WO2000065052A1 - «bridge-1», un facteur de transcription - Google Patents

«bridge-1», un facteur de transcription Download PDF

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WO2000065052A1
WO2000065052A1 PCT/US2000/010616 US0010616W WO0065052A1 WO 2000065052 A1 WO2000065052 A1 WO 2000065052A1 US 0010616 W US0010616 W US 0010616W WO 0065052 A1 WO0065052 A1 WO 0065052A1
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bridge
seq
protein
polynucleotide
polypeptide
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WO2000065052A9 (fr
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Joel F. Habener
Melissa K. Thomas
Gordon G. Wong
Kwok-Ming Yao
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Habener Joel F
Thomas Melissa K
Wong Gordon G
Yao Kwok Ming
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Priority to AU46496/00A priority Critical patent/AU4649600A/en
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Publication of WO2000065052A9 publication Critical patent/WO2000065052A9/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to the field of transcription factors. More specifically, the present invention relates to a novel mammalian transcription factor, to the nucleotide and amino acids sequences thereof, and to the use of this novel transcription factor in the diagnosis and treatment of developmental disorders including diabetes.
  • Basic helix-loop-helix (bHLH) transcription factors regulate a diverse array of physiologic processes in developing and adult organisms. Myogenesis, lymphocyte differentiation, neurogenesis, sex determination, and the development and functions of pancreatic ⁇ -cells are dependent on the regulated action of both ubiquitous and tissue-specific bHLH proteins (Murre, C., etal, Biochim. Biophys. Ada 1218:129-135 (1994), Naya, F. J., etal., Genes Development 77:2323-2334 ( 1997)).
  • E2A proteins function either as homodimers or as heterodimers with tissue-specific Class B bHLH proteins to bind and transactivate promoters via conserved sequence elements known as E boxes.
  • E2A proteins are best characterized in their interaction with other bHLH proteins, other types of protein-protein interactions have been described (Eckner, R. et al., Genes and Development 10:2418-2490 (1996); Kho, C.-J., et al., J. Biol. Chem. 272:3845-3851 (1997); Kho, C.-J., et al. J. Biol. Chem. 272:13426-13431 (1997); Loveys, D. A., etal, Gene 20T ⁇ 69-
  • E2A is the pancreatic ⁇ -cell, in which the rat insulin I gene is regulated by glucose-responsive minienhancers consisting of E box binding sites for E2A (Nelson, C, et al., Genes Dev. 4:1035-1043 (1990)) and A boxes that bind homeoproteins (German, M. S. et al., Genes Dev. 6:2X65-2X16 (1992)). At the E boxes, E2A forms heterodimers with tissue-specific bHLH transcription factors such as Beta-2/NeuroD (Naya, F. J., et al, Genes Dev. 9: 1009-1019 (1995)).
  • tissue-specific bHLH transcription factors such as Beta-2/NeuroD
  • Homeoproteins such as PDX-1, Lmxl, and Isl-1, bind to the A boxes and act in synergy with E2A heterodimers on adjacent E boxes to activate transcription of the insulin gene (German, M. S. etal., Genes Dev. 6:2X65-2X16 (1992); Johnson, J. D., et al., Mol. Cell. Biol. 7:3488-3496 (1997); Peers, B., et al., Mol. Endo.
  • CBP and p300 may act as E2A coactivators (Eckner, R. et al, Genes and Development 70:2478-2490 (1996); Qiu, Y., et al, Mol. Cell. Biol 18:2951-2964 (1998)).
  • p300 serves as a coactivator for both E2A and Beta-2 NeuroD in insulin gene transcription (Qiu, Y., et al, Mol. Cell. Biol. 18:2951-2964 (1998)).
  • PDZ domains named for three proteins in which the motif was initially noted (post-synaptic density protein PSD-95 (Cho, K.-O., et al, Neuron 9:929- 942 (1992)), Drosophila discs-large tumor suppressor protein DlgA (Yun, C. H. C, etal, Proc. Natl. Acad. Sci. USA 94:3010-3015 (1997))), and the mammalian tight junction protein ZO-1 (Itoh, M., et al, J. Cell Biol. 727:491-502 (1993))), are conserved domains that mediate protein-protein interactions in a variety of intracellular signaling processes (Saras, J., and C.-H.
  • PDZ domains have been implicated in protein-protein interactions required for post-synaptic density ion channel and receptor clustering, signal transduction pathways regulating cell growth, visual signal transduction cascade regulation, and Fas-mediated regulation of apoptosis (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)).
  • PDZ domains appear in proteins with a diverse range of functions, including protein tyrosine phosphatases, proteases, ion channels, and signal transduction scaffolding molecules (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)).
  • Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination.
  • E2A proteins in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for ⁇ -cell function.
  • Diabetes mellitus type I results from a genetically conferred vulnerability that causes a primary deficiency of insulin.
