex identified in accordance with the present invention which comprises AW755252 and a member of the GROUPl.
  • methods are also provided for reducing in a patient the level and/or activity of a AW755252-interacting protein selected from the GROUPl.
  • an antibody may be administered to a patient.
  • the antibody administered may be immunoreactive with AW755252 or a member of the GROUPl .
  • Suitable antibodies may be monoclonal or polyclonal that fall within any antibody classes, e.g., IgG, IgM, IgA, etc.
  • the antibody suitable for this invention may also take a form of various antibody fragments including, but not limited to, Fab and F(ab')2, single-chain fragments (scFv), and the like.
  • an antibody selectively immunoreactive with the protein complex formed from AW755252 and a AW755252-interacting protein in accordance with the present invention is administered to a patient.
  • an antibody specific to a AW755252-interacting protein selected from the GROUPl is administered to a patient.
  • Methods for making the antibodies of the present invention should be apparent to a person of skill in the art, especially in view of the discussions in Section 3 above.
  • the antibodies can be administered in any suitable form and route as described in Section 8 below.
  • the antibodies are administered in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the antibodies may be delivered by a gene-therapy approach.
  • nucleic acids encoding the antibodies may be introduced into a patient such that desirable antibodies may be produced recombinantly in vivo from the nucleic acids.
  • the nucleic acids with appropriate transcriptional and translation regulatory sequences can be directly administered into the patient.
  • the nucleic acids can be incorporated into a suitable vector as described in Sections 2.2 and 5.3.1.1 and delivered into a patient along with the vector.
  • the expression vector containing the nucleic acids can be administered directly to a patient. It can also be introduced into cells, preferably cells derived from a patient to be treated, and subsequently delivered into the patient by cell transplantation. See Section 6.3.2 below.
  • antisense compounds specific to nucleic acids encoding one or more interacting protein members of a protein complex identified in the present invention is administered to a patient to be therapeutically or prophylactically teeated.
  • the antisense compounds should specifically inhibit the expression of the one or more interacting protein members.
  • antisense drugs generally act by hybridizing to a particular target nucleic acid thus blocking gene expression. Methods for designing antisense compounds and using such compounds in treating diseases are well known and well developed in the art. For example, the antisense drug Viteavene ® (fomivirsen), a 21 -base long oligonucleotide, has been successfully developed and marketed by Isis Pharmaceuticals, Inc. for treating cytomegalovirus (CMV)-induced retinitis.
  • CMV cytomegalovirus
  • antisense compounds are oligonucleotides designed based on the nucleotide sequence of the mRNA or gene of one or more of the interacting protein members of a particular protein complex of the present invention.
  • antisense compounds can be designed to specifically hybridize to a particular region of the gene sequence or mRNA of one or more of the interacting protein members to modulate (increase or decrease), replication, teanscription, or translation.
  • the term "specifically hybridize” or paraphrases thereof means a sufficient degree of complementarity or pairing between an antisense oligo and a target DNA or mRNA such that stable and specific binding occurs therebetween. In particular, 100% complementary or pairing is not required. Specific hybridization takes place when sufficient hybridization occurs between the antisense compound and its intended target nucleic acids in substantially absence of non-specific binding of the antisense compound to non-target sequences under predetermined conditions, e.g., for purposes of in vivo treatment, preferably under physiological conditions. Preferably, specific hybridization results in the interference with normal expression of the target DNA or mRNA.
  • an antisense oligo can be designed to specifically hybridize to the replication or transcription regulatory regions of a target gene, or the translation regulatory regions such as translation initiation region and exon/intron junctions, or the coding regions of a target mRNA.
  • the antisense compounds preferably contain modified backbones or non-natural internucleoside linkages, including but not limited to, modified phosphorous-containing backbones and non-phosphorous backbones such as mo holino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones; formacetyl and thioformacetyl backbones; alkene-containing backbones; methyleneimino and methylenehydrazino backbones; amide backbones, and the like.
  • modified phosphorous-containing backbones and non-phosphorous backbones such as mo holino backbones
  • siloxane, sulfide, sulfoxide, sulfone, sulfonate, sulfonamide, and sulfamate backbones formacetyl and thioformacetyl backbones
  • modified phosphorous-containing backbones include, but are not limited to phosphorothioates, phosphorodithioates, chiral phosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphotriesters, and boranophosphates and various salt forms thereof. See e.g, U.S. Pat. Nos.
  • non-phosphorous containing backbones described above are disclosed in, e.g., U.S. Pat. Nos. 5,034,506; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
  • PNA peptide nucleic acid
  • amide containing backbone e.g., an aminoethylglycine backbone.
  • PNA antisense compounds are resistant to RNAse H digest and thus exhibit longer half-life.
  • various modifications may be made in PNA backbones to impart desirable drug profiles such as better stability, increased drug uptake, higher affinity to target nucleic acid, etc.
  • oligonucleotides with substituted or modified sugar moieties may also be used.
  • an antisense compound may have one or more 2'-O-methoxyethyl sugar moieties. See e.g., U.S. Pat. Nos. 4,981 ,957; 5,118,800; 5,319,080; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,567,811; 5,576,427; 5,591,722; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference.
  • oligonucleotide modifications are also useful including linking an oligonucleotide to a lipid, phospholipid or cholesterol moiety, cholic acid, thioether, aliphatic chain, polyamine, polyethylene glycol (PEG), or a protein or peptide.
  • the modified oligonucleotides may exhibit increased uptake into cells, improved stability, i.e., resistance to nuclease digestion and other biodegradations. See e.g., U.S. PatentNo.4,522,811; Burnham, Am. J. Hosp. Phartn., 15:210-218 (1994).
  • Antisense compounds can be synthesized using any suitable methods known in the art. In fact, antisense compounds may be custom made by commercial suppliers. Alternatively, antisense compounds may be prepared using DNA synthesizers commercially from various vendors, e.g., Applied Biosystems Group of Norwalk, CT.
  • the antisense compounds can be formulated into a pharmaceutical composition with suitable carriers and admimstered into a patient using any suitable route of administration.
