WO2000065340A1 - Interactions proteine-proteine - Google Patents

Interactions proteine-proteine Download PDF

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WO2000065340A1
WO2000065340A1 PCT/US2000/010651 US0010651W WO0065340A1 WO 2000065340 A1 WO2000065340 A1 WO 2000065340A1 US 0010651 W US0010651 W US 0010651W WO 0065340 A1 WO0065340 A1 WO 0065340A1
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complex
set forth
protein
complex set
proteins
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PCT/US2000/010651
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English (en)
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Karen Heichman
Paul L. Bartel
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Myriad Genetics, Inc.
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Priority to EP00926188A priority Critical patent/EP1181549A1/fr
Priority to CA002371006A priority patent/CA2371006A1/fr
Priority to AU44754/00A priority patent/AU4475400A/en
Priority to JP2000614029A priority patent/JP2002542774A/ja
Publication of WO2000065340A1 publication Critical patent/WO2000065340A1/fr

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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
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

Definitions

  • the present invention relates to the discovery of novel protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases.
  • physiological disorders and diseases include non-insulin dependent diabetes mellitus (NIDDM), neurodegenerative disorders, such as Alzheimer's Disease (AD), and the like.
  • NIDDM non-insulin dependent diabetes mellitus
  • AD Alzheimer's Disease
  • the present invention is directed to complexes of these proteins and/or their fragments, antibodies to the complexes, diagnosis of physiological generative disorders (including diagnosis of a predisposition to and diagnosis of the existence of the disorder), drug screening for agents which modulate the interaction of proteins described herein, and identification of additional proteins in the pathway common to the proteins described herein.
  • a first step in defining the function of a novel gene is to determine its interactions with other gene products in appropriate context. That is, since proteins make specific interactions with other proteins or other biopolymers as part of functional assemblies or physiological pathways, an appropriate way to examine function of a gene is to determine its physical relationship with other genes.
  • proteins make specific interactions with other proteins or other biopolymers as part of functional assemblies or physiological pathways
  • an appropriate way to examine function of a gene is to determine its physical relationship with other genes.
  • the present invention relates to the discovery of protein-protein interactions that are involved in mammalian physiological pathways, including physiological disorders or diseases, and to the use of this discovery.
  • the identification of the interacting proteins described herein provide new targets for the identification of useful pharmaceuticals, new targets for diagnostic tools in the identification of individuals at risk, sequences for production of transformed cell lines, cellular models and animal models, and new bases for therapeutic intervention in such physiological pathways
  • one aspect of the present invention is protein complexes.
  • the protein complexes are a complex of (a) two interacting proteins, (b) a first interacting protein and a fragment of a second interacting protein, (c) a fragment of a first interacting protein and a second interacting protein, or (d) a fragment of a first interacting protein and a fragment of a second interacting protein.
  • the fragments of the interacting proteins include those parts of the proteins, which interact to form a complex.
  • This aspect of the invention includes the detection of protein interactions and the production of proteins by recombinant techniques. The latter embodiment also includes cloned sequences, vectors, transfected or transformed host cells and transgenic animals.
  • a second aspect of the present invention is an antibody that is immunoreactive with the above complex.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. While the antibody is immunoreactive with the complex, it is not immunoreactive with the component parts j -*
  • a third aspect of the present invention is a method for diagnosing a predisposition for physiological disorders or diseases in a human or other animal.
  • the diagnosis of such disorders includes a diagnosis of a predisposition to the disorders and a diagnosis for the existence of the disorders.
  • the ability of a first interacting protein or fragment thereof to form a complex with a second interacting protein or a fragment thereof is assayed, or the genes encoding interacting proteins are screened for mutations in interacting portions of the protein molecules.
  • the inability of a first interacting protein or fragment thereof to form a complex, or the presence of mutations in a gene within the interacting domain, is indicative of a predisposition to, or existence of a disorder.
  • the ability to form a complex is assayed in a two-hybrid assay.
  • the ability to form a complex is assayed by a yeast two-hybrid assay.
  • the ability to form a complex is assayed by a mammalian two-hybrid assay.
  • the ability to form a complex is assayed by measuring in vitro a complex formed by combining said first protein and said . second protein.
  • the proteins are isolated from a human or other animal.
  • the ability to form a complex is assayed by measuring the binding of an antibody, which is specific for the complex.
  • the ability to form a complex is assayed by measuring the binding of an antibody that is specific for the complex with a tissue extract from a human or other animal.
  • coding sequences of the interacting proteins described herein are screened for mutations.
  • a fourth aspect of the present invention is a method for screening for drug candidates which are capable of modulating the interaction of a first interacting protein and a second interacting protein.
  • the amount of the complex formed in the presence of a drug is compared with the amount of the complex formed in the absence of the drug. If the amount of complex formed in the presence of the drug is greater than or less than the amount of complex formed in the absence of the drug, the drug is a candidate for modulating the interaction of the first and second interacting proteins.
  • the drug promotes the interaction if the complex formed in the presence of the drug is greater and inhibits (or disrupts) the interaction if the complex formed in the presence of the drug is less.
  • the drug may affect the interaction directly, i.e., by modulating the binding of the two proteins, or indirectly, e.g., by modulating the expression of one or both of the proteins.
  • a fifth aspect of the present invention is a model for such physiological pathways, disorders or diseases.
  • the model may be a cellular model or an animal model, as further described herein.
  • an animal model is prepared by creating transgenic or "knock-out" animals.
  • the knock-out may be a total knock-out, i.e., the desired gene is deleted, or a conditional knock-out, i.e., the gene is active until it is knocked out at a determined time.
  • a cell line is derived from such animals for use as a model.
  • an animal model is prepared in which the biological activity of a protein complex of the present invention has been altered.
  • the biological activity is altered by disrupting the formation of the protein complex, such as by the binding of an antibody or small molecule to one of the proteins which prevents the formation of the protein complex.
  • the biological activity of a protein complex is altered by disrupting the action of the complex, such as by the binding of an antibody or small molecule to the protein complex which interferes with the action of the protein complex as described herein.
  • a cell model is prepared by altering the genome of the cells in a cell line.
  • the genome of the cells is modified to produce at least one protein complex described herein.
  • the genome of the cells is modified to eliminate at least one protein of the protein complexes described herein.
  • a sixth aspect of the present invention are nucleic acids coding for novel proteins discovered in accordance with the present invention.
  • a seventh aspect of the present invention is a method of screening for drug candidates useful for treating a physiological disorder.
  • drugs are screened on the basis of the association of a protein with a particular physiological disorder. This association is established in accordance with the present invention by identifying a relationship of the protein with a particular physiological disorder.
  • the drugs are screened by comparing the activity of the protein in the presence and absence of the drug. If a difference in activity is found, then the drug is a drug candidate for the physiological disorder.
  • the activity of the protein can be assayed in vitro or in vivo using conventional techniques, including transgenic animals and cell lines of the present invention. 5
  • the present invention is the discover)' of novel interactions between proteins described herein.
  • the genes coding for some of these proteins may have been cloned previously, but their potential interaction in a physiological pathway or with a particular protein was unknown. Alternatively, the genes coding for some of these proteins have not been cloned previously and represent novel genes. These proteins are identified using the yeast two-hybrid method and searching a human total brain library, as more fully described below.