  • This deficiency of insulin is believed to be the consequence of destruction of a specialized population of cells that produce insulin in the body, i.e., pancreatic ⁇ -cells.
  • An autoimmune process may also contribute to ⁇ -cell damage.
  • glucose production is augmented, and the efficiency of peripheral glucose use is reduced until a new equilibrium between these processes is reached at a very high plasma glucose level. Because of the high plasma glucose levels, the filtered load of glucose exceeds the renal tubular capacity for reabsorption.
  • Glucose therefore is excreted in the urine in large quantities, causing, by its osmotic effect, increased excretion of water and salts and frequent urination.
  • the goal of insulin treatment is to systematically lower plasma levels of glucose, free fatty acids and ketoacids to normal and reduce nitrogen losses. This result is achieved by direct actions of insulin and also by diminishing the secretion of the insulin antagonist glucagon.
  • diabetes mellitus noninsulin-dependent or type II
  • type II diabetes Another more common form of diabetes mellitus, noninsulin-dependent or type II, often is associated with obesity.
  • this disease there appears to be both a deficit in insulin production in combination with a resistance to the action of insulin on major target tissues.
  • the locus of resistance is distal to the insulin receptor binding site, but defects in receptor tyrosine kinase activity, glucose transport, and activities of insulin-sensitive enzymes have been found.
  • ⁇ -cell recognition of glucose there is a derangement in ⁇ -cell recognition of glucose as a stimulus, so that first phase insulin secretion is lost, though a delayed release does occur.
  • Treatment of type II diabetes does not normally require insulin administration.
  • Caloric regulation, weight reduction if obesity is present, and the use of sulfony lurea drugs simultaneously improve tissue responsiveness to endogenous insulin and ⁇ -cell responsiveness to glucose. In late stages, insulin administration is usually required.
  • pancreatic beta cell transcriptional regulators may cause diabetes mellitus.
  • Maturity onset diabetes of the young (MODY) is amonogenic heritable subgroup of type II diabetes with early onset (age 30 or less).
  • MODY Maturity onset diabetes of the young
  • PDX-1 transcription factor-1
  • Mutations in PDX-1 may lead to diabetes in humans via disrupting the regulation of the insulin gene and/or altering the development of the pancreas and of pancreatic beta cell mass.
  • Transcriptional regulators of the insulin gene and of pancreas development may be important targets for novel therapeutic agents of diabetes mellitus.
  • Insulin excess is usually caused by tumors of the ⁇ -cells.
  • the cardinal manifestation is a low plasma glucose level in the fasting state.
  • Chronic insulin excess and persistent hypoglycemia disturbed central nervous system function results in unusual behavior, defects in cerebration, loss of consciousness, or convulsions. Removal of the tumor may cure the condition.
  • drugs that inhibit insulin secretion may ameliorate the condition.
  • Bridge-1 is a novel coactivator for members of the E2A transcription factor family isolated from pancreatic insulinoma cells. Bridge-1 represents a novel PDZ-like domain coactivator for E2A and participates in the regulation of insulin gene transcription in pancreatic ⁇ -cells.
  • Bridge-1 interacts with E2A proteins to function as a coactivator of gene transcription mediated by E12 and E47.
  • Rat Bridge-1 was isolated by yeast two- hybrid screening of a cDNA library prepared from rat insulinoma (INS- 1 ) cells, on the basis of its specific interaction with E12.
  • INS- 1 rat insulinoma
  • Bridge-1 is homologous to proteins of unknown function from several species and contains a truncated PDZ-like domain, a domain known to be involved in protein-protein interactions.
  • Bridge- 1 RNA is widely expressed in pancreatic islet cell lines and in a variety of murine and human tissues. By immunocytochemistry, the Bridge-1 protein expression pattern is primarily nuclear, with marked expression in pancreatic islets and coexpression with insulin.
  • Bridge-1 The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro translated Bridge- 1 and El 2 and E47 and by mammalian two-hybrid studies. In contrast, Bridge-1 does not interact with the pancreas-specific basic helix-loop-helix protein Beta-
  • the PDZ-like domain of Bridge- 1 is required for interaction with E 12, as deletion mutants of Bridge- 1 that lack the PDZ-like domain interact poorly with El 2 in mammalian two-hybrid studies.
  • the carboxy terminus of E12 participates in this interaction, as demonstrated by an impaired interaction with Bridge-1 of a truncated E 12 mutant (E 12 ⁇ C) in which the carboxy-terminal 9 amino acids were deleted.
  • Bridge-1 has transactivation potential, as a Gal4 DNA-binding domain/Bridge- 1 fusion protein transactivates a Gal4C AT reporter.
  • Bridge-1 also functions as a coactivator by enhancing El 2- or E47-mediated activation of the rat insulin I gene minienhancer promoter-reporter constructs in transient transfection experiments. Substitution of the mutant El 2 ⁇ C for El 2 reduces the coactivation of rat insulin I promoter-reporter constructs by Bridge-1. Inactivation of endogenous Bridge- 1 in insulinoma (INS- 1 ) cells by expression of a Bridge- 1 antisense RNA diminishes rat insulin I promoter activity . Bridge- 1 , by utilizing its PDZ-like domain to interact with E 12, provides a new mechanism for the coactivation and regulation of transcription of the insulin gene.