  • the antisense compounds may also be used in a "gene-therapy" approach. That is, the oligonucleotide is subcloned into a suitable vector and transformed into human cells. The antisense oligonucleotide is then produced in vivo through transcription. Methods for gene therapy are disclosed in Section 6.3.2 below.
  • an enzymatic RNA or ribozyme is designed to target the nucleic acids encoding one or more of the interacting protein members of the protein complex of the present invention.
  • Ribozymes are RNA molecules, which have an enzymatic activity and are capable of repeatedly cleaving other separate RNA molecules in a nucleotide base sequence specific manner. See Kim et al., Proc. Natl. Acad. of Sci. USA, 84:8788 (1987); Haseloff and Gerlach, Nature, 334:585 (1988); and Jefferies et al, Nucleic Acid Res., 17:1371 (1989).
  • a ribozyme typically has two portions: a catalytic portion and a binding sequence that guides the binding of ribozymes to a target RNA through complementary base-pairing.
  • a catalytic portion Once the ribozyme is bound to a target RNA, it enzymatically cleaves the target RNA, typically destroying its ability to direct translation of an encoded protein. After a ribozyme has cleaved its RNA target, it is released from that target RNA and thereafter can bind and cleave another target. That is, a single ribozyme molecule can repeatedly bind and cleave new targets. Therefore, one advantage of ribozyme treatment is that a lower amount of exogenous RNA is required as compared to conventional antisense therapies. In addition, ribozymes exhibit less affinity to mRNA targets than DNA-based antisense oligos, and therefore are less prone to bind to wrong targets.
  • a ribozyme may target any portions of the mRNA of one or more interacting protein members including AW755252, and GROUP 1 proteins.
  • Methods for selecting a ribozyme target sequence and designing and making ribozymes are generally known in the art. See e.g., U.S. Patent Nos. 4,987,071; 5,496,698; 5,525,468; 5,631,359; 5,646,020; 5,672,511; and 6,140,491, each of which is incorporated herein by reference in its entirety.
  • suitable ribozymes may be designed in various configurations such as hammerhead motifs, hairpin motifs, hepatitis delta virus motifs, group I intron motifs, or RNase P RNA motifs. See e.g., U.S. Patent Nos. 4,987,071; 5,496,698; 5,525,468; 5,631,359; 5,646,020; 5,672,511; and 6,140,491; Rossi et al, AIDS Res. Human Retroviruses 8:183 (1992); Hampel and Tritz, Biochemistry 28:4929 (1989); Hampel et al, Nucleic Acids Res. , 18:299 (1990); Pe ⁇ otta and Been, Biochemistry 31:16 (1992); and Guerrier-Takada et al, Cell, 35:849 (1983).
  • Ribozymes can be synthesized by the same methods used for normal RNA synthesis. For example, such methods are disclosed in Usman et al, J. Am. Chem. Soc, 109:7845-7854 (1987) and Scaringe et al, Nucleic Acids Res., 18:5433-5441 (1990). Modified ribozymes may be synthesized by the methods disclosed in, e.g., U.S. Pat. No. 5,652,094; International Publication Nos. WO 91/03162; WO 92/07065 and WO 93/15187; European Patent Application No.
  • Ribozymes of the present invention may be administered to cells by any known methods, e.g., disclosed in International Publication No. WO 94/02595.
  • they can be administered directly to a patient through any suitable route, e.g., intravenous injection.
  • they may be delivered in encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • they may also be delivered by gene therapy approach, using a DNA vector from which the ribozyme RNA can be transcribed directly. Gene therapy methods are disclosed in detail below in Section 6.3.2.
  • the patient level and activity of a particular protein complex and the interacting protein members thereof identified in accordance with the present invention may also be inhibited by various other methods.
  • compounds identified in accordance with the methods described in Section 5 that are capable of interfering with or dissociating protein-protein interactions between the interacting protein members of a protein complex may be administered to a patient.
  • Compounds identified in in vitro binding assays described in Section 5.2 that bind to the AW755252-containing protein complex or the interacting members thereof may also be used in the treatment.
  • useful agents also include incomplete proteins, i.e., fragments of the interacting protein members that are capable of binding to their respective binding partners in a protein complex but are defective of its normal cellular functions.
  • binding domains of the interacting member proteins of a protein complex may be used as competitive inhibitors of the activities of the protein complex.
  • derivatives or homologues of the binding domains may also be used.
  • the gene therapy methods discussed in Section 6.2.2 below are used to "knock out" the gene encoding an interacting protein member of a protein complex, or to reduce the gene expression level.
  • the gene may be replaced with a different gene sequence or a non-functional sequence or simply deleted by homologous recombination.
  • the method disclosed in U.S. Patent No. 5,641,670, which is incorporated herein by reference, may be used to reduce the expression of the genes for the interacting protein members.
  • an exogenous DNA having at least a regulatory sequence, an exon and a splice donor site can be introduced into an endogenous gene encoding an interacting protein member by homologous recombination such that the regulatory sequence, the exon and the splice donor site present in the DNA construct become operatively linked to the endogenous gene.
  • the expression of the endogenous gene is controlled by the newly introduced exogenous regulatory sequence. Therefore, when the exogenous regulatory sequence is a strong gene expression repressor, the expression of the endogenous gene encoding the interacting protein member is reduced or blocked. See U.S. Patent No. 5,641,670.
  • the present invention also provides methods for increasing in a patient the level and/or activity of a protein complex or of an individual protein member thereof identified in accordance with the present invention. Such methods can be particularly useful in instances where a reduced level and/or activity of a protein complex or a protein member thereof are associated with a particular disease or disorder to be teeated, or where an increased level and/or activity of a protein complex or a protein member thereof would be beneficial to the improvement of a cellular function or disease state.
  • the disease or disorder may be treated or prevented.
  • the protem complex or the AW755252-interacting protein may be administered directly to the patient to increase the level and/or activity of the protein complex or the AW755252-interacting protein.
  • protem complexes prepared by any one of the methods described in Section 2.2 may be administered to the patient, preferably in a pharmaceutical composition as described below.
  • one or more individual interacting protein members of the protein complex may also be administered to the patient in need of treatment.
  • one or more proteins such as AW755252, GROUPl may be given to a patient.