  • Glucose Transporter 1 (Glutl) and DRAL(FHL2) A fragment of Glutl and DRAL(FHL2) Glutl and a fragment of DRAL(FHL2) A fragment of Glutl and a fragment of DRAL(FHL2)
  • Glucose Transporter 1 (Glutl) and myosin heavy chain A fragment of Glutl and myosin heavy chain
  • Glucose Transporter 1 Glutl
  • HSS human sperm surface protein
  • OGTase/Mvosin Heavy Chain Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and myosin heavy chain
  • Insulin-Regulated Membrane- Spanning Aminopeptidase (IRAP) and 14-3-3 beta A fragment of IRAP and 14-3-3 beta IRAP and a fragment of 14-3-3 beta A fragment of IRAP and a fragment of 14-3-3 beta
  • INP Insulin-Regulated Membrane-Spanning Aminopeptidase
  • HSS human sperm surface protein
  • PI-3 Kinase pi 10 subunit PI-3K110
  • Complement Protein C4 A fragment of PI-3K110 and Complement Protein C4
  • PI-3K110 and a fragment of Complement Protein C4 A fragment of PI-3K110 and a fragment of Complement Protein C4 TABLE 9
  • PI-3 Kinase pi 10 subunit PI-3K110
  • Tenascin XB A fragment of PI-3K110 and Tenascin XB PI-3K110 and a fragment of Tenascin XB
  • a fragment of PI-3K110 and GAA PI-3K110 and a fragment of GAA A fragment of PI-3K110 and a fragment of GAA
  • C-myc Binding Protein (MM-1) and C-Napl A fragment of MM-1 and C-Napl MM-1 and a fragment of C-Napl A fragment of MM- 1 and a fragment of C-Nap 1
  • MM-1 C-myc Binding Protein (MM-1) and Beta Spectrin A fragment of MM-1 and Beta Spectrin
  • MM-1 and a fragment of Beta Spectrin A fragment of MM-1 and a fragment of Beta Spectrin
  • Nef- Associated Factor 1 beta (Naflb) and I-TRAF A fragment of Naflb and I-TRAF Naflb and a fragment of I-TRAF A fragment of Naflb and a fragment of I-TRAF
  • Akt kinase 1 (Aktl) and NuMAl A fragment of Aktl and NuMAl
  • Akt2 Akt kinase 2
  • BAP31 Akt kinase 2 (Akt2)
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and desmin A fragment of OGTase and desmin OGTase and a fragment of desmin A fragment of OGTase and a fragment of desmin
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and alpha-karyopherin A fragment of OGTase and alpha-karyopherin
  • OGTase and a fragment of alpha-karyopherin A fragment of OGTase and a fragment of alpha-karyopherin
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and clone 25100
  • OGTase O-linked N-acetylglucosaminyltransferase
  • clone 25100 A fragment of OGTase and clone 25100 OGTase and a fragment of clone 25100
  • Rab4 and alpha-cantein-like protein A fragment of Rab4 and alpha-cantein-like protein Rab4 and a fragment of alpha-cantein-like protein A fragment of Rab4 and a fragment of alpha-cantein-like protein
  • Rab4 and Rab2 A fragment of Rab4 and Rab2
  • Rab4 and a fragment of Rab2 A fragment of Rab4 and a fragment of Rab2
  • Glucose Transporter 4 Glucose Transporter 4
  • PN7065 Novel Protein Fragment PN7065
  • Glucose Transporter 4 Glut4
  • Novel Protein Fragment PN7386 PN7386
  • OGTase/PN6931 Interaction O-linked N-acetylglucosaminyltransferase (OGTase) and Novel Protein Fragment PN6931
  • PN6931 A fragment of OGTase and PN6931 OGTase and a fragment of PN6931 A fragment of OGTase and a fragment of PN6931
  • Nef- Associated Factor 1 beta Naflb
  • Novel Protein Fragment PN7582 PN7582
  • OGTase O-linked N-acetylglucosaminyltransferase
  • MOP2 A fragment of OGTase and MOP2 OGTase and a fragment of MOP2 A fragment of OGTase and a fragment of MOP2 TABLE 34
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and Clone 25100
  • OGTase and Clone 25100 A fragment of OGTase and Clone 25100 OGTase and a fragment of Clone 25100
  • OGTase O-linked N-acetylglucosaminyltransferase
  • OGTase O-linked N-acetylglucosaminyltransferase
  • EGR1 A fragment of OGTase and EGR1 OGTase and a fragment of EGR1 A fragment of OGTase and a fragment of EGR1
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and Dynamin II A fragment of OGTase and Dynamin II
  • OGTase and a fragment of Dynamin II A fragment of OGTase and a fragment of Dynamin II
  • OHTase O-linked N-acetylglucosaminyltransferase (OGTase) and INT-6
  • OGTase O-linked N-acetylglucosaminyltransferase
  • HSPC028 A fragment of OGTase and HSPC028 OGTase and a fragment of HSPC028
  • OGTase O-linked N-acetylglucosaminyltransferase
  • B AP31 O-linked N-acetylglucosaminyltransferase
  • OGTase O-linked N-acetylglucosaminyltransferase (OGTase) and Interferon-Ind Protein A fragment of OGTase and Interferon-Ind Protein OGTase and a fragment of Interferon-Ind Protein A fragment of OGTase and a fragment of Interferon-Ind Protein
  • Glucose Transporter 4 Glut4
  • Beta-catenin A fragment of Glut4 and Beta-catenin
  • Glucose Transporter 4 (Glut4) and Alpha-SNAP
  • Glucose Transporter 4 Glut4
  • MAPKKK6 A fragment of Glut4 and MAPKKK6 Glut4 and a fragment of MAPKKK6
  • Glucose Transporter 1 Glutl
  • DRAL/FHL2 A fragment of Glutl and DRAL/FHL2 Glutl and a fragment of DRAL/FHL2 Glutl and a fragment of DRAL/FHL2 A fragment of Glutl and a fragment of DRAL/FHL2
  • Glucose Transporter 1 Glutl
  • MYSA cardiac muscle myosin heavy chain
  • Glutl and a fragment of MYSA A fragment of Glutl and a fragment of MYSA
  • Insulin-Regulated Membrane-Spanning Aminopeptidase (IRAP) and SG2NA A fragment of IRAP and SG2NA IRAP and a fragment of SG2NA
  • PI-3 Kinase p85 subunit PI-3K85
  • PI-3 Kinase p85 subunit PI-3K85
  • SLP-76 A fragment of PI-3K85 and SLP-76
  • PI-3 Kinase p85 subunit PI-3K85
  • 14-3-3-eta A fragment of PI-3K85 and 14-3-3-eta PI-3K85 and a fragment of 14-3-3-eta
  • a fragment of PI-3K85 and TACC2 PI-3K85 and a fragment of TACC2 A fragment of PI-3K85 and a fragment of TACC2
  • Glucose Transporter 4 Glut4
  • MM-1 C-Myc Binding Protein
  • Glucose Transporter 1 (Glutl) and KIAA0144 (KIAA) A fragment of Glut 1 and KIAA
  • VAP-A Associated Protein A
  • OGTase O-Linked- ⁇ -AcetylglucosaminylTransferase
  • ⁇ afla ⁇ EF-Associated Factor 1 Alpha
  • OGTase and a fragment of ⁇ afla A fragment of OGTase and a fragment of ⁇ afla
  • OHTase O-Linked- ⁇ -AcetylglucosaminylTransferase
  • Alpha-2-Catenin O-Linked- ⁇ -AcetylglucosaminylTransferase
  • PI-3 Kinase pi 10 subunit PI-3K110
  • TRIP15 A fragment of PI-3K110 and TRIP 15 PI-3K110 and a fragment of TRIP15
  • Glucose Transporter 4 (Glut4) and Tankyrase A fragment of Glut4 and Tankyrase Glut4 and a fragment of Tankyrase A fragment of Glut4 and a fragment of Tankyrase
  • IRAP Insulin-Regulated Membrane-Spanning Aminopeptidase
  • PDPZ protein tyrosine phosphatase zeta
  • Insulin-Regulated Membrane- Spanning Aminopeptidase (IRAP) and ⁇ -spectrin A fragment of IRAP and ⁇ -spectrin IRAP and a fragment of ⁇ -spectrin A fragment of IRAP and a fragment of ⁇ -spectrin TABLE 69
  • IRAP Insulin-Regulated Membrane-Spanning Aminopeptidase
  • PI-3K85 PI-3 Kinase p85 subunit
  • IRAP and a fragment of PI-3K85 A fragment of IRAP and a fragment of PI-3K85
  • PI-3 Kinase pi 10 subunit PI-3K110
  • APP Amyloid Precursor Protein
  • NIDDM non- insulin dependent diabetes mellitus
  • Glut4 is a cytokine- inducible gene that reportedly acts as a negative regulator of cardiac-specific genes (Jeyaseelan et al., 1997). This interaction may serve as a tie between heart disease and diabetes.
  • PN7065 bears a striking similarity to a rat salt-induced protein kinase (GenBank accession AB020480). Experiments in rats have shown that this salt-induced kinase may play an important role in the regulation of adrenocortical functions in response to high plasma salt and ACTH stimulation (Wang et al, 1999). It is possible that Glut4 may act as a substrate for the kinase.
  • PN7386 is identical to a human chromosome 20 clone called 850H21 (GenBank accession AL031680) that is uncharacterized in the literature. It is possible that the protein product of this clone may participate in protein trafficking or in the signal transduction mechanism that regulates this process.
  • Beta-catenin is a protein containing so-called Armadillo repeats that is involved in two important cellular processes: signal transduction via the Wingless pathway and cell adhesion (Ben-Ze'ev et al., 1998). This interaction between Glut4 and beta-catenin may shed light on the regulation of insulin-responsive glucose transport since it links the transporter to an important signaling pathway.
  • the alpha-SNAP is an important mediator in the cellular process of intracellular transport (St-Denis, et al., 1998).
  • alpha-SNAP and Glut4 interact provides a link between glucose transportation and the machinery required to perform movement of the glucose transporter between the outside and the interior of the cell.
  • Glut4 has been demonstrated to interact with tropomyosin 3, a protein involved in muscle contraction (Squire et al., 1998). This interaction may represent a link between glucose uptake and muscle function.
  • MAPKKK6 putative protein kinase
  • 14-3-3 zeta is a signal transduction protein which has been shown to interact specifically with phospherine residues (Thorson et al., 1998). 14-3-3 zeta is part of a pathway that links the insulin receptor molecule at the cell surface to the Glut4 protein located either in the interior of the cell or also at the cell surface.
  • Glut4 that interacts with 14-3-3 zeta has been shown to be phosphorylated on a critical serine residue by the kinase Akt-2 that has also been implicated in glucose uptake (Kupriyanova et al., 1999).
  • the efp-like protein is a putative transcription factor (Orimo et al., 1995). Its function is not well described but the Glut4 protein could influence the transcriptional activation of various genes, some of which might be involved in cellular metabolism.
  • Tankyrase is a known telomere-asscoiated protein (Smith et al., 1998).
  • MM-1 MM-1 and was identified by virtue of its ability to interact with the proto-oncogene c-myc (Mori et al., 1998). Other than this original characterization, there is not much else known about MM-1 but on the basis of the interaction found here, MM-1 may play critical roles in both cancer and diabetes.
  • MM-1 has been used in two- hybrid assays.