  • Bridge-1 signaling is not restricted to the fully developed pancreatic beta cell, but is also operative during pancreas development. Bridge-1 expression can be detected in the developing mouse pancreas at el 0.5 by rt-PCT and at el 5 by immunocytochemistry. Analysis of additional Bridge-1 mutants in transient transfection studies indicates that both the intact PDZ-like domain and the carboxyl terminal domain of Bridge-1 are required to mediate transcriptional transactivation. Bridge-1 also interacts with and appears to regulate other transcriptional activators.
  • the pancreatic beta cell-specific transcription factor PDX- 1 interacts with Bridge- 1 in both yeast and mammalian two-hybrid systems.
  • Bridge- 1 transactivation activity is enhanced by addition of the coactivator p300, in a dose-dependent manner.
  • the protein- protein interaction and transactivation functions of Bridge-1 may also be modulated by the multiple forms of Bridge-1 observed on Northern blots, in cDNA expression libraries, and on Western blots.
  • the present invention is directed to isolated nucleic acid molecules comprising a polynucleotide encoding Bridge-1 whose amino acid sequence is shown in Figure 1 (SEQ ID NO:2), or a fragment thereof.
  • the invention provides isolated nucleic acid molecules encoding Bridge- 1 having an amino acid sequence as encoded by the cDNA in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture ("ATCC"), 10801 University Boulevard. Manassas, V A, on April 20, 1999, and assigned accession number 203947.
  • ATCC American Type Tissue Culture
  • the invention is directed to an isolated nucleic acid molecule that hybridizes under stringent conditions to the above-described nucleic acid molecules.
  • the present invention is also directed to variants of the nucleic acid molecules of the present invention, which encode fragments, analogs or derivatives of the Bridge-1 protein, e.g., polypeptides having at least one biological activity that is substantially similar to at least one biological activity of the Bridge-1 protein.
  • the present invention is further directed to isolated nucleic acid molecules that encode a Bridge-1 polypeptide as well as methods for generating nucleic acid molecules that encode a Bridge-1 polypeptide.
  • nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 80% identical, more preferably at least 85% identical, more preferably at least 90% identical, and more preferably at least 95%, 96%, 97%, 98%, or 99% identical to the above described nucleic acid molecules.
  • the present invention also relates to vectors which contain the above- described isolated nucleic acid molecules, host cells transformed with the vectors and the production of Bridge-1 polypeptides by recombinant methods.
  • the present invention further provides isolated Bridge-1 polypeptides having the amino acid sequence shown in Figure 1 (SEQ ID NO:2).
  • isolated Bridge-1 polypeptides are provided having an amino acid sequence as encoded by the cDNA in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture ("ATCC"), 10801 University Boulevard. Manassas, NA, on April 20, 1999, and assigned accession number 203947.
  • ATCC American Type Tissue Culture
  • the invention is directed to screening methods for identifying proteins, proteins fragments, biological and chemical compounds and other small molecules that bind to the full-length Bridge-1 protein or Bridge-1 polypeptide fragments.
  • the invention is directed to methods for identifying proteins, protein fragments, biological and chemical compounds, or other small molecules that enhance or inhibit Bridge-1 activity.
  • the invention is directed to a method for stimulating gene expression in a cell comprising transfecting said cell with a recombinant vector, said vector comprising an isolated polynucleotide having the nucleotide sequence set forth in FIG. 1 (SEQ ID NOT).
  • the invention is directed to a method for enhancing transcription activation of a Bridge-1 target gene.
  • Bridge-1 target gene is meant any gene transcription of which is mediated either directly or indirectly by Bridge-1, either as a transactivator, coactivator or otherwise.
  • the invention is directed to a method for enhancing or inhibiting transcriptional activation of a Bridge-1 target gene by administering an effective amount of a protein, protein fragment, biological or chemical compound or other molecule that enhances or inhibits Bridge- 1 activity.
  • the invention is directed to a method of treating diabetes comprising administering to a patient in need thereof a therapeutically effective amount of Bridge- 1 in a pharmaceutically acceptable carrier or excipient for a time sufficient to provide an effective level of endogenous insulin in the patient.
  • the invention is directed to anti-Bridge- 1 antibodies.
  • FIG. 1 Sequence of rat Bridge-1 cDNA and encoded protein.
  • Figure 1 shows the sequence of rat Bridge-1 cDNA (SEQ ID NO: l) and the corresponding encoded protein (SEQ ID NO:2). Positions of in-frame stop codons are designated by asterisks under the corresponding nucleotide sequences. The arrowhead is placed below the corresponding nucleotide sequence to indicate the starting position of the two-hybrid clone # 18. The PDZ-like domain (i. e. , amino acids 138-178) is underlined.