  • Proteins isolated or purified from normal individuals or recombinantly produced can all be used in this respect.
  • two or more interacting protein members of a protein complex are admimstered.
  • the proteins or protein complexes may be administered to a patient needing treatment in any methods described in Section 8. 6.3.2. Gene Therapy
  • the patient level and/or activity of a particular AW755252-containing protein complex or a AW755252-interacting protein member thereof is increased or restored by the gene therapy approach.
  • nucleic acids encoding one or more protein members of a AW755252-containing protein complex of the present invention, or portions or fragments of the protein members are inteoduced into tissue cells of a patient needing treatment such that the one or more protein members are expressed from the introduced nucleic acids.
  • nucleic acids encoding one or more of AW755252, GROUPl, or fragments, homologues or derivatives thereof can be used in the gene therapy in accordance with the present invention.
  • a nucleic acid encoding a wild-type protein can be introduced into tissue cells of the patient.
  • the exogenous nucleic acid can be used to replace the co ⁇ esponding endogenous defective gene by, e.g., homologous recombination. See U.S. Patent No. 6,010,908, which is incorporated herein by reference.
  • the exogenous nucleic acid is simply used to express a wild-type protein in addition to the endogenous mutant protein.
  • the method disclosed in U.S. Patent No. 6,077,705 may be employed in gene therapy.
  • the patient is administered both a nucleic acid construct encoding a ribozyme and a nucleic acid construct comprising a ribozyme resistant gene encoding a wild type form of the gene product.
  • undesirable expression of the endogenous gene is inhibited and a desirable wild-type exogenous gene is introduced.
  • additional copies of wild-type exogenous genes may be inteoduced into the patient by gene therapy, or alternatively, a gene activation method such as that disclosed in U.S. Patent No. 5,641,670 may be used.
  • a nucleic acid encoding a desirable protein e.g., one selected from AW755252, GROUPl is incorporated into a suitable expression vector and is operably linked to a promoter in the vector.
  • Suitable promoters include but are not limited to viral transcription promoters derived from adenovirus, simian virus 40 (SV40) (e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) (e.g., CMV immediate-early promoter), human immunodeficiency virus (HIV) (e.g., long terminal repeat (LTR)), vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSV) (e.g., thymidine kinase promoter).
  • SV40 simian virus 40
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • HMV herpes simplex virus
  • HSV herpes simplex virus
  • tissue-specific promoters may be operably linked to the exogenous gene.
  • selection markers may also be included in the vector for purposes of selecting, in vitro, those cells that contain the exogenous gene.
  • selection markers known in the art may be used including, but not limited to, e.g., genes conferring resistance to neomycin, hygromycin, zeocin, and the like.
  • the exogenous nucleic acid is incorporated into a plasmid DNA vector.
  • a plasmid DNA vector Many commercially available expression vectors may be useful for the present invention, including, e.g., pCEP4, pcDNAI, pIND, pSecTag2, pVAXl, pcDNA3.1, and pBI-EGFP, and pDisplay.
  • viral vectors may also be used.
  • the viral genome is engineered to eliminate the disease-causing capability, e.g., the ability to replicate in the host cells.
  • the exogenous nucleic acid to be introduced into a patient may be incorporated into the engineered viral genome, e.g., by inserting it into a viral gene that is non-essential to the viral infectivity.
  • Viral vectors are convenient to use as they can be easily introduced into tissue cells by way of infection.
  • the recombinant virus typically is integrated into the genome of the host cell. In rare instances, the recombinant virus may also replicate and remain as extrachromosomal elements.
  • reteoviral vectors have been developed for gene therapy. These include vectors derived from adenovirus, oncoreteoviruses (e.g., MLV), lentiviruses (e.g., HIV and SIV) and other reteoviruses.
  • gene therapy vectors have been developed based on murine leukemia virus (See, Cepko, et al, Cell, 37:1053-1062 (1984), Cone and Mulligan, Proc Natl. Acad. Sci. U.S.A., 81:6349-6353 (1984)), mouse mammary tumor virus (See, Salmons et al, Biochem. Biophys. Res.
  • Adeno-associated virus (AAV) vectors have been successfully tested in clinical trials. See e.g., Kay et al, Nature Genet. 24:257-61 (2000). AAV is a naturally occurring defective virus that requires other viruses such as adenoviruses or herpes viruses as helper viruses. See Muzyczka, Curr. Top. Microbiol Immun., 158:97 (1992). A recombinant AAV virus useful as a gene therapy vector is disclosed in U.S. Patent No. 6,153,436, which is incorporated herein by reference.
  • Adenoviral vectors can also be useful for purposes of gene therapy in accordance with the present invention.
  • U.S. Patent No. 6,001,816 discloses an adenoviral vector, which is used to deliver a leptin gene intravenously to a mammal to treat obesity.
  • Other recombinant adenoviral vectors may also be used, which include those disclosed in U.S. Patent Nos. 6,171,855; 6,140,087; 6,063,622; 6,033,908; and 5,932,210, and Rosenfeld et al, Science, 252:431-434 (1991); and Rosenfeld et al, Cell, 68:143-155 (1992).
  • viral vectors include recombinant hepatitis viral vectors (See, e.g., U.S. Patent No. 5,981,274), and recombinant entomopox vectors (See, e.g., U.S. Patent Nos. 5,721,352 and 5,753,258).
  • WO 94/18834 discloses a method of delivering DNA into mammalian cells by conjugating the DNA to be delivered with a polyelectrolyte to form a complex.
  • the complex may be microinjected into or uptaken by cells.
  • exogenous gene fragment or plasmid DNA vector containing the exogenous gene may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Patent No. 6,090,619; Wu and Wu, J. Biol Chem., 263:14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991).
  • U.S. Patent No. 6,090,619 Wu and Wu, J. Biol Chem., 263:14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991).
  • 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine having 3-100 lysine residues), which is itself coupled to an integrin receptor binding moiety (e.g., a cyclic peptide having the sequence RGD).
  • a polycation moiety e.g., poly-L-lysine having 3-100 lysine residues
  • an integrin receptor binding moiety e.g., a cyclic peptide having the sequence RGD
  • the exogenous nucleic acid or vectors containing it can also be delivered into cells via amphiphiles. See e.g., U.S. Patent No. 6,071,890.