  • C-Napl was originally identified as a protein that could interact with the Nek2 cell cycle-regulated protein kinase (Fry et al., 1998).
  • the finding that MM-1 can interact with C-Napl serves to tie Glut4 and glucose transport in general to the control of the cell cycle.
  • the second protein shown to interact with MM-1 is beta spectrin.
  • Spectrins give flexibility to the cell and also act as a scaffold for other cellular proteins (Grum et al, 1999).
  • beta spectrin could interact with the vesicle-associated protein IRAP.
  • the finding that MM-1 can bind to beta spectrin further strengthens the argument that beta spectrin plays a role in glucose transport.
  • MM-1 was shown to bind to a third protein, KIAA0477, which has no known function. KIAA0477 was originally isolated from brain but its tissue distribution is not known. The finding that KIAA0477 interacts with MM-1 suggests that KIAA0477 plays a role in glucose transport or in some cellular function associated with vesicular transport.
  • DRAL is a LIM domain-containing protein that is also known as FHL-2 or SLIM3. DRAL was identified as a protein that was expressed in normal muscle tissue culture cells but was down-regulated in cancerous rhabdomyosarcoma cells (Genini et al., 1997).
  • HSS HSS is a testis-specific protein that has no known function (Shankar et al., 1998). It does contain a putative transmembrane domain and a leucine-zipper dimerization domain. Since it interacts with Glutl and it is presumably membrane-bound, HSS could potentially act with Glutl or Glut4 to affect glucose transport in the testis.
  • Glutl has also been demonstrated to interact with a form of the myosin heavy chain. Myosin heavy chain plays a key role in muscle structure and contraction (Eddinger et al., 1998). The interaction between Glutl and myosin suggests that glucose uptake and muscle function may be interrelated via the association of these two proteins.
  • dynamin As an interactor of Glutl. Dynamin has been implicated in the movement of glucose transporters via vesicular trafficking and likely plays a critical role in endocytosis (or movement from the cell surface to the interior of the cell) (Kao et al., 1998). Because of dynamin' s link to Diabetes and glucose transport, we used it in two- hybrid assays and found two proteins that could interact with it. The first protein is called CALM, and it is a clathrin assembly protein similar to the AP-3 family of adaptor proteins.
  • CALM was originally found in a lymphoid myeloid leukemia cell line containing a chromosome translocation resulting in the fusion of the AF10 gene with CALM (Dreyling et al., 1996). Clathrin and its associated proteins have a long history of involvement in the transport of vesicles from the cell surface to the interior of the cell. The association of dynamin and CALM further supports this role and ties CALM to glucose transport. Dynamin also binds to a proteosome activator subunit termed Psme3.
  • the human Psme3 gene maps to the region of the BRCA1 gene, and its function was deduced by its similarity to the mouse gene that is also referred to as the Ki antigen (Kohda et al., 1998). Since the proteosome is required for the post-translational processing and the specific degradation of certain proteins, the finding that Psme3 can bind to dynamin implies that this type of protease activity may play a key role in glucose transport.
  • DRAL/FHL2 is a protein shown to be down-regulated in rhabdomyosarcoma (Genini et al., 1997). It is entirely composed of LIM domains, polypeptide motifs that form double zinc fingers and may function by facilitating binding to nucleic acid or other proteins.
  • the same region of Glutl has been shown to bind to a cardiac muscle myosin heavy chain (MYSA)(Metzger et al., 1999).
  • KIAA0144 Although the KIAA0144 gene is uncharacterized to date, the region of it that contacts Glutl is highly enriched for serine, threonine and proline residues, possibly providing a clue to its function. Other proteins with similar "STP" domains include the extracellular portions of cell surface receptors. Finally, a potential translation product of clone 25204 bears a striking resemblance to a previously identified mouse gene called SEZ-6. This gene was found by virtue of its increased transcript levels in brain tissue following exposure to a seizure producing drug (Shimizu-Nishikawa et al., 1995).
  • IRAP insulin-regulated membranse-spanning aminopeptidase
  • IRAP also known as vpl65, gpl60 and oxytocinase
  • Glut4 transporter co-localizes with the Glut4 transporter in specified endocytic vesicles (Keller et al., 1995; Malide et al., 1997). Since expression of the N-terminal fragment of IRAP has been shown to result in the translocation of Glut4 to the plasma membrane, IRAP is thought to play a key role in glucose transport (Waters et al., 1997). Using the two-hybrid system, we have detected the interaction of IRAP with two proteins, 14-3-3 beta and HSS.
  • the 14-3-3 family of proteins are critical signal transduction proteins that bind to phosphoserine residues (Jin et al., 1996).
  • the interaction of IRAP with 14-3-3 beta strongly suggests that the function of IRAP could be regulated by phosphorylation and by the subsequent binding by 14-3-3 family members.
  • IRAP and the Glut4 glucose transporter co-localize in the same intracellular vesicles, it is possible that Glut4 may participate in signal transduction mechanisms mediated by the 14-3-3 proteins such as 14-3-3 beta.
  • IRAP has also been shown to bind to the HSS protein described above. Like Glutl and Glut4, IRAP is membrane-bound and could potentially bind to HSS in the membrane. This finding points to HSS as playing a role in glucose transport or in other important functions performed in intracellular vesicles.
  • IRAP IRAP with two more proteins.
  • the N-terminal portion of IRAP has been shown to interact with SLAP-2.
  • the rabbit homolog of SLAP-2 has been demonstrated to localize to the sarcolemma or the membrane of muscle cells although its function has not been elucidated (Wigle et al., 1997).
  • SLAP-2 may play a role in vesicular transport or may at least participate in it since it has been shown to be membrane- associated and localizes to both the cell membrane as well as to intracellular stores in the endoplasmic reticulum.
  • the C-terminal extracellular portion of IRAP has been demonstrated to interact with SG2NA.
  • SG2NA is a cell cycle nuclear autoantigen that contains so-called WD-40 repeats that are present in a variety of signal transduction proteins (Muro et al., 1995). Once again, the significance of this interaction is unclear however it is possible that SG2NA binding to IRAP is part of a more complex regulatory mechanism.
  • VAP-A or VAP-33 VAMP-associated protein A
  • the N-terminal portion of IRAP has been shown to interact with another signal transduction protein, the zeta polypeptide of protein tyrosine phosphatase. This protein is not well characterized but could play a role in regulating glucose transport by dephosphorylating critical proteins that cause or prevent glucose transport (Nishiwaki et al., 1998).
  • the C-terminal portion of IRAP has been shown to interact with non-erythrocytic beta-spectrin. This protein is thought to be involve in secretion and could play a role in the movement of Glut 4 vesicles through IRAP (Hu et al., 1992).
  • PI-3 kinase phosphatidylinositol-3
  • This protein is a central player in cellular signal transduction and participates in transmitting signals from the outside of the cell into the interior.
  • PI-3 kinase p85 involves the insulin receptor (Martin et al., 1996).
  • Glut 4 it is well known that the movement of Glut 4 between the plasma membrane and the interior of the cell depends on the action of the PI-3 kinase signal transduction pathway since inhibitors of this kinase prevent the cycling of Glut4.
  • PI-3 kinase p85 interacts with tankyrase.
  • Two-hybrid interactions have been detected using the pi 10 catalytic subunit of PI-3 kinase, and these include interactions with the p85 and p55 subunits of the same enzyme complex as well as non-subunit interactions.
  • Protein phosphatase 5 (PP5) is a TPR domain containing protein that seems to be part of larger multiprotein complexes that possess several cellular functions (Silverstein et al., 1997).
  • Our two-hybrid studies have confirmed the biochemical interaction between protein phosphatase 5 and Hsp90, and the association between these proteins has been previously demonstrated using biochemical methods.
  • PP5 has also been demonstrated using the two-hybrid assay herein to interact with another related heat shock protein, Hsp89, and also with tankyrase.
  • Phosphatidyl inositol-3 kinase is a very important signal transduction protein and likely plays a critical role in Glut4-mediated glucose uptake (Shankar et al.. 1998). This protein participates in transmitting signals from the outside of the cell into the interior. It is composed of two subunits, the p85 regulatory subunit and the pi 10 catalytic subunit, and functions by facilitating the transmission of signals from the outside of the cell into the interior.
  • PI-3 kinase has been implicated in insulin-regulation and glucose uptake since one of its functions involves the insulin receptor (Martin et al., 1996). Further, the movement of Glut4 between the plasma membrane and the interior of the cell depends on the action of the PI-3 kinase signal transduction pathway since inhibitors of this kinase prevent the cycling of Glut4.
  • SLP-76 is a tyrosine phosphoprotein that participates in T cell signaling (Clements et al., 1998; Jackman et al., 1995). It is thought that SLP-76 acts as a so-called adaptor protein since it plays a role in intermediate steps of signal transduction. This is achieved by bridging factors that act at the plasma membrane with other molecules that perform functions within the interior of the cell.
  • SLP-76 may play a critical role in the PI-3 kinase signal transduction pathway by virtue of its ability to bind the p85 regulatory subunit.
  • the p85 subunit of PI-3 kinase has also been demonstrated to bind to two more important signal transduction proteins: 14-3-3 zeta and 14-3-3 eta. These proteins bind specifically to phosphoserine residues in a number of proteins (Oghira et al., 1997; Thorson et al., 1998; Yaffe et al., 1997).