  • FIG. 2A Homologies between Bridge-1 protein sequence and sequences from other species.
  • FIG. 2B PDZ-like domain homologies between Bridge-1 and other PDZ-domain containing proteins.
  • the Bridge-1 protein (SEQ ID NO: 2) is schematically depicted to illustrate the PDZ-like domain (PDZ) identified by homology with other proteins. Alignment positions with conserved similar or identical residues by Blast analysis in at least 50 percent of the aligned sequences are indicated with asterisks and grey boxes. Identical amino acids are designated in bold print. Protein sequences represented are the following: BRIDGE, rat Bridge-1, aa 138-178); SIP1 (SEQ ID NO: 6), interacting protein with human SRY (Poulat, F., etal, J. Biol. Chem.
  • Ada 727(5:321-324 (1993)) (Genbank ace. no. 631508), aa 76-116 (SEQ ID NO:10); NHERF, rabbit protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na+/H+ exchanger (Wilson, R., et al, Nature 368:32-38 (1994); Zhuang, Y., et al, Cell 79:875-884 (1994)) (Genbank ace. no.
  • FIG. 2C Comparison of the Bridge-1 PDZ-like domain with typical
  • PDZ domain sequences Alignment of typical PDZ domains and designation of regions of secondary structure (boxed) are depicted according to Doyle et al. (Doyle, D. A., et al, Cell 55: 1067-1076 (1996)).
  • a segment of Bridge-1 sequence is aligned for comparison. Amino acids within Bridge-1 sequence identified as conserved among sequences aligned in FIG.2b are designated (*) for reference.
  • similarities or identities by Blast analysis between the Bridge-1 sequence and any of the three ZO-1, PSD95-3, or DLG-1 sequences are indicated (+).
  • PSD95-3 rat presynaptic density protein 95, PDZ domain 3 (Cho, K.-O., et al, Neuron 9:929-942 (1992)) (Genbank ace. no. P31016), aa 312-391 (SEQ ID NO: 19); DLG-1, D. melanogaster lethal (Anand, G., X. et al, J. Biol. Chem. 272:19140-19151 (1997)) discs large- 1 tumor suppressor protein (Yun, C. H. C, etal, Proc. Natl. Acad. Sci. USA 94:3010-3015 (1997)) (Genbank ace. no.
  • FIG. 3 (Panels A-D). Characterization of Bridge-1 Expression.
  • FIG. 4 panels A-D. Immunocytochemical staining of Bridge-1 in RIN1027-B2 cells and mouse pancreas. Fluorescent immunostaining of
  • RIN 1027-B2 cells was conducted with rabbit polyclonal anti-Bridge- 1 antisera (A) or preimmune antisera (C) and photographed under identical conditions.
  • a phase contrast view of the field of cells stained with anti-Bridge- 1 antisera (B) is shown for comparison.
  • Mouse embryonic day 19 pancreas (D) was stained with rabbit polyclonal anti-Bridge- 1 antisera.
  • An islet (*), ducts (d), and adjacent exocrine pancreas are shown. Examples of exocrine cell nuclei with positive Bridge-1 immunostaining are indicated with arrows.
  • FIG. 5 (Panels A-D). Bridge-1 is co-expressed with insulin in murine pancreas. Fluorescent immunostaining of adult murine pancreas was conducted by co-staining with rabbit polyclonal anti-Bridge- 1 antiserum (A) and guinea pig anti-insulin antiserum (B). Co-staining of an adjacent section of murine pancreas with rabbit preimmune antiserum (C) and guinea pig anti-insulin antiserum (D), photographed under identical conditions, is shown for comparison. Arrows point to ⁇ cells that coexpress Bridge-1 and insulin within an islet of Langerhans.
  • FIG. 6 (Panels A-B). Coimmunoprecipitation of Bridge-1 and E2A proteins.
  • FIG. 7 Bridge-1 interacts with El 2 in a mammalian two-hybrid system.
  • FIG. 8 Bridge-1 and Beta-2/NeuroD do not interact in a mammalian two-hybrid system.
  • HeLa cells were transiently transfected with 5 ⁇ g Gal4CAT reporter and 10 ⁇ g of pM (Gal4DBD).
  • pM-Beta-2 Gal4DBD-Beta-2
  • NP 16AD pNP 16-E 12
  • pNP 16-Bridge- 1 NP 16AD-Bridge
  • FIG. 9 The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1.
  • HeLa cells were transiently transfected with Gal4CAT reporter, pM, pM-Bridge-1, pNP16 (NP16AD) or pNP16-E12 (NP16AD-E12), as in FIG. 7.
  • pM-Bridge-l(l-72), pM-Bridge-l(l-133), and pM-Bridge-l(l-184) were substituted for pM-Bridge-1.
  • FIG. 10 (Panels A-B).
  • the carboxy-terminus of El 2 contributes to the Bridge-1/E12 interaction.