  • the exogenous nucleic acid or a vector containing the nucleic acid forms a complex with the cationic amphiphile. Mammalian cells contacted with the complex can readily take the complex up.
  • the exogenous gene can be introduced into a patient for purposes of gene therapy by various methods known in the art.
  • the exogenous gene sequences alone or in a conjugated or complex form described above, or incorporated into viral or DNA vectors may be administered directly by injection into an appropriate tissue or organ of a patient.
  • catheters or like devices may be used for delivery into a target organ or tissue. Suitable catheters are disclosed in, e.g., U.S. Patent Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705, all of which are incorporated herein by reference.
  • these vectors be administered in a pharmaceutically acceptable carrier for injection such as a sterile aqueous solution or dispersion, preferably isotonic. Dose and duration of teeatment is determined individually depending on the degree and rate of improvement. Such determinations are performed routinely by physicians in the art.
  • the exogenous gene or vectors containing the gene can be inteoduced into isolated cells using any known techniques such as calcium phosphate precipitation, microinjection, lipofection, electroporation, gene gun, receptor-mediated endocytosis, and the like.
  • Cells expressing the exogenous gene may be selected and redelivered back to the patient by, e.g., injection or cell transplantation.
  • the appropriate amount of cells delivered to a patient will vary with patient conditions, and desired effect, which can be determined by a skilled artisan. See e.g, U.S. Patent Nos. 6,054,288; 6,048,524; and 6,048,729.
  • the cells used are autologous, i.e., cells obtained from the patient being teeated.
  • Defective conditions or disorders in a patient associated with decreased level or activity of a AW755252-containing protein complex or a AW755252-interacting protein identified in accordance with the present invention can also be ameliorated by administering to the patient a compound identified by the methods described in Sections 5.3.1.4, 5.2, and Section 5.4, which is capable of modulating the functions of the protein complex or the AW755252-interacting protein, e.g., by triggering or initiating, enhancing or stabilizing protein-protein interaction between the interacting protem members of the protein complex, or the mutant forms of such interacting protein members found in the patient.
  • cell and animal models are provided in which one or more of the AW755252-containing protein complexes identified in the present invention are in an abe ⁇ ant form, e.g., increased or decreased level of the protein complexes, altered interaction between interacting protein members of the protein complexes, and/or altered distribution or localization (e.g., in organs, tissues, cells, or cellular compartments) of the protein complexes.
  • Such cell and animal models are useful tools for studying the disorders and diseases caused by the protein complex abe ⁇ ation and for testing various methods for treating the diseases and disorders.
  • Cell models having an abe ⁇ ant form of one or more of the protein complexes of the present invention are provided in accordance with the present invention.
  • the cell models may be established by isolating, from a patient, cells having an abe ⁇ ant form of one or more of the protein complexes of the present invention.
  • the isolated cells may be cultured in vitro as a primary cell culture.
  • the cells obtained from the primary cell culture or directly from the patient may be immortalized to establish a human cell line. Any methods for constructing immortalized human cell lines may be used in this respect. See generally Yeager and Reddel, Curr. Opini. Biotech., 10:465-469 (1999).
  • the human cells may be immortalized by transfection of plasmids expressing the SV40 early region genes (See e.g., Jha etal, Exp.
  • the human cells may be immortalized by recombinantly expressing the gene for the human telomerase catalytic subunit hTERT in the human cells. See Bodnar et al, Science, 279:349-352 (1998).
  • cell models are provided by recombinantly manipulating appropriate host cells.
  • the host cells may be bacteria cells, yeast cells, insect cells, plant cells, animal cells, and the like.
  • the cells are derived from mammals, preferably humans.
  • the host cells may be obtained directly from an individual, or a primary cell culture, or preferably an immortal stable human cell line.
  • human embryonic stem cells or pluripotent cell lines derived from human stem cells are used as host cells. Methods for obtaining such cells are disclosed in, e.g., Shamblott, et al, Proc. Natl. Acad. Sci. USA, 95:13726-13731 (1998) and Thomson et al, Science, 282:1145-1147 (1998).
  • a cell model is provided by recombinantly expressing one or more of the protein complexes of the present invention in cells that do not normally express such protein complexes.
  • cells that do not contain a particular protein complex may be engineered to express the protein complex.
  • a particular human protein complex is expressed in non-human cells.
  • the cell model may be prepared by introducing into host cells nucleic acids encoding all interacting protein members required for the formation of a particular protein complex, and expressing the protein members in the host cells.
  • the recombination expression methods described in Section 2.2 may be used.
  • the methods for introducing nucleic acids into host cells disclosed in the context of gene therapy in Section 6.2.2 may also be used.
  • a cell model over-expressing one or more of the protein complexes of the present invention is provided.
  • the cell model may be established by increasing the expression level of one or more of the interacting protein members of the protein complexes.
  • all interacting protem members of a particular protein complex are over-expressed.
  • the over-expression may be achieved by introducing into host cells exogenous nucleic acids encoding the proteins to be over-expressed, and selecting those cells that over-express the proteins.
  • the expression of the exogenous nucleic acids may be transient or, preferably stable.
  • the recombinant expression methods described in Section 2.2, and the methods for introducing nucleic acids into host cells disclosed in the context of gene therapy in Section 6.2.2 may be used.
  • any host cells may be employed for establishing the cell model.
  • human cells lacking a protein complex to be over-expressed or having a normal level of the protein complex are used as host cells.
  • the host cells may be obtained directly from an individual, or a primary cell culture, or preferably an immortal stable human cell line.
  • human embryonic stem cells or pluripotent cell lines derived from human stem cells are used as host cells. Methods for obtaining such cells are disclosed in, e.g., Shamblott, et al, Proc. Natl. Acad. Sci. USA, 95:13726-13731 (1998), and Thomson et al, Science, 282:1145-1147 (1998).
  • mutations that can cause reduced expression level may also be inteoduced into the gene by homologous recombination.