  • our studies have shown that the Glut4 glucose transporter can also interact with 14-3-3 zeta.
  • PI-3 kinase can also be connected to glucose uptake mechanisms by its interaction with the 14-3-3 signal transduction proteins.
  • PI-3 kinase p85 was shown to interact with chromogranin C, a neuroendocrine secretory granule protein in the granin family (Ozawa et al., 1995). Members of the granin family localize to specialized secretory vesicles and are thought to serve an important function in protein sorting and secretion (Leitner et al., 1999).
  • TACC2 has been shown to interact with PI-3 kinase p85 in the yeast two-hybrid assay.
  • TACC2 is a member of a family of "transforming coiled coil” proteins that have been implicated in cellular growth control and cancer (Still et al., 1999). Although the function of TACC2 remains unknown, its interaction with p85 demonstrates that it may also be a part of an important signal transduction pathway. The pi 10 subunit of this protein has been shown to interact with complement protein C4, tenascin XB and alpha acid glucosidase (GAA). The complement C4 protein plays a key role in acitvating the classical complement pathway, and it is involved in evoking histamine release from basophils and mast cells.
  • Naturally occurring deficiencies of C4 have been correlated with a number of immune-related human diseases such as Systemic Lupus Erythmatosus, kidney disease, hepatitis, dementia and the propensity for recurrent infectiions (Mascart-Lemone et al., 1983; Vergani et al., 1985; Waters et al., 1997; Nerl et al., 1984; Lhotta et al, 1990).
  • the finding that C4 interacts with the pi 10 subunit of PI-3 kinase suggests that the function of C4 is somehow regulated by this signal transduction, perhaps in response to some immunologic stimulus.
  • the pi 10 catalytic subunit of PI- 3 kinase has been demonstrated to interact with tenascin XB.
  • Tenascin XB is an extracellular structural protein. Deficiency of tenascin XB is linked to the connective tissue disorder Ehler-Danlos syndrome (Burch et al., 1997). This finding that pi 10 can bind to tenascin XB suggest that the function of tenascin XB could be modified or regulated by the PI-3 kinase signal trasduction pathway.
  • PI-3 kinase pi 10 has also been shown to bind to GAA or alph acid glucosidase.
  • GAA is a lysosomal enzyme that catalyzes the release of glucose from glycosylated substrates, and defects in GAA result in a glycogen storage disease (Raben et al., 1995).
  • the finding that PI-3 kinase can bind to GAA suggests that GAA activity may be influenced by the PI-3 kinase signaling mechanism.
  • Our previous two-hybrid results have linked the PI-3 kinase to human diseases such as Diabetes and Alzheimer's disease. These new findings therefore link complement protein C4, tenascin XB and alpha acid glucosidase to these diseases as well as to the other diseases already described.
  • TRIP 15 is part of a larger multiprotein complex termed the signalsome that is likely to be involved in cell signaling (Seeger et al., 1998).
  • Our two- hybrid results have linked the PI-3 kinase to human diseases such as Diabetes and Alzheimer's disease.
  • O-linked N-acetylglucosaminyltransferase or OGTase is an enzyme implicated in intracellular signal transduction (Kreppel et al., 1997). It has been speculated that OGTase may play a key role in glucose uptake and may therefore participate in the Diabetes related pathways (Cooksey et al., 1999). OGTase has been shown to also interact with myosin heavy chain. The binding of myosin heavy chain to OGTase suggests that the function of myosin in muscle structure or function could be influenced by OGTase in its signal transductive capacity. This could potentially affect many cellular processes in the muscle cell, including glucose transport.
  • OGTase has been shown to bind to a novel protein termed PN6931.
  • PN6931 is very similar to the mouse kinesin light chain gene (GenBank accession AF055666). Kinesin is a molecular motor involved in cellular transport and chromosome movement (Kirchner et al., 1999). Perhaps by post- translationally modifying this novel kinesin-like protein, OGTase can influence its function.
  • Amino acids 250 to 450 of OGTase has been shown to interact with a member of the 14-3-3 protein family, 14-3-3 epsilon.
  • the 14-3-3 proteins function in intracellular signal transduction pathways by specifically binding to phosphoserine residues on other critical signaling molecules (Ogihara et al., 1997; Yaffe et al., 1997).
  • the interaction between OGTase and 14-3-3 epsilon may serve to link two different signal transduction pathways, one which involves phosphorylation and a second which involves another type of protein modification.
  • Amino acids 250 to 450 of OGTase has been shown to interact with two proteins, alpha-2 catenin and Naf 1 a.
  • Alpha-catenin is a protein related to vinculins that functions in cell-cell contact by binding to cadherins (Rudiger et al., 1998).
  • the same region of OGTase can interact with Naflb, a protein identified by virtue of its ability to bind the Nef gene product of HIV in the two-hybrid assay (Fukushi et al., 1999).
  • Over-expression of Nafla was observed to cause an increase in cell surface expression of the CD4 antigen, therefore this protein may also function in intracellular trafficking and could potentially participate in a number of diseases related to this general process such as Diabetes and Alzheimer's.
  • Naflb is a protein which was identified by virtue of its ability to bind the Nef gene product of HIV in the two-hybrid assay (Fukushi et al., 1999). Over-expression of Naflb was observed to cause an increase in cell surface expression of the CD4 antigen, therefore this protein may also function in intracellular trafficking and could potentially participate in a number of diseases related to this general process such as Diabetes and Alzheimer's.
  • the TRAF-interacting protein I-TRAF was found to be an interactor.
  • I-TRAF appears to act as a regulator of the TRAF signal transduction pathway that transmits signals from TNF (tumor necrosis factor) receptor family members (Rothe et al., 1996).
  • TNF tumor necrosis factor
  • a two-hybrid search using Naflb as a bait has also identified a novel protein fragment called PN7582. This small protein fragment does not appear to have any strong similarity to known proteins therefore its function is not readily apparent. It is very possible that it may participate in protein trafficking or signal transduction by its association with Naflb.
  • the first interactor, desmin is a cytoplasmic intermediate filament protein found in muscle (Capetanaki et al., 1998). It functions in striated muscle by connecting myofibrils with themselves and with the plasma membrane. Desmin is broken down into three functional domains, the head, rod and tail, and OGTase can bind to the rod structure.
  • the second protein shown to interact with OGTase is called alpha-karyopherin.
  • Alpha-karyopherin also known as importin and SRP1 is a ubiquitously expressed protein that plays a role in trafficking nuclear localization signal-containing proteins into the nucleus through the nuclear pore (Moroianu, 1997).
  • the third protein that OGTase has been shown to bind is glutanimyl-tRNA synthetase.
  • the aminoacyl-tRNA synthetases not only play a key role in protein synthesis, but recent studies have shown that they also impact a number of other cellular processes such as tRNA processing, RNA splicing, RNA trafficking, apoptosis, and transcriptional and translational regulation (Martinis et al, 1999).
  • the fourth protein shown to bind to OGTase in the yeast two-hybrid assay is called clone 25100 and it has no known function.
  • the gene for clone 25100 was isolated from human infant brain and appears to encode a small protein with no structural characteristics that shed light on its function. All four of these OGTase- interacting proteins may act as substrates for OGTase or may affect its function in some way. The finding that they interact with OGTase suggests that they may play a role in glucose transport or in the pathogenesis of Diabetes.
  • OGTase can interact with a variety of proteins that can fall into a number of functional categories.
  • Two proteins that have been implicated in vesicular transport bind to OGTase.
  • the first is called B AP31 , and it likely plays a critical role in sorting and transporting membrane proteins between intracellular compartments (Annaert et al., 1997).
  • the second protein is called dynamin II, and it is implicated in the movement of transport vesicles from the plasma membrane to sites within the interior of the cell (Sontag et al., 1994).
  • OGTase has been demonstrated to interact with two proteins that function in transcription, as well.
  • EGR1 early growth response protein 1
  • EGR1 early growth response protein 1
  • MOP2 a basic helix-loop-helix containing transcription factor that is involved in the induction of oxygen regulated genes (Hogenesch et al, 1997).
  • OGTase binds to a structural protein called talin.
  • Talin is a cytoplasmic protein that serves to link integrins with the actin cytoskeleton (Calderwood etal., 1999).
  • OGTase has been shown in the yeast two-hybrid assay to bind to five proteins of unknown or poorly characterized function.
  • OGTase binds to Int-6, a protein that has also been demonstrated to bind to the HTLV-I Tax transactivator and it is a component of promyelocytic leukemia nuclear bodies (Desbois et al., 1996).
  • OGTase binds to interferon-induced protein 54, an uncharacterized protein that shows a large increase in its transcript following treatment with alpha- and beta-interferons (Levy et al, 1986).
  • HSPC028, KIAA0443, and clone 25100 interact with OGTase.
  • Akt kinase is a serine/threonine protein kinase that has been implicated in insulin-regulated glucose transport and the development of non-insulin dependent diabetes mellitus (Krook et al., 1998). Because of this link, Akt kinase has been used in two-hybrid assays to determine what proteins interact with it either because they are substrates of Akt kinase or because they are regulators of the kinase. Two closely related Akt proteins were used: Aktl and its closely related family-member Akt2. Aktl and Akt2 were both shown to interact with the nuclear mitotic apparatus protein NuMAl . NuMAl displays a distinct pattern of immunofluorescent staining that varies throughout the cell cycle.