  • FIG. 1 A) Schematic diagrams of E 12 (aa 1 -649), the E 12 fragment utilized as bait in the yeast two-hybrid screening that identified Bridge- 1 (aa 521 -649), and the E12 mutant E12 ⁇ C (aa 1-640).
  • the two activation domains are designated as AD 1 and AD2 and the basic helix-loop-helix domain as bHLH (modeled after a published schematic diagram ) (Spicer, D. B., et al, Twist. Science 272:1416- 1480 (1996)).
  • the carboxy terminal sequences of E12, E12 bait, and E12 ⁇ C are shown below the respective schematic diagrams.
  • the asterisk designates the carboxy terminus of E12 ⁇ C, truncated by nine amino acids (EAH ⁇ PAGHL), due to the introduction of a TAG stop codon corresponding to amino acid position 641 in E12.
  • HeLa cells were transiently transfected with Gal4CAT reporter, pM, pM-Bridge- 1 , pNP 16 (NP 16 AD) or pNP 16-E 12 (NP 16 AD-E 12), as in FIG.
  • FIG. 12 panels A-D). Bridge-1 co-activation of rat insulin I minienhancer FarFlat reporter with E47 or E12.
  • HeLa cells were transiently transfected with 2 ⁇ g 5FF1CAT reporter in the presence of4 ⁇ g pcDN A3 -Bridge-1 and 1 ⁇ g pcDNA3-E12 orpcDNA3-E12 ⁇ C.
  • FIG. 13 Bridge-1 is expressed in developing mouse pancreas. Polyclonal rabbit anti-Bridge- 1 antisera was used in immunostaining of paraffin sections of mouse embryonic day 15 pancreas. At embryonic day 15, Bridge-1 is expressed in a nuclear pattern throughout the branching, developing ductal tree destined to become pancreas, as well as within structures budding from the ducts that likely represent developing islets. The arrowhead indicates nuclear Bridge-1 staining in one of the buds of the developing pancreatic ductal tree.
  • FIG. 14 (Panels A and B). Bridge-1 interacts with PDX-1.
  • El 2 interacting clones identified by yeast two-hybrid screening were tested for their interaction with human interleukin-1 receptor (IL-R), D. melanogaster bicoid, PDX-1, and rat E 12 fusion proteins by semi-quantitative yeast two-hybrid interaction assay.
  • IL-R human interleukin-1 receptor
  • D. melanogaster bicoid D. melanogaster bicoid
  • PDX-1 D. melanogaster bicoid
  • rat E 12 fusion proteins by semi-quantitative yeast two-hybrid interaction assay.
  • Strength of interaction was measured by growth on leucine dropout plates to follow LEU reporter gene activity and by the intensity of blue color on X-gal plates to assess lacZ reporter gene activity, after 30 °C incubation for 72 hours.
  • Bridge-1 interacts with PDX-1 in a mammalian two- hybrid system. HeLa cells were transiently transfected with 5 ⁇ g Gal4CAT reporter and 10 ⁇ g of pM (GaWDBD), pM-Bridge (GaWDBD-Bridge- 1 ), pNP 16
  • NP16AD pNP16-PDX-l
  • CAT activity was enhanced relative to the controls, consistent with a Bridge-1 /PDX-1 interaction.
  • FIG. 15 The PDZ-like domain is required for Bridge-1 transactivation activity. Proline residues were substituted for highly conserved amino acids in a series of point mutations constructed within the PDZ-like domain of the Bridge- 1 /GaW D ⁇ A-binding domain fusion protein construct. BHK cells were transiently transfected, in duplicate, with 1 ⁇ g GaWCAT reporter and 4 ⁇ g pM (EMPTY VECTOR) or 4 ⁇ g pM-Bridge-1 (BRIDGE-1) or its mutants (N159P, N164P, N175P, D165P, G151P), as designated.
  • EMPY VECTOR EPTY VECTOR
  • BRIDGE-1 4 ⁇ g pM-Bridge-1
  • a fluorimager scan of thin layer chromatography of a representative fluorimetric chloramphenicol acetyltransferase activity assay is shown.
  • Asterisks (*) represent acetylated forms of the fluorescent substrate that results from chloramphenicol acetyltransferase activity. Quantitation of these transactivation activities relative to empty vector activity is as follows: Bridge-1, 854%; VI 59P, 96%; N164P, 335%; N175P, 122%; D156P, 44%; G151P, 185%. Note that each of the five Bridge-1 point mutants tested have diminished transactivation activity relative to the wild-type Bridge-1.
  • FIG. 16 p300 coactivates Bridge-1 transactivation activity.
  • BHK cells were transiently transfected, in duplicate, with 1 ⁇ g GaWCAT reporter, 0.5 ⁇ g of empty vector pM or 0.5 ⁇ g pM-Bridge-1 , and 0.0 to 3.0 ⁇ g p300 expression plasmid, as indicated. Results shown are the mean of two transfections.