  • a gene encoding a ribozyme or antisense compound specific to the mRNA encoding an interacting protem member may also be inteoduced into the host cells, preferably stably integrated into the genome of the host cells.
  • a gene encoding an antibody or fragment thereof specific to an interacting protein member may also be introduced into the host cells.
  • the recombination expression methods described in Sections 2.2, 6.1 and 6.2 can all be used for purposes of manipulating the host cells.
  • the cell models of the present invention containing an abe ⁇ ant form of a AW755252-containing protein complex of the present invention are useful in screening assays for identifying compounds useful in treating diseases and disorders involving ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris. In addition, they may also be used in in vitro pre-clinical assays for testing compounds, such as those identified in the screening assays of the present invention. A variety of parameters relevant to particularly physiological disorders or diseases may be analyzed.
  • the first kind of cell populations may be those derived from ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris tissues.
  • transgenic non-human animals are provided expressing an abe ⁇ ant form of one or more of the AW755252-containing protein complexes of the present invention.
  • Animals of any species may be used to generate the transgenic animal models, including but not limited to, mice, rats, hamsters, sheep, pigs, rabbits, guinea pigs, preferably non-human primates such as monkeys, chimpanzees, baboons, and the like.
  • the transgenic animals are produced to over-express one or more protein complexes formed from AW755252 or a derivative or homologue thereof (including the animal counterpart of AW755252) and a AW755252-interacting protein selected from the group of GROUPl, or a derivative or homologue thereof (including an animal counterpart thereof).
  • Over-expression may be exhibited in a tissue or cell that normally express the animal counterparts of such protein complexes. That is the level the protem complexes is elevated and is higher than the normal level.
  • the one or more protein complexes are expressed in tissues or cells that do not normally express such protein complexes (including the animal counterpart of the human protein complexes).
  • human AW755252 and at least one human protein selected from the group of GROUPl are expressed in the transgenic animals.
  • the transgenic animals are made such that they contain and express exogenous genes encoding AW755252 or a homologue or derivative thereof and one or more of the AW755252-interacting proteins or a homologue or derivative thereof.
  • both exogenous genes are human genes.
  • exogenous genes may be operably linked to a native or non-native promoter, preferably a non-native promoter.
  • an exogenous AW755252 gene may be operably linked to a promoter that is not the native AW755252 promoter. If the expression of the exogenous gene is desired to be limited to a particular tissue, an appropriate tissue-specific promoter may be used.
  • Over-expression may also be achieved by manipulating the native promoter to create mutations that lead to gene over-expression, or by a gene activation method such as that disclosed in U.S. Patent No. 5,641,670 as described above.
  • the transgenic animal endogenous genes encoding the animal homologues of AW755252 and the animal homologues of a AW755252-interacting protein are both knocked out. Instead, the transgenic animal expresses a human version of AW755252 and a protein selected from the group of proteins in GROUPl.
  • a transgenic mouse can be generated which can be knock out for the endogenous AW755252 (AW755252-null) but expresses a wild type human AW755252 gene. Because of the homology human AW755252 gene can compensate for the endogenous AW755252 gene.
  • the transgenic animal with specific mutations in the human AW755252 transgene provide an excellent test model to predict onset and progression of the ischemic heart disease, myocardial infarction, cardiac failure, dilated cardiomyopathy, hypertrophia cordis and angina pectoris and to design and test drug formulations for treatment of AW755252 related disorders resulting from a specific mutation in humans.
  • the transgenic animal of this invention exhibits abe ⁇ ant interactions between AW755252 and a AW755252-interacting protein selected from the group of GROUPl.
  • variants of AW755252 and its interaction partners exhibiting altered protein-protein interaction properties and the nucleic acid variants encoding such variant proteins may be obtained by random or site-specific mutagenesis in combination with a protein-protein interaction assay system, particularly the yeast two-hybrid system described in Section 5.3.1.
  • variants of AW755252 and its interaction partners exhibiting increased or decreased or abolished binding affinity to each other may be identified and isolated.
  • the transgenic animal of the present invention may be made to express such protein variants by modifying the endogenous genes.
  • the nucleic acid variants may be introduced exogenously into the transgenic animal genome to express the protein variants therein.
  • the exogenous nucleic acid variants are derived from human and the co ⁇ esponding endogenous genes are knocked out.
  • the founder lines may be established by introducing appropriate exogenous nucleic acids into, or modifying an endogenous gene in, germ lines, embryonic stem cells, embryos, or sperms which are then in producing a transgenic animal.
  • the gene introduction may be conducted by various methods including those described in Sections 2.2, 6.1 and 6.2. See also, Van der Putten et al, Proc. Natl Acad. Sci. USA, 82:6148-6152 (1985); Thompson et al, Cell, 56:313-321 (1989); Lo, Mol. Cell. Biol, 3:1803-1814 (1983); Gordon, Trangenic Animals, Intl. Rev. Cytol.
  • the exogenous gene is incorporated into an appropriate vector, such as those described in Sections 2.2 and 6.2, and is transformed into embryonic stem (ES) cells.
  • ES embryonic stem
  • the transformed ES cells are then injected into a blastocyst.
  • the blastocyst with the transformed ES cells is then implanted into a su ⁇ ogate mother animal. In this manner, a chimeric founder line animal containing the exogenous nucleic acid (transgene) may be produced.
  • site-specific recombination is employed to integrate the exogenous gene into a specific predetermined site in the animal genome, or to replace an endogenous gene or a portion thereof with the exogenous sequence.
  • site-specific recombination systems may be used including those disclosed in Sauer, Curr. Opin. Biotechnol, 5:521-527 (1994); and Capecchi, et al, Science, 244:1288-1291 (1989). and Gu et al, Science, 265:103-106 (1994).
  • the Cre/lox site-specific recombination system known in the art may be conveniently used which employs the bacteriophage PI protein Cre recombinase and its recognition sequence loxP.
  • the transgenic animals of the present invention may be transgenic animals that carry a transgene in all cells or mosaic transgenic animals carrying a transgene only in certain cells, e.g., somatic cells.
  • the transgenic animals may have a single copy or multiple copies of a particular transgene.