  • Aktl and Akt2 may be capable of phosphorylating NuMA, especially since the region of NuMA that interacts with Akt2 (amino acids 98 to 365) contains 23 serines and 9 threonines. Akt2 was also shown to interact with two proteins involved in vesicular transport.
  • B AP31 The first protein, B AP31 , likely plays a role in the movement of membrane-bound proteins from the Golgi appartus to the plasma membrane (Annaert et al., 1997). Since BAP31 was also shown to interact with OGTase in our previous experiments, two signal transduction proteins implicated in glucose transport have been tied to BAP31. Akt2 was also shown to bind to another vesicular transport protein termed beta-adaptin or AP2-beta. Beta-adaptin is a part of the AP2 coat assembly complex that links clathrin and to transmembrane receptors resident in coated vesicles (Pearse, 1989).
  • PTP lb is a protein tyrosine phosphatase that plays a critical role in signal transduction. It has been implicated as a negative regulator of insulin-responsive glucose transport (Chen et al., 1997), and therefore it has been used in yeast two-hybrid assays in an attempt to find more proteins involved in this function, perhaps by acting as substrates. PTP lb was shown to interact with VAMP-associated protein A (VAP-A or VAP-33). This protein has been implicated in intracellular transport (specifically exocytosis or movement to the cell surface) in A. californica and likely plays a similar role in humans (Skehel et al., 1995; weir et al., 1998).
  • the interaction between PTPlb and VAP-A serves as a potential tie between PTPlb and IRAP since IRAP was also shown to bind to VAP-A in two-hybrid experiments.
  • IRAP insulin-regulated membrane-spanning aminopeptidase (also known as vpl65, gpl60 and oxytocinase) co-localizes with the Glut4 transporter in specified endocytic vesicles (Keller et al., 1995; Malide et al., 1997). Since expression of the N-terminal fragment of IRAP has been shown to result in the translocation of Glut4 to the plasma membrane, IRAP is thought to play a key role in glucose transport (Waters et al., 1997). Thus, the result that PTPlb interacts with VAP-A serves to strengthen the tie between PTPlb and VAP-A to glucose transport and Diabetes.
  • the small GTP -binding protein Rab4 is another signal transduction factor that has been implicated in the regulation of insulin-stimulated glucose uptake (Vollenweider et al., 1997; Cormont et al., 1996). Rab4, therefore, has also been used in the yeast two-hybrid assay to find additional proteins involved in glucose transport and in Diabetes. Two proteins have been shown to bind to Rab4, an alpha-catenin-like protein (sometimes called alpha-catulin) and another small GTP-binding protein called Rab2. The alpha-catenin-like protein resembles alpha-catenin and vinculin however its function has not yet been well-characterized (Janssens et al., 1999).
  • Alpha- catenin itself is a protein related to vinculins that functions in cell-cell contact by binding to cadherins (Rudinger et al., 1998).
  • alpha-catenin was found in our previous studies to bind to OGTase and, as a consequence, has been implicated in glucose transport.
  • the second protein shown to bind to Rab4 is Rab2.
  • Rab2 has not been thought to play a role in insulin- stimulated glucose transport (Uphues et al., 1994) although it does play an important role in vesicular transport in the cell (Tisdale et al., 1998).
  • the finding that Rab4 and Rab2 interact suggests that they may be capable of influencing each others cellular functions, thus Rab2 could potentially affect Rab4's role in glucose transport.
  • the proteins disclosed in the present invention were found to interact with their corresponding proteins in the yeast two-hybrid system. Because of the involvement of the corresponding proteins in the physiological pathways disclosed herein, the proteins disclosed herein also participate in the same physiological pathways. Therefore, the present invention provides a list of uses of these proteins and DNA encoding these proteins for the development of diagnostic and therapeutic tools useful in the physiological pathways. This list includes, but is not limited to, the following examples. Two-Hybrid System
  • yeast two-hybrid system The principles and methods of the yeast two-hybrid system have been described in detail elsewhere (e.g., Bartel and Fields, 1997; Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992). The following is a description of the use of this system to identify proteins that interact with a protein of interest.
  • the target protein is expressed in yeast as a fusion to the DNA-binding domain of the yeast Gal4p.
  • DNA encoding the target protein or a fragment of this protein is amplified from cDNA by PCR or prepared from an available clone.
  • the resulting DNA fragment is cloned by ligation or recombination into a DNA-binding domain vector (e.g., pGBT9, pGBT.C, pAS2-l) such that an in- frame fusion between the Gal4p and target protein sequences is created.
  • a DNA-binding domain vector e.g., pGBT9, pGBT.C, pAS2-l
  • the target gene construct is introduced, by transformation, into a haploid yeast strain.
  • a library of activation domain fusions i.e., adult brain cDNA cloned into an activation domain vector
  • the yeast strain that carries the activation domain constructs contains one or more Gal4p-responsive reporter gene(s), whose expression can be monitored. Examples of some yeast reporter strains include Yl 90, PJ69, and CBY14a.
  • An aliquot of yeast carrying the target gene construct is combined with an aliquot of yeast carrying the activation domain library. The two yeast strains mate to form diploid yeast and are plated on media that selects for expression of one or more Gal4p-responsive reporter genes.
  • Colonies that arise after incubation are selected for further characterization.
  • the activation domain plasmid is isolated from each colony obtained in the two-hybrid search.
  • the sequence of the insert in this construct is obtained by the dideoxy nucleotide chain termination method. Sequence information is used to identify the gene/protein encoded by the activation domain insert via analysis of the public nucleotide and protein databases. Interaction of the activation domain fusion with the target protein is confirmed by testing for the specificity of the interaction.
  • the activation domain construct is co-transformed into a yeast reporter strain with either the original target protein construct or a variety of other DNA-binding domain constructs. Expression of the reporter genes in the presence of the target protein but not with other test proteins indicates that the interaction is genuine.
  • yeast two-hybrid system In addition to the yeast two-hybrid system, other genetic methodologies are available for the discovery or detection of protein-protein interactions. For example, a mammalian two-hybrid system is available commercially (Clontech, Inc.) that operates on the same principle as the yeast two- hybrid system. Instead of transforming a yeast reporter strain, plasmids encoding DNA-binding and activation domain fusions are transfected along with an appropriate reporter gene (e.g., lacZ) into a mammalian tissue culture cell line.
  • an appropriate reporter gene e.g., lacZ
  • transcription factors such as the Saccharomyces cerevisiae Gal4p are functional in a variety of different eukaryotic cell types, it would be expected that a two-hybrid assay could be performed in virtually any cell line of eukaryotic origin (e.g., insect cells (SF9), fungal cells, worm cells, etc.).
  • SF9 insect cells
  • SF9 fungal cells
  • worm cells etc.
  • Other genetic systems for the detection of protein-protein interactions include the so-called SOS recruitment system (Aronheim et al., 1997).
  • Protein interactions are detected in various systems including the yeast two-hybrid system, affinity chromatography, co-immunoprecipitation, subcellular fractionation and isolation of large molecular complexes.
  • affinity chromatography affinity chromatography
  • co-immunoprecipitation subcellular fractionation and isolation of large molecular complexes.
  • WO 99/65939 PCT published application No. WO 99/65939, each of which are incorporated herein by reference.
  • the protein of interest (or an interacting domain thereof) can be produced in eukaryotic or prokaryotic systems.
  • a cDNA encoding the desired protein is introduced in an appropriate expression vector and transfected in a host cell (which could be bacteria, yeast cells, insect cells, or mammalian cells). Purification of the expressed protein is achieved by conventional biochemical and immunochemical methods well known to those skilled in the art. The purified protein is then used for affinity chromatography studies: it is immobilized on a matrix and loaded on a column.
  • a host cell which could be bacteria, yeast cells, insect cells, or mammalian cells.
  • Extracts from cultured cells or homogenized tissue samples are then loaded on the column in appropriate buffer, and non-binding proteins are eluted. After extensive washing, binding proteins or protein complexes are eluted using various methods such as a gradient of pH or a gradient of salt concentration. Eluted proteins can then be separated by two-dimensional gel electrophoresis, eluted from the gel, and identified by micro-sequencing. The purified proteins can also be used for affinity chromatography to purify interacting proteins disclosed herein. All of these methods are well known to those skilled in the art.
  • both proteins of the complex of interest can be produced in eukaryotic or prokaryotic systems.
  • the proteins (or interacting domains) can be under control of separate promoters or can be produced as a fusion protein.
  • the fusion protein may include a peptide linker between the proteins (or interacting domains) which, in one embodiment, serves to promote the interaction of the proteins (or interacting domains). All of these methods are also well known to those skilled in the art.
  • Purified proteins of interest can also be used to generate antibodies in rabbit, mouse, rat, chicken, goat, sheep, pig, guinea pig, bovine, and horse.
  • the methods used for antibody generation and characterization are well known to those skilled in the art.
  • Monoclonal antibodies are also generated by conventional techniques. Single chain antibodies are further produced by conventional techniques. "
  • DNA molecules encoding proteins of interest can be inserted in the appropriate expression vector and used for transfection of eukaryotic cells such as bacteria, yeast, insect cells, or mammalian cells, following methods well known to those skilled in the art.