  • p300 increases the Bridge- 1/Gal4 DNA-binding domain fusion protein transactivation of the GaWCAT reporter construct in a dose dependent manner.
  • FIG. 17 A "small form" of Bridge-1 is found on Western blots.
  • Western blot analysis utilizing rabbit polyclonal anti-Bridge- 1 antisera, of whole cell extracts derived from HeLa, BHK, and mouse insulinoma MIN-6 cells following SDS-PAGE fractionation and electroblotting onto a PNDF membrane.
  • Enhanced chemiluminescence was utilized to visualize the protein antisera complexes.
  • Migration positions for the full length Bridge- 1 (29kD) and the " small form” of Bridge-1 (19kD) are indicated. Note that the proportion of full length to "small form” Bridge-1 varies among the different cell types analyzed.
  • FIG. 18 The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1.
  • HeLa cells were transiently transfected with GaWDNA-binding domain Bridge- 1 or GaWDNA-binding domain/Bridge 1 deletion mutant (amino acids as indicated) fusion protein and
  • E12/NP16 activation domain fusion protein expression plasmids and a GaWCAT reporter The interaction of each mutant with the E12/NP16 activation domain fusion protein was assessed and normalized to the interaction observed with the full length GaWDNA-binding domain/Bridge- 1 fusion protein. Data shown are the mean ⁇ SEM of four transfections, conducted in duplicate. Bridge- 1 mutants that lacked an intact PDZ domain interacted poorly with El 2 while mutants with an intact PDZ domain retained the ability to interact with El 2.
  • FIG. 19 The Bridge-1/E12 interaction requires the PDZ-like domain of Bridge-1.
  • Bridge-1 mutant fusion proteins amino acids as indicated
  • a rat El 2 fragment amino acids 521-649
  • Results shown are the mean ⁇ SEM of three independent determinations, conducted in duplicate.
  • Bridge-1 mutants that lacked an intact PDZ domain interacted poorly with E12 while mutants with an intact PDZ domain retained the ability to interact with El 2.
  • FIG. 20 The Bridge-1 PDZ domain is required for interaction with El 2.
  • HeLa cells were transiently transfected with expression plasmids encoding fusion proteins of GaWDNA-binding domain (GaW), GaWDNA-binding domain/Bridge- 1 (Bridge) or GaWDNA-binding domain/Bridge- 1 proline substitution mutants (amino acid positions and changes as indicated) in conjunction with an expression plasmid encoding an E 12NP 16 activation domain fusion protein and a GaWCAT reporter.
  • GaWCAT assay A representative fluorometric CAT assay, conducted in duplicate, is shown. Asterisks indicate the CAT activity as demonstrated by acetylated forms of the fluorescent substrate.
  • FIG. 21 Schematic diagram of El 2 mutant constructs tested in mammalian two-hybrid experiments for interaction with Bridge-1.
  • the two activation domains are designated as AD1 and AD2 and the D ⁇ A-binding and dimerization bHLH domain is designated bHLH.
  • the carboxy-terminal sequences of E12, E12 bait, and E12 ⁇ C are shown belowthe respective schematic diagrams.
  • the asterisk designates the carboxy terminus of E12 ⁇ C, truncated by 9 amino acids (EAH ⁇ PAGHL), due to the introduction of a TAG stop codon by site- directed mutagenesis corresponding to amino acid position 641 in El 2.
  • FIG.22 Carboxy-terminal amino acids ofE12 mediate interaction with Bridge-1.
  • HeLa cells were transiently transfected with GaWDNA-binding domain/Bridge- 1 fusion protein and E12NP16 activation domain or El 2 mutant/NP16 activation domain fusion protein expression plasmids and a GaWCAT reporter.
  • the observed interactions in the mammalian two-hybrid assay were normalized to that seen for GaWDNA-binding domain/Bridge- 1 and E 12/NP 16 activation domain fusion proteins. Results shown are the mean ⁇ SEM of three to five transfections conducted in duplicate. Deletion of 9 carboxy- terminal amino acids in E12 reduced its interaction with Bridge-1 by over 50 percent, and a larger deletion of E12 that included the bHLH domain eliminated the remaining interaction with Bridge-1.
  • FIG. 23 The E12 ⁇ C mutant diminishes Bridge-1 coactivation of the rat insulin I promoter minienhancer FarFlat.
  • HeLa cells were transiently transfected with a rat insulin I promoter-reporter construct encoding pentamerized FarFlat enhancer sequences and a CAT reporter and expression plasmids encoding Bridge- 1 , E 12 or the E 12 ⁇ C mutant in which the carboxy-terminal 9 amino acids were deleted by mutagenesis.
  • Data shown are the mean ⁇ SEM of four transfections conducted in duplicate and normalized to the activity of Bridge-1 combined with E 12.
  • the E 12 ⁇ C mutation that impairs Bridge- 1 -E 12 interaction see FIG.