  • founder transgenic animals thus produced may be bred to produce various offspring. For example, they can be inbred, outbred, and crossbred to establish homozygous lines, heterozygous lines, and compound homozygous or heterozygous lines.
  • Animal models used for the present invention may include myocardial infarction models, which are, for example, prepared by the following method:
  • an active compound identified in accordance with the present invention can be in any pharmaceutically acceptable salt form.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, organic or inorganic salts of the compounds of the present invention, including inorganic or organic acid addition salts of the compound.
  • salts include, but are not limited to, hydrochloride salts, sulfate salts, bisulfate salts, borate salts, nitrate salts, acetate salts, phosphate salts, hydrobromide salts, laurylsulfonate salts, glucoheptonate salts, oxalate salts, oleate salts, laurate salts, stearate salts, palmitate salts, valerate salts, benzoate salts, naththylate salts, mesylate salts, tosylate salts, citrate salts, lactate salts, maleate salts, succinate salts, tartrate salts, fumarate salts, and the like. See, e.g., Berge, et al, J. Pharm. Sci, 66:1-19 (1977).
  • the active compounds can be incorporated into a formulation that includes pharmaceutically acceptable carriers such as binders (e.g., gelatin, cellulose, gum tragacanth), excipients (e.g., starch, lactose), lubricants (e.g., magnesium stearate, silicon dioxide), disintegrating agents (e.g., alginate, Primogel, and corn starch), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint).
  • binders e.g., gelatin, cellulose, gum tragacanth
  • excipients e.g., starch, lactose
  • lubricants e.g., magnesium stearate, silicon dioxide
  • disintegrating agents e.g., alginate, Primogel, and corn starch
  • sweetening or flavoring agents e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint
  • Suitable oral formulations can also be in the form of suspension, syrup, chewing gum, wafer, elixir, and the like. If desired, conventional agents for modifying flavors, tastes, colors, and shapes of the special forms can also be included.
  • the active compounds can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil.
  • the active compounds can also be administered parenterally in the form of solution or suspension, or in lyophilized form capable of conversion into a solution or suspension form before use.
  • diluents or pharmaceutically acceptable carriers such as sterile water and physiological saline buffer can be used.
  • Other conventional solvents, pH buffers, stabilizers, anti-bacteria agents, surfactants, and antioxidants can all be included.
  • useful components include sodium chloride, acetates, citrates or phosphates buffers, glycerin, dextrose, fixed oils, methyl parabens, polyethylene glycol, propylene glycol, sodium bisulfate, benzyl alcohol, ascorbic acid, and the like.
  • the parenteral formulations can be stored in any conventional containers such as vials and ampoules.
  • Topical administration examples include nasal, buccal, mucosaL rectal, or vaginal applications.
  • the active compounds can be formulated into lotions, creams, ointments, gels, powders, pastes, sprays, suspensions, drops and aerosols.
  • one or more thickening agents, humectants, and stabilizing agents can be included in the formulations. Examples of such agents include, but are not limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum, beeswax, or mineral oil, lanolin, squalene, and the like.
  • a special form of topical administration is delivery by a transdermal patch. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al, Annual Review of Medicine, 39:221-229 (1988), which is inco ⁇ orated herein by reference.
  • hydrogels made of polyethylene glycols, collagen, or poly(glycolic-co-L-lactic acid) may be useful. See, e.g., Phillips et al., J. Pharmaceut Sci. 73:1718-1720 (1984).
  • the active compounds can also be conjugated, to a water soluble non-immunogenic non-peptidic high molecular weight polymer to form a polymer conjugate.
  • an active compound is covalently linked to polyethylene glycol to form a conjugate.
  • a conjugate exhibits improved solubility, stability, and reduced toxicity and immunogenicity.
  • the active compound in the conjugate can have a longer half-life in the body, and exhibit better efficacy. See generally, Burnham, Am. J. Hosp. Pharm., 15:210-218 (1994).
  • PEGylated proteins are cu ⁇ ently being used in protem replacement therapies and for other therapeutic uses.
  • Liposomes can also be used as carriers for the active compounds of the present invention.
  • Liposomes are micelles made of various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Various modified lipids can also be used. Liposomes can reduce the toxicity of the active compounds, and increase their stability. Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art. See, e.g., U.S. Patent No. 4,522,811; Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976).
  • the active compounds can also be admimstered in combination with another active agent that synergistically treats or prevents the same symptoms or is effective for another disease or symptom in the patient treated so long as the other active agent does not interfere with or adversely affect the effects of the active compounds of this invention.
  • additional active agents include but are not limited to anti-inflammation agents, antiviral agents, antibiotics, antifungal agents, antithrombotic agents, cardiovascular drugs, cholesterol lowering agents, anti-cancer drugs, hypertension drugs, and the like.
  • the toxicity profile and therapeutic efficacy of the therapeutic agents can be determined by standard pharmaceutical procedures in cell models or animal models, e.g., those provided in Section 7.
  • the LD 50 represents the dose lethal to about 50% of a tested population.
  • the ED 50 is a parameter indicating the dose therapeutically effective in about 50% of a tested population.
  • Both LD 50 and ED 50 can be determined in cell models and animal models.
  • the IC 50 may also be obtained in cell models and animal models, which stands for the circulating plasma concentration that is effective in achieving about 50% of the maximal inhibition of the symptoms of a disease or disorder. Such data may be used in designing a dosage range for clinical trials in humans.
  • the dosage range for human use should be designed such that the range centers around the ED 50 and/or IC 50 , but significantly below the LD 50 obtained from cell or animal models.
  • therapeutically effective amount for each active compound to be included in a pharmaceutical composition of the present invention can vary with factors including but not limited to the activity of the compound used, stability of the active compound in the patient's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of abso ⁇ tion, distribution, and excretion of the active compound by the body, the age and sensitivity of the patient to be treated, and the like.
  • the amount of administeation can also be adjusted as the various factors change over time.
  • the present invention also provides for isolated nucleic acid molecules and their fragments encoding one or more interacting protein members of a protein complex identified in the present invention or portions of these polypeptides that are capable of interacting with other protein(s) of the present protein-protein interactions.
  • nucleic acid is intended to include both DNA (e.g., cDNA or genomic DNA) and RNA (e.g., mRNA).