  • eukaryotic cells such as bacteria, yeast, insect cells, or mammalian cells
  • Transfected cells expressing both proteins of interest are then lysed in appropriate conditions, one of the two proteins is immunoprecipitated using a specific antibody, and analyzed by polyacrylamide gel electrophoresis. The presence of the binding protein (co-immunoprecipitated) is detected by immunoblotting using an antibody directed against the other protein. Co-immunoprecipitation is a method well known to those skilled in the art.
  • Transfected eukaryotic cells or biological tissue samples can be homogenized and fractionated in appropriate conditions that will separate the different cellular components. Typically, cell lysates are run on sucrose gradients, or other materials that will separate cellular components based on size and density. Subcellular fractions are analyzed for the presence of proteins of interest with appropriate antibodies, using immunoblotting or immunoprecipitation methods. These methods are all well known to those skilled in the art.
  • agents that disrupt protein-protein interactions can be beneficial in many physiological disorders, including, but not-limited to NIDDM, AD and others disclosed herein.
  • Each of the methods described above for the detection of a positive protein-protein interaction can also be used to identify drugs that will disrupt said interaction.
  • cells transfected with DNAs coding for proteins of interest can be treated with various drugs, and co- immunoprecipitations can be performed.
  • a derivative of the yeast two-hybrid system called the reverse yeast two-hybrid system (Leanna and Hannink, 1996), can be used, provided that the two proteins interact in the straight yeast two-hybrid system. Modulation of protein-protein interactions
  • agents which are capable of modulating the interaction will provide agents which can be used to track the physiological disorder or to use as lead compounds for development of therapeutic agents.
  • An agent may modulate expression of the genes of interacting proteins, thus affecting interaction of the proteins.
  • the agent may modulate the interaction of the proteins.
  • the agent may modulate the interaction of wild-type with wild-type proteins, wild-type with mutant proteins, or mutant with mutant proteins.
  • Agents can be tested using transfected host cells, cell lines, cell models or animals, such as described herein, by techniques well known to those of ordinary skill in the art, such as disclosed in U.S. Patents No. 5,622,852 and 5,773,218, PCT published application No.
  • the modulating effect of the agent can be screened in vivo or in vitro.
  • Exemplary of a method to screen agents is to measure the effect that the agent has on the formation of the protein complex.
  • the proteins disclosed in the present invention interact with one or more proteins known to be involved in a physiological pathway, such as in NIDDM or AD. Mutations in interacting proteins could also be involved in the development of the physiological disorder, such as NIDDM or AD, for example, through a modification of protein-protein interaction, or a modification of enzymatic activity, modification of receptor activity, or through an unknown mechanism. Therefore, mutations can be found by sequencing the genes for the proteins of interest in patients having the physiological disorder, such as insulin, and non-affected controls. A mutation in these genes, especially in that portion of the gene involved in protein interactions in the physiological pathway, can be used as a diagnostic tool, and the mechanistic understanding the mutation provides can help develop a therapeutic tool.
  • Individuals can be screened to identify those at risk by screening for mutations in the protein disclosed herein and identified as described above. Alternatively, individuals can be screened by analyzing the ability of the proteins of said individual disclosed herein to form natural complexes.
  • individuals can be screened by analyzing the levels of the complexes or individual proteins of the complexes or the mRNA encoding the protein members of the complexes.
  • Techniques to detect the formation of complexes including those described above, are known to those skilled in the art.
  • Techniques and methods to detect mutations are well known to those skilled in the art.
  • Techniques to detect the level of the complexes, proteins or mRNA are well known to those skilled in the art.
  • a number of cellular models of many physiological disorders or diseases have been generated. The presence and the use of these models are familiar to those skilled in the art.
  • primary cell cultures or established cell lines can be transfected with expression vectors encoding the proteins of interest, either wild-type proteins or mutant proteins.
  • the effect of the proteins disclosed herein on parameters relevant to their particular physiological disorder or disease can be readily measured.
  • these cellular systems can be used to screen drugs that will influence those parameters, and thus be potential therapeutic tools for the particular physiological disorder or disease.
  • the purified protein of interest can be added to the culture medium of the cells under examination, and the relevant parameters measured.
  • the DNA encoding the protein of interest can be used to create animals that overexpress said protein, with wild-type or mutant sequences (such animals are referred to as "transgenic"), or animals which do not express the native gene but express the gene of a second animal (referred to as “transplacement”), or animals that do not express said protein (referred to as “knock-out”).
  • transgenic wild-type or mutant sequences
  • transplacement animals which do not express the native gene but express the gene of a second animal
  • knock-out animals that do not express said protein
  • the knock-out animal may be an animal in which the gene is knocked out at a determined time.
  • the generation of transgenic, transplacement and knock-out animals uses methods well known to those skilled in the art.
  • parameters relevant to the particular physiological disorder can be measured.
  • These parametes may include receptor function, protein secretion in vivo or in vitro, survival rate of cultured cells, concentration of particular protein in tissue homogenates, signal transduction, behavioral analysis, protein synthesis, cell cycle regulation, transport of compounds across cell or nuclear membranes, enzyme activity, oxidative stress, production of pathological products, and the like.
  • the measurements of biochemical and pathological parameters, and of behavioral parameters, where appropriate, are performed using methods well known to those skilled in the art.
  • These transgenic, transplacement and knock-out animals can also be used to screen drugs that may influence the biochemical, pathological, and behavioral parameters relevant to the particular physiological disorder being studied.
  • Cell lines can also be derived from these animals for use as cellular models of the physiological disorder, or in drug screening.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo.
  • Several approaches for use in rational drug design include analysis of three-dimensional structure, alanine scans, molecular modeling and use of anti-id antibodies. These techniques are well known to those skilled in the art.
  • the substance may be further investigated. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • a substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature.
  • Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This approach might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a range of sources e.g., spectroscopic techniques, x-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modeling process.
  • a template molecule is then selected, onto which chemical groups that mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted thereon can be conveniently selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent it is exhibited. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • one of the proteins of the interaction is used to detect the presence of a "normal" second protein (i.e., normal with respect to its ability to interact with the first protein) in a cell extract or a biological fluid, and further, if desired, to detect the quantitative level of the second protein in the extract or biological fluid.
  • a "normal" second protein i.e., normal with respect to its ability to interact with the first protein
  • an antibody against the protein complex is used to detect the presence and/or quantitative level of the protein complex. The absence of the protein complex would be indicative of a predisposition or existence of the physiological disorder.
  • the cDNA encoding the bait protein was generated by PCR from brain cDNA.
  • Gene-specific primers were synthesized with appropriate tails added at their 5' ends to allow recombination into the vector pGBTQ.
  • the tail for the forward primer was 5'- GCAGGAAACAGCTATGACCATACAGTCAGCGGCCGCCACC-3' (SEQ ID NO:l) and the tail for the reverse primer was 5'-ACGGCCAGTCGCGTGGAGTGTTATGTCATGCGGCCGCTA-3' (SEQ ID NO:2).
  • the tailed PCR product was then introduced by recombination into the yeast expression vector pGBTQ, which is a close derivative of pGBTC (Bartel et al., 1996) in which the polylinker site has been modified to include Ml 3 sequencing sites.
  • the new construct was selected directly in the yeast J693 for its ability to drive tryptophane synthesis (genotype of this strain: Mat ⁇ , ade2, his3, leu2, trpl, URA3::GALl-lacZ LYS2::GAL1-HIS3 gal4del gal ⁇ Odel cyhR2).
  • the bait is produced as a C-terminal fusion protein with the DNA binding domain of the transcription factor Gal4 (amino acids 1 to 147).
  • a total human brain (37 year-old male Caucasian) cDNA library cloned into the yeast expression vector pACT2 was purchased from Clontech (human brain MATCHMAKER cDNA, cat. # HL4004AH), transformed into the yeast strain J692 (genotype of this strain: Mat a, ade2, his3, leu2, trpl, URA3::GALl-lacZ LYS2::GAL1-HIS3 gal4del gal80del cyhR2), and selected for the ability to drive leucine synthesis.
  • each cDNA is expressed as a fusion protein with the transcription activation domain of the transcription factor Gal4 (amino acids 768 to 881) and a 9 amino acid hemagglutinin epitope tag.
  • J693 cells (Mat ot type) expressing the bait were then mated with J692 cells (Mat a type) expressing proteins from the brain library.
  • the resulting diploid yeast cells expressing proteins interacting with the bait protein were selected for the ability to synthesize tryptophane, leucine, histidine, and ⁇ -galactosidase.
  • DNA was prepared from each clone, transformed by electroporation into E.
  • coli strain KC8 (Clontech KC8 electrocompetent cells, cat # 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 brain library plasmid).
  • DNA for both plasmids was prepared and sequenced by di- deoxynucleotide chain termination method. The identity of the bait cDNA insert was confirmed and the cDNA insert from the brain library plasmid was identified using BLAST program against public nucleotides and protein databases.
  • Plasmids from the brain library were then individually transformed into yeast cells together with a plasmid driving the synthesis of lamin fused to the Gal4 DNA binding domain. Clones that gave a positive signal after ⁇ -galactosidase assay were considered false-positives and discarded. Plasmids for the remaining clones were transformed into yeast cells together with plasmid for the original bait. Clones that gave a positive signal after ⁇ -galactosidase assay were considered true positives.
  • EXAMPLE 4 Identification of GLUT 1 /Myosin Heavy Chain Interaction
  • EXAMPLE 5 Identification of GLUT1/HSS Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl (SP accession no. PI 1166) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1-? of HSS (GB accession no. X91879).