  • FIG. 24 Inactivation of Bridge-1 in insulin-producing cells reduces insulin promoter activity.
  • Rat insulinoma (I ⁇ S-1) cells were transiently transfected with an antisense Bridge-1 cDNA expression construct (antisense- Bridge-1) or the empty expression plasmid pcDNA3 (empty vector) and a rat insulin I promoter-reporter construct spanning residues -410 to +47 of the rat insulin I promoter sequence, including the minienhancer FarFlat. Expression of the Bridge-1 antisense construct decreased insulin promoter activity by 45%), indicating that endogenous Bridge- 1 contributes to insulin promoter activation in insulin-producing cells.
  • FIG. 25 Bridge-1 interacts with PDX-1.
  • Recombinant glutathione-S- transferase/Bridge-1 fusion protein (GST-Bridge) or glutathione-S-transferase (GST) were incubated with [ 35 S]-radiolabeled in vitro translated rat PDX-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS- polyacrylamide electrophoresis. The corresponding autoradiogram demonstrating that GST-Bridge-I interacted with radiolabeled PDX-1 is shown.
  • FIG. 26 Bridge-1 activates the insulin promoter enhancer FarFlat in conjunction with PDX-1 and E2A proteins.
  • FIG. 27 Bridge-1 interacts with multiple domains within the nuclear receptor coactivator p300.
  • Recombinant glutathione-S-transferase fusion proteins including fragments of human p300 (GST-p300, amino acids as indicated) were incubated with [ 5 S]-radiolabeled in vitro translated rat Bridge-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS- polyacrylamide electrophoresis.
  • the corresponding autoradiogram demonstrates that Bridge- 1 interacts with two distinct segments of p300, amino acids 1 -595 and 741-1571, but not with a third segment, amino acids 1572-2370.
  • FIG 28 Bridge-1 interacts with multiple domains within the nuclear receptor corepressor NcoR.
  • Recombinant glutathione-S-transferase fusion proteins including the two nuclear receptor binding domains of the nuclear receptor corepressor NCoR (GST-NCoR, NBDl or NBD2) were incubated with [ 35 S]-radiolabeled in vitro translated rat Bridge-1 protein and Glutathione Sepharose 4B (Pharmacia) beads. After multiple washes, proteins adherent to the glutathione sepharose beads were subjected to SDS-polyacrylamide electrophoresis. The corresponding autoradiogram demonstrates that Bridge-1 interacts with both nuclear receptor binding domains of NCoR.
  • FIG. 29 Bridge-1 conform multimers. Recombinant glutathione-S- transferase/Bridge-1 fusion protein (GST-Bridge) or glutathione-S-transferase
  • FIG. 30 The carboxy-terminal domain within Bridge-1 is required for transactivation.
  • the PDZ domain may also participate in Bridge-1 transactivation because, in an independent series of experiments, proline-substitution mutagenesis of conserved amino acids within the PDZ domain substantially reduced Bridge-1 transactivation activity, indicating that the PDZ domain within Bridge-1 is also required for its transactivation activity.
  • the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a novel mammalian transcription factor (Bridge-1) whose amino acid sequence is shown in Figure 1 (SEQ ID NO:2).
  • Bridge-1 novel mammalian transcription factor
  • the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1 ) was obtained by sequencing a cDNA clone, which was deposited with the American Type Tissue Culture (“ATCC”) and assigned accession number 203947.
  • ATCC American Type Tissue Culture
  • a Bridge-1 "polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NOT .
  • the Bridge-1 polynucleotide can contain the nucleotide sequence of the full-length Bridge-1 cDNA sequence shown in FIG. 1 (SEQ ID NOT), including the 5' and 3 ' untranslated sequences, the coding region, with or without any signal sequence, the protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a Bridge-1 "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • isolated polynucleotides are provided which encode the Bridge- 1 protein.
  • a nucleic acid molecule of the present invention encoding a Bridge- 1 polypeptide may be obtained using standard cloning and screening procedures.
  • the nucleic acid molecule described in Figure 1 (SEQ ID NOT) was obtained from a cDNA expression library from rat pancreatic islet cells.
  • the Bridge- 1 cDNA of the present invention encodes a protein of about 222 amino acids, which includes a PDZ-like domain.
  • Bridge-1 is widely expressed, since the corresponding transcript was found in several human tissues, including pancreas, testes, kidney, and liver.
  • Isolated nucleic acids of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • isolated nucleic acidmolecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention.
  • Additional illustrative examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells and purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention as well as partially or substantially purified mRNA molecules.
  • Purified as it refers to preparations made from biological cells or hosts should be understood to mean any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest.
  • a purified preparation can be obtained by following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations.
  • the procedures may include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, density gradient centrifugation and electrophoresis.
  • “Substantially pure” should be understood to mean a "highly" purified preparation that contains at least 95% of the DNA or protein of interest.
  • Isolated nucleic acid molecules according to the present invention further include nucleic acid molecules produced synthetically.