  • This aspect of the invention also pertains to isolated nucleic acid fragments sufficient for use as hybridization probes to identify nucleic acids encoding polypeptides capable of interacting with other protein(s) of the protein-protein interactions disclosed herein, and to isolated nucleic acid fragments for use as PCR primers for the amplification or mutation of nucleic acids encoding polypeptides capable of interacting with the other proteins.
  • the nucleic acid fragment encoding a polypeptide capable of interacting with other protein(s) of the protein-protein interactions disclosed herein can be prepared by isolating a fragment, sequencing the fragment (optional), expressing the fragment (e.g., by recombinant expression in vitro) and assessing the protein interacting property of the encoded polypeptide.
  • the isolated polynucleotide may be 100% identical or less than 100% identical to a reference sequence (i.e., a specific nucleic acid sequence disclosed herein) or to a fragment of the reference sequence depending on the number of nucleotide alterations or variations in the isolated polynucleotide.
  • the isolated polynucleotide which, over its entire length, is less than 100% identical to the reference sequence or to the fragment of the reference sequence is a variant nucleic acid.
  • the number of nucleotide alterations or variations (A nt ) needed for a given % identity is determined by first multiplying (x) the total number of nucleotides (T n t) in the reference sequence by a number (n) which is obtained by dividing the percent identity by 100 (for example 0.80 for 80%, 0.90 for 90% 0.92 for 92%, 0.95 for 95%, 0.97 for 97% and so on) and then subtracting that product from said total number of nucleotides (T nt ) in the reference sequence. After this calculation, any non-integer value may be rounded off to the nearest integer to obtain the approximate number without decimal values.
  • the first decimal number is rounded off, to approximate the number of nucleic acid alterations to an integer to obtain a polynucleotide of a given % identity. If the first decimal number is 5 or greater than 5, then the number preceding the decimal point is increased by "one" and all the decimal numbers are dropped (rounded up). If the first decimal number is less than 5, then the number preceding the decimal point is unchanged and all the decimal numbers are dropped (rounded down).
  • a variant nucleic acid encoding a polypeptide capable of interacting with other protein(s) of the protein-protein interactions disclosed herein can be prepared by isolating a nucleic acid, determining the sequence identities with the reference sequences or fragments thereof, expressing the variant nucleic acid (e.g., by recombinant expression in vitro) and assessing the protein interacting property of the encoded polypeptide.
  • isolated nucleic acids, their fragments or variants encoding polypeptides of the present invention may be mouse sequences or their homologues (e.g. human proteins).
  • nucleic acid sequences of the present invention can be isolated from an appropriate biological source or library using methods known to one skilled in the art and the sequence information disclosed herein. For example, using all or a portion of a nucleic acid sequence disclosed herein as a hybridization probe, the nucleic acid such as a cDNA clone is isolated from a cDNA library of human origin. Further, utilizing the sequence information provided by the cDNA sequence, human genomic clones encoding a polypeptide identical to that set forth herein or variants thereof can be isolated.
  • nucleic acids having the appropriate level of sequence relatedness with the reference polynucleotide sequences may be identified by using hybridization and washing conditions of appropriate stringency.
  • stringent conditions and “stringent hybridization conditions” mean hybridization occurring only if there is at least 90% preferably at least 95%> and more preferably at least 97% and most preferably 100%> identity between the sequences. It is well known that during nucleic acid hybridizations, conditions can be set up so that hybridizations only occur between the probe and the target nucleic acid of interest that is highly complementary to the probe.
  • the T, fee (melting temperature; a measure of the stability of a nucleic acid duplex) of perfect hybrids formed by DNA, RNA or oligonucleotide probes can be determined according to the art known formula which is as follows:
  • the DNA denatures with a TRON for mammalian genomes, with a base composition of about 40% GC, the DNA denatures with a TRON, of about 87°C.
  • a specific example of stringent hybridization conditions is as follows: an overnight incubation at 42°C in a solution having: 5x SSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA, 50% formamide, followed by washing the hybridization support in 0. Ix SSC at about 65°C.
  • 5x SSC 150mM NaCl, 15mM trisodium citrate
  • 50 mM sodium phosphate pH7.6
  • 5x Denhardt's solution 10% dextran sulfate
  • the [Na j M of different strengths of SSC are as follows: For 20X, 10X, 5X, 2X, IX. 0.1X are 3.3, 1.65, 0.825, 0.33, 0.165 and 0.0165, respectively.
  • Hybridization and wash conditions are well known and exemplified in laboratory manuals. See, Sambrook, et al, Molecular Cloning: A Laboratory Manual, particularly Chapter 10 (third edition) therein. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
  • novel polynucleotides of the present invention may also be obtained from an appropriate library or nucleic acid containing samples (e.g. cell samples) by selective amplification of target sequences using PCR.
  • sequence information disclosed in the present application can be used to design oligonucleotide primers.
  • Primers co ⁇ esponding to regions immediately upstream and downstream of the nucleic encoding a given polypeptide can be used to amplify the sequences encoding one or more interacting protein members of a protein complex identified in the present invention or portions of these polypeptides that are capable of interacting with other protein(s) of the present protein-protein interactions.
  • the oligonucleotide primers can be from 15 to about 25 nucleotides long.
  • primers of about 20 nucleotides long are used.
  • stringency of annealing between the primer and the target very high, the formation of spurious products (i.e., those that do not encode polypeptides capable of interacting with bait polypetides) can be avoided.
  • spurious products i.e., those that do not encode polypeptides capable of interacting with bait polypetides
  • steategies known in the prior art e.g., choosing suitable length of primers, avoiding substantial tandem repeats of one or more nucleotides in the primer, avoiding sequences prone to secondary structure, nested primers etc., may be applied to achieve specificity.
  • the PCR primers specific to a given nucleic acid can be used to isolate polynucleotides (e.g., from mouse and/or other mammalian samples including humans), the polynucleotides encoding polypeptides identical to the disclosed polypeptides and variants thereof.
  • the polynucleotides may then be subject to various prior art known techniques for elucidation of the polynucleotide sequence. In this way, variants of (or mutations in) the polynucleotide sequence can be detected. This information can be used in the protein-protein interaction of the invention.