  • EXAMPLE 6 Identification of OGTase/Mvosin Heavy Chain Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1773-2283 of myosin heavy chain (GB accession no. AF111785).
  • EXAMPLE 9 Identification of PI-3K110/Complement Protein C4 Interaction
  • EXAMPLE 13 Identification of MM-1 /Beta Spectrin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-175 of MM-1 (SP accession no. Q99471) as bait was performed. One clone that was identified by this procedure included amino acids 1545-1789 of beta spectrin (SP accession no. Q01082).
  • EXAMPLE 14 Identification of MM-1/KIAA0477 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 27-175 of MM-1 (SP accession no. Q99471) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 2448-3207 of KIAA0477 (GB accession no. AB007946).
  • a yeast two-hybrid system as described in Example 1 using amino acids 250-700 of dynamin (SP accession no. Q05193) as bait was performed.
  • One clone that was identified by this procedure included amino acids encoded by nucleotides 948-1599 of CALM (GB accession no. U45976).
  • a yeast two-hybrid system as described in Example 1 using amino acids 250-700 of dynamin (SP accession no. Q05193) as bait was performed.
  • One clone that was identified by this procedure included amino acids encoded by nucleotides 378-966 of Psme3 (GB accession no.
  • EXAMPLE 17 Identification of Naflb/I-TRAF Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 590-1610 of Naflb (GB accession no. Q05193) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 209-1420 of I-TRAF (GB accession no. U59863).
  • EXAMPLE 18 Identification of Aktl /NuMAl Interaction
  • EXAMPLE 20 Identification of Akt2/B AP31 Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 915-131 1 of Akt2 (GB accession no. M95936) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 469-877 of BAP31 (GB accession no. NM00574).
  • OGTase/Desmin Interaction A yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1349 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 219-449 of desmin (SP accession no. P17661).
  • EXAMPLE 23 Identification of OGTase/ Alpha-karyopherin Interaction
  • EXAMPLE 26 Identification of PTPlb/VAP-A Interaction A yeast two-hybrid system as described in Example 1 using amino acids 280-436of PTPlb (SP accession no. PI 8031) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 9-? of VAP-A (GB accession no. AF086627).
  • EXAMPLE 27 Identification of Rab4/ Alpha-catenin-like Protein Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-214 of Rab4 (SP accession no. P20338) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1573-1987 of alpha-catenin-like protein (GB accession no. U97067).
  • EXAMPLE 29 Identification of Glut4/PN7065 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. PI 4672) as bait was performed. One clone that was identified by this procedure included novel protein fragment PN7065. The DNA sequence and the predicted protein sequence for PN7065 are set forth in 75 and 76, respetively.
  • a yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 1016-1618 of OGTase (GenBank (GB) accession no. U77413) as bait was performed.
  • One clone that was identified by this procedure included novel protein fragment PN6931.
  • the DNA sequence and the predicted protein sequence for PN6931 are set forth in Tables 79 and 80, respectively.
  • a yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 233-1568 of Naflb (GB accession no. AJOl 1896) as bait was performed.
  • One clone that was identified by this procedure included novel protein fragment PN7582.
  • the DNA sequence and the predicted protein sequence for PN7582 are set forth in Tables 81 and 82, respectively.
  • EXAMPLE 33 Identification of OGTase/Talin Interaction
  • EXAMPLE 35 Identification of OGTase/Clone 25100 Interaction
  • EXAMPLE 36 Identification of OGTase/KIAA0443 Interaction
  • EXAMPLE 38 Identification of OGTase/Dynamin II Interaction
  • EXAMPLE 45 Identification of Glut4/MAPKKK6 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (SP accession no. PI 4672) as bait was performed. One clone that was identified by this procedure included amino acids 824-1012 of MAPKKK6 (GenBank (GB) accession no. AF 100318).
  • EXAMPLE 46 Identification of GLUT4/Tropomvosin 3 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 434-509 of Glut4 (SP accession no. PI 4672) as bait was performed. One clone that was identified by this procedure included amino acids 171-286 of tropomyosin 3 (SP accession no. P06753).
  • EXAMPLE 47 Identification of GLUT1/DRAL/FHL2 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl (SP accession no. P 11166) as bait was performed. One clone that was identified by this procedure included amino acids 1-280 of DRAL/FHL2 (SP accession no. Q 13229).
  • EXAMPLE 48 Identification of GLUT 1 /MYSA Interaction A yeast two-hybrid system as described in Example 1 using amino acids 448-492 of Glutl
  • a yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed.
  • One clone that was identified by this procedure included amino acids 623-713 of SG2NA (SP accession no. P70483).
  • EXAMPLE 50 Identification of IRAP/SLAP-2 Interaction
  • EXAMPLE 51 Identification of OGTase/14-3-3 Epsilon Interaction
  • EXAMPLE 52 Identification of PI-3K85/Chromogranin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-250 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 2-322 of chromogranin (SP accession no. PI 3521).
  • EXAMPLE 55 Identification of PI-3K85/14-3-3-zeta Interaction A yeast two-hybrid system as described in Example 1 using amino acids 425-630 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids 79-246 of 14-3-3-zeta (AP accession no. P29213).
  • EXAMPLE 56 Identification of PI-3K85/14-3-3-eta Interaction A yeast two-hybrid system as described in Example 1 using amino acids 425-630 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. One clone that was identified by this procedure included amino acids beginning with residue 91 of 14-3-3-eta (SP accession no. Q04917).
  • EXAMPLE 57 Identification of PI-3K85/TACC2 Interaction
  • a yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. PI 4672) as bait was performed.
  • One clone that was identified by this procedure included amino acids 27-168 of MM-1 (GenBank (GB) accession no. D89667).
  • EXAMPLE 60 Identification of Glutl /Dynamin Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-492 of Glutl (Swiss Protein (SP) accession no. PI 1166) as bait was performed. One clone that was identified by this procedure included amino acids 104-365 of dynamin (GB accession no. L07807).
  • EXAMPLE 61 Identification of Glut 1 /Clone 25204 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-492 of Glutl (Swiss Protein (SP) accession no. PI 1166) as bait was performed. One clone that was identified by this procedure included undetermined amino acids residues of Clone 25204 (GB accession no. AF131749).
  • EXAMPLE 64 Identification of OGTasePP5/Alpha-2-Catenin A yeast two-hybrid system as described in Example 1 using acids encoded by nucleotides 1016-1618 of OGTase (GB accession no. U77413) as bait was performed. One clone that was identified by this procedure included amino acids 366-469 of Alpha-2-Catenin (GB accession no. M94151).
  • EXAMPLE 65 Identification of PI-3K110/TRIP15 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3K110
  • a yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. PI 4672) as bait was performed.
  • One clone that was identified by this procedure included amino acids 229-379 of KIAA0282 (GenBank (GB) accession no. D87458), an efp-like protein.
  • EXAMPLE 68 Identification of Glut4/Tankyrase Interaction A yeast two-hybrid system as described in Example 1 using amino acids 463-509 of Glut4 (Swiss Protein (SP) accession no. PI 4672) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1110-1750 of tankyrase (GB accession no. AF082557).
  • a yeast two-hybrid system as described in Example 1 using amino acids encoded by nucleotides 2311-3136 of IRAP (GB accession no. U62768) as bait was performed.
  • One clone that was identified by this procedure included amino acids 1487-1993 of ⁇ -spectrin (SP accession no. Q01082).
  • EXAMPLE 72 Identification of PP5/HSP89 Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of PP5 (SP accession no. P53041) as bait was performed. One clone that was identified by this procedure included amino acids 517-681 of Hsp89 (SP accession no. P07900).
  • EXAMPLE 73 Identification of PP5/Tankyrase Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-150 of PP5 (SP accession no. P53041) as bait was performed. One clone that was identified by this procedure included amino acids encoded by nucleotides 1110-1750 and nucleotides 1809-2257 of tankyrase (GB accession no. AF082557).
  • a yeast two-hybrid system as described in Example 1 using amino acids 320-440 of PI-3 kinase p85 subunit (SP accession no. P27986) as bait was performed. Two clones that were identified by this procedure included amino acids encoded by nucleotides 1110-1750 of tankyrase (GB accession no. AF082557).
  • EXAMPLE 75 Identification of PI-3K110/APP Interaction A yeast two-hybrid system as described in Example 1 using amino acids 1-300 of PI-3 kinase pi 10 subunit (SP accession no. P42338) as bait was performed. One clone that was identified by this procedure included amino acids 374-546 of APP (SP accession no. P05067).
  • EXAMPLE 76 Generation of Polyclonal Antibody against Protein Complexes
  • Glut4 interacts with CARP to form a complex.
  • a complex of the two proteins is prepared, e.g., by mixing purified preparations of each of the two proteins.
  • the protein complex can be stabilized by cross-linking the proteins in the complex, by methods known to those of skill in the art.
  • the protein complex is used to immunize rabbits and mice using a procedure similar to that described by Harlow et al. (1988). This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al., 1993). Briefly, purified protein complex is used as immunogen in rabbits.
  • Rabbits are immunized with 100 ⁇ g of the protein in complete Freund's adjuvant and boosted twice in three- week intervals, first with 100 ⁇ g of immunogen in incomplete Freund's adjuvant, and followed by 100 ⁇ g of immunogen in PBS.