  • Isolated polynucleotides of the present invention include DNA molecules comprising an open reading frame (ORF), i.e. coding region, with an initiation codon at position 495 of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1); and DNA molecules which comprise a sequence substantially different than that described above but which, due to the degeneracy of the genetic code, still encode the Bridge-1 protein.
  • ORF open reading frame
  • SEQ ID NO: 1 DNA molecules which comprise a sequence substantially different than that described above but which, due to the degeneracy of the genetic code, still encode the Bridge-1 protein.
  • the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants described above.
  • the invention provides isolated nucleic acid molecules encoding the Bridge- 1 polypeptide having an amino acid sequence as encoded by the cDNA clone in the plasmid pcDN A3 -Bridge-1 deposited with the American
  • the invention further provides an isolated polynucleotide having the nucleotide sequence of the Bridge-1 coding region shown in Figure 1 (SEQ ID NO: 1 ) or the nucleotide sequence of the Bridge-1 cDNA contained in the above-described clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • isolated nucleic acid molecules preferably DNA molecules, are useful as probes for gene mapping by in situ hybridization with chromosomes and for detecting expression of the Bridge-1 gene, or genes homologous to the Bridge- 1 gene, in human tissue, for instance, by Northern blot analysis.
  • the invention provides an isolated nucleic acid molecule that hybridizes under stringent conditions to the above-described nucleic acid molecules.
  • stringent conditions is intended to mean, as a non- limiting example, overnight incubation at 42 °C in a solution comprising 50% formamide, 5xSSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lxSSC at about 65°C.
  • such "an isolated nucleic acid molecule that hybridizes under stringent conditions” will be at least 15 bp, preferably at least 20 bp, more preferably at least 30 bp, more preferably at least 40 bp, and most preferably, at least 50 bp in length.
  • fragments of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA clone described above or the nucleotide sequence as shown in Figure 1 (SEQ ID NOT) or the nucleotide sequence of the ORF, i.e.
  • the coding region as shown in Figure 1 (SEQ ID NO: 1), is intended to mean DNA fragments at least 15 bp, more preferably at least 20 bp, more preferably at least 30 bp, more preferably at least 40 bp, more preferably at least 50 bp, more preferably at least 60 bp, more preferably at least 70 bp, more preferably at least 80 bp, more preferably at least 90 bp, more preferably at least 100 bp length, and most preferably at least 200 bp, in length.
  • Such fragments are useful, wter alia, as diagnostic probes and primers. Larger DNA fragments, up to, for example, 500 bp in length, are also useful as probes according to the present invention.
  • a fragment of at least 20 bp in length is intended to mean fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence of the ORF, i. e. coding region, as shown in FIG. 1 (SEQ ID NO: 1 ).
  • fragments are useful diagnostically wter alia as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR).
  • polynucleotide fragments of the invention comprise at least 15 contiguous nucleotides of the Bridge- 1 coding sequence shown in Fig. 1 (SEQ ID NOT), but do not comprise all or a portion of any Bridge-1 intron.
  • the nucleic acid comprising Bridge-1 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3 ' to the Bridge-1 gene in the genome.)
  • EST sequences are publicly available and accessible through sequence databases.
  • the EST sequences referred to below were identified in a BLAST search of the EST database. These sequences are believed to be partial sequences of the cDNA insert identified in the recited GenBank accession numbers.
  • sequences are related to the coding region of SEQ ID NO: 1, GenBank Accession Nos: AW140997 (SEQ ID NO:29); AI410372 (SEQ ID NO:30); AI710949 (SEQ ID NO.31); AI410370 (SEQ ID NO:
  • AA764187 (SEQ ID NO:46); AA530067 (SEQ IDNO:47); AA760338 (SEQ ID NO:48); AV239440 (SEQ ID NO:49); AA230657 (SEQ ID NO:50); AA832760 (SEQ ID NO.51); AN018936 (SEQ ID ⁇ O:52); AA0331 11 (SEQ ID NO:53); W41542 (SEQ ID NO:54); AI426803 (SEQ ID NO:55); AVI 17212 (SEQ ID NO:56); AI853315 (SEQ ID NO:57); W61442 (SEQ ID NO:58); AV043811
  • W88749 (SEQ ID NOT 10); AI698667 (SEQ ID NOT 11); AI439894 (SEQ ID NOT 12); AI421551 (SEQ ID NOT 13); H63468 (SEQ ID NO: 114); AI082760 (SEQ ID NOT 15); W73843 (SEQ ID NOT 16); W73699 (SEQ ID NOT 17); AA535984 (SEQ ID NO: 118); AW000865 (SEQ ID NO: 119); R25346 (SEQ ID NO:120); R26538 (SEQ ID NO:121); AA936901 (SEQ ID NOT22); AI350558
  • AA374147 (SEQ IDNO:136); AW368137 (SEQ IDNO:137); AW389910 (SEQ