  • probes and primers based on the nucleic acid sequences disclosed herein can be used to detect and isolate transcripts or genomic sequences encoding polypeptides of interest from mouse samples or homologous polypeptides from human samples.
  • an isolated nucleic acid molecule of the invention consists essentially of nucleotide sequence shown in SEQ ID NO: 13.
  • an isolated nucleic acid molecule of the invention consists essentially of a nucleic acid molecule which is a complement of the nucleic acid sequence shown in SEQ ID NO: 13 or a portion of any of these nucleic acid sequences.
  • An isolated nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO: 13 is either fully complementary or sufficiently complementary to the nucleotide sequence shown in SEQ ID NO: 13, respectively, so that it can hybridize to the nucleotide sequence shown in SEQ ID NO: 13, respectively, under stringent hybridization conditions.
  • the nucleic acid fragments of the invention consist essentially of contiguous nucleotides 1 to 1458 set forth in SEQ ID NO: 13.
  • the hybridization probe used to detect and isolate these fragments can be a segment of 15-mer to 30-mer, 50-mer, 100-mer or more of the nucleic acid set forth in SEQ ID NO:13.
  • yeast two-hybrid system The principles and methods of the yeast two-hybrid system have been described in detail (Bartel and Fields, 1997). The following is thus a description of the particular procedure that we used, which was applied to all proteins.
  • the cDNA encoding the bait protein was generated by PCR from cDNA prepared from a desired tissue.
  • the cDNA product was then inteoduced by recombination into the yeast expression vector pGBT.Q, which is a close derivative of pGBT.C (See Bartel et al., Nat Genet., 12:72-77 (1996)) in which the polylinker site has been modified to include Ml 3 sequencing sites.
  • the new construct was selected directly in the yeast strain PNY200 for its ability to drive tryptophane synthesis (genotype of this strain: MATalpha te ⁇ l-901 leu2-3,112 ura3-52 his3-200 ade2 gal4delta gal80).
  • the bait was produced as a C-terminal fusion protein with the DNA binding domain of the transcription factor Gal4 (amino acids 1 to 147).
  • Prey libraries were transformed into the yeast strain BK100 (genotype of this strain: MATa tepl-901 leu2-3,112 ura3-52 his3-200 gal4delta gal80 LYS2::GAL-HIS3 GAL2-ADE2 met2::GAL7-lacZ), and selected for the ability to drive leucine synthesis.
  • each cDNA was expressed as a fusion protein with the teanscription activation domain of the transcription factor Gal4 (amino acids 768 to 881) and a 9 amino acid hemagglutinin epitope tag.
  • PNY200 cells (MATalpha mating type), expressing the bait, were then mated with BK100 cells (MATa mating type), expressing prey proteins from a prey library.
  • the resulting diploid yeast cells expressing proteins interacting with the bait protein were selected for the ability to synthesize tryptophan, leucine, histidine, and adenine.
  • DNA was prepared from each clone, transformed by electroporation into E. coli strain KC8 (Clontech KC8 electeocompetent cells, Catalog No. C2023-1), and the cells were selected on ampicillin-containing plates in the absence of either tryptophane (selection for the bait plasmid) or leucine (selection for the library plasmid).
  • DNA for both plasmids was prepared and sequenced by the dideoxynucleotide chain termination method. The identity of the bait cDNA insert was confirmed and the cDNA insert from the prey library plasmid was identified using the BLAST program to search against public nucleotide and protein databases. Plasmids from the prey library were then individually transformed into yeast cells together with a plasmid driving the synthesis of lamin and 5 other test proteins, respectively, fused to the Gal4 DNA binding domain. Clones that gave a positive signal in the beta-galactosidase assay were considered false-positives and discarded. Plasmids for the remaining clones were transformed into yeast cells together with the original bait plasmid. Clones that gave a positive signal in the beta-galactosidase assay were considered true positives.
  • the amplified V H and V K genes are ligated together and subcloned into a phagemid vector for the construction of a phage display library.
  • E. coli. cells are transformed with the ligation mixtures, and thus a phage display library is established.
  • the ligated V H and V genes are subcloned into a vector suitable for ribosome display in which the VH-V k sequence is under the control of a T7 promoter. See Schaffitzel et al, J. Immun. Meth., 231:119-135 (1999).
  • the libraries are screened with the AW755252-PROTEIN2 complex and individual AW755252 and PROTEIN2. Several rounds of screening are preferably performed. Clones co ⁇ esponding to scFv fragments that bind the
  • AW755252-PROTEIN2 complex but not the individual AW755252 and PROTEIN2 are selected and purified.
  • a single purified clone is used to prepare an antibody selectively immunoreactive with the AW755252-PROTEIN2 complex.
  • the antibody is then verified by an immunochemistry method such as RIA and ELISA.
  • the clones co ⁇ esponding to scFv fragments that bind the AW755252-PROTEIN2 complex and also binds AW755252 and/or PROTEIN2 may be selected.
  • the scFv genes in the clones are diversified by mutagenesis methods such as oligonucleotide-directed mutagenesis, error-prone PCR (See Lin-Goerke et al, Biotechniques, 23:409 (1997)), dNTP analogues (See Zaccolo et al, J. Mol. Biol, 255:589 (1996)), and other methods.
  • the diversified clones are further screened in phage display or ribosome display libraries. In this manner, scFv fragments selectively immunoreactive with the AW755252-PROTEIN2 complex may be obtained.

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Abstract

L'invention concerne des complexes protéiques comprenant AW755252 et une ou plusieurs protéines choisies dans le groupe constitué par GROUP1. L'invention concerne également des méthodes d'utilisation de ces complexes protéiques dans le diagnostic de maladies et de troubles. En outre, lesdits complexes protéiques sont également utiles dans des analyses de criblage destinées à identifier des composés permettant de traiter et/ou prévenir efficacement des maladies et des troubles associés à AW755252 et ses interacteurs.
PCT/US2003/026997 2002-08-28 2003-08-26 Proteines interagissant avec aw755252 et leur utilisation Ceased WO2004019880A2 (fr)

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