  • Antibody-containing serum is collected two weeks thereafter.
  • the antisera is preadsorbed with Glut4 and CARP, such that the remaining antisera comprises antibodies which bind conformational epitopes, i.e., complex-specific epitopes, present on the Glut4-CARP complex but not on the monomers.
  • Polyclonal antibodies against each of the complexes set forth in Tables 1-73 are prepared in a similar manner by mixing the specified proteins together, immunizing an animal and isolating antibodies specific for the protein complex, but not for the individual proteins.
  • Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising Glut4/CARP complexes conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known in the art. The complexes can be prepared as described in Example 76, and may also be stabilized by cross-linking. The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 ⁇ g of immunogen, and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RJA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.
  • immunogen comprising Glut4/CARP complexes conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known in the art.
  • the complexes can be prepared as described in Example 76, and may also be stabilized by cross
  • Spleens are removed from immune mice and a single-cell suspension is prepared (Harlow et al., 1988). Cell fusions are performed essentially as described by Kohler et al. (1975). Briefly,
  • myeloma cells American Type Culture Collection, Rockville, MD
  • NS-1 myeloma cells are fused with immune spleen cells using polyethylene glycol as described by Harlow et al. (1988).
  • Cells are plated at a density of 2x10 5 cells/well in 96-well tissue culture plates. Individual wells are examined for growth, and the supernatants of wells with growth are tested for the presence of
  • Glut4/CARP complex-specific antibodies by ELISA or RIA using Glut4/CARP complex as target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
  • Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibodies for characterization and assay development. Antibodies are tested for binding to Glut4 alone or to CARP alone, to determine which are specific for the Glut4/CARP complex as opposed to those that bind to the individual proteins.
  • Monoclonal antibodies against each of the complexes set forth in Tables 1-73 are prepared in a similar manner by mixing the specified proteins together, immunizing an animal, fusing spleen cells with myeloma cells and isolating clones which produce antibodies specific for the protein complex, but not for the individual proteins.
  • the present invention is useful in screening for agents that modulate the interaction of Glut4 and CARP.
  • the knowledge that Glut4 and CARP form a complex is useful in designing such assays.
  • Candidate agents are screened by mixing Glut4 and CARP (a) in the presence of a candidate agent, and (b) in the absence of the candidate agent. The amount of complex formed is measured for each sample.
  • An agent modulates the interaction of Glut4 and CARP if the amount of complex formed in the presence of the agent is greater than (promoting the interaction), or less than (inhibiting the interaction) the amount of complex formed in the absence of the agent.
  • the amount of complex is measured by a binding assay, which shows the formation of the complex, or by using antibodies immunoreactive to the complex.
  • a binding assay is performed in which immobilized Glut4 is used to bind labeled CARP.
  • the labeled CARP is contacted with the immobilized Glut4 under aqueous conditions that permit specific binding of the two proteins to form an Glut4/CARP complex in the absence of an added test agent.
  • Particular aqueous conditions may be selected according to conventional methods. Any reaction condition can be used as long as specific binding of Glut4/CARP occurs in the control reaction.
  • a parallel binding assay is performed in which the test agent is added to the reaction mixture.
  • the amount of labeled CARP bound to the immobilized Glut4 is determined for the reactions in the absence or presence of the test agent. If the amount of bound, labeled CARP in the presence of the test agent is different than the amount of bound labeled CARP in the absence of the test agent, the test agent is a modulator of the interaction of Glut4 and CARP.
  • Candidate agents for modulating the interaction of each of the protein complexes set forth in Tables 1-73 are screened in vitro in a similar manner.
  • EXAMPLE 79 In vivo Identification of Modulators for Protein-Protein Interactions
  • an in vivo assay can also be used to screen for agents which modulate the interaction of Glut4 and CARP.
  • a yeast two-hybrid system in which the yeast cells express (1) a first fusion protein comprising Glut4 or a fragment thereof and a first transcriptional regulatory protein sequence, e.g., GAL4 activation domain, (2) a second fusion protein comprising CARP or a fragment thereof and a second transcriptional regulatory protein sequence, e.g., GAL4 DNA-binding domain, and (3) a reporter gene, e.g., ⁇ -galactosidase, which is transcribed when an intermolecular complex comprising the first fusion protein and the second fusion protein is formed.
  • Parallel reactions are performed in the absence of a test agent as the control and in the presence of the test agent.
  • a functional Glut4/CARP complex is detected by detecting the amount of reporter gene expressed. If the amount of reporter gene expression in the presence of the test agent is different than the amount of reporter gene expression in the absence of the test agent, the test agent is a modulator of the interaction of Glut4 and CARP.
  • Candidate agents for modulating the interaction of each of the protein complexes set forth in Tables 1-73 are screened in vivo in a similar manner.

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Abstract

L'invention concerne la découverte de nouvelles interactions protéine-protéine impliquées dans les voies physiologiques mammaliennes, dont les troubles ou les maladies physiologiques. Les troubles et les maladies physiologiques sont, part exemple, le diabète sucré insulino-dépendant, les troubles neurodégénératifs, tels que la maladie d'Alzheimer, et similaire. Ainsi, l'invention concerne des complexes de ces protéines et/ou de leurs fragments, des anticorps dirigés contre lesdits complexes, le diagnostic de troubles dégénératifs physiologiques (dont le diagnostic de la prédisposition au trouble et le diagnostic de la présence de ce dernier), le criblage de médicaments à la recherche d'agents qui modulent l'interaction des protéines décrites, et l'identification de protéines additionnelles dans la voie commune aux protéines décrites.
PCT/US2000/010651 1999-04-22 2000-04-21 Interactions proteine-proteine WO2000065340A1 (fr)

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AU44754/00A AU4475400A (en) 1999-04-22 2000-04-21 Protein-protein interactions
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073050A2 (fr) * 2000-03-24 2001-10-04 Millennium Pharmaceuticals, Inc. 3714, 16742, 23546, et 13887 nouvelles molecules de proteine kinase et leurs utilisations
WO2001085942A2 (fr) * 2000-05-05 2001-11-15 Incyte Genomics, Inc. Proteines associees au cytosquelette
WO2003087403A2 (fr) * 2002-04-16 2003-10-23 Evotec Neurosciences Gmbh Diagnostic et utilisation therapeutique d'une proteine golgi pour des maladies neurodegeneratives
EP1420800A1 (fr) * 2001-08-02 2004-05-26 Howard Florey Institute Of Experimental Physiology And Medicine Modulation de l'activite du recepteur (at 4) d'aminopeptidase (irap)/d'angiotensine iv regulee par l'insuline

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101723514B1 (ko) * 2016-07-26 2017-04-05 아주대학교산학협력단 Sno-ogt 억제제를 포함하는 알츠하이머병, 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AUSUBEL ET AL.: "Short Protocols in Molecular Biology", 1995, WILEY AND SONS, INC, US, XP002930868 *
GUNSTER ET AL.: "Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic", MOLECULAR CELL BIOL.,, vol. 17, no. 4, April 1997 (1997-04-01), pages 2326 - 2335, XP002930869 *
NAYA ET AL.: "Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor", vol. 9, 15 April 1995 (1995-04-15), pages 1009 - 1019, XP002930870 *
ROMANOWSKI ET AL.: "XMXM7, a novel member of the xenopus MCM family, interacts with XMCM3 and colocalizes with it throughout replication", PROC. NATL. ACAD. SCI. USA,, vol. 93, September 1996 (1996-09-01), pages 10189 - 10194, XP002930871 *
ZILBERMAN ET AL.: "Evolutionarily conserved promoter region containing CArG*like elements is crucial for smooth muscle myosin heavy chain gene expression", CIRC. RES.,, vol. 82, no. 5, 23 March 1998 (1998-03-23), pages 566 - 575, XP002930872 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073050A2 (fr) * 2000-03-24 2001-10-04 Millennium Pharmaceuticals, Inc. 3714, 16742, 23546, et 13887 nouvelles molecules de proteine kinase et leurs utilisations
WO2001073050A3 (fr) * 2000-03-24 2002-06-20 Millennium Pharm Inc 3714, 16742, 23546, et 13887 nouvelles molecules de proteine kinase et leurs utilisations
WO2001085942A2 (fr) * 2000-05-05 2001-11-15 Incyte Genomics, Inc. Proteines associees au cytosquelette
WO2001085942A3 (fr) * 2000-05-05 2002-06-20 Incyte Genomics Inc Proteines associees au cytosquelette
EP1420800A1 (fr) * 2001-08-02 2004-05-26 Howard Florey Institute Of Experimental Physiology And Medicine Modulation de l'activite du recepteur (at 4) d'aminopeptidase (irap)/d'angiotensine iv regulee par l'insuline
EP1420800A4 (fr) * 2001-08-02 2008-01-23 Florey Howard Inst Modulation de l'activite du recepteur (at 4) d'aminopeptidase (irap)/d'angiotensine iv regulee par l'insuline
WO2003087403A2 (fr) * 2002-04-16 2003-10-23 Evotec Neurosciences Gmbh Diagnostic et utilisation therapeutique d'une proteine golgi pour des maladies neurodegeneratives
WO2003087403A3 (fr) * 2002-04-16 2004-05-21 Evotec Neurosciences Gmbh Diagnostic et utilisation therapeutique d'une proteine golgi pour des maladies neurodegeneratives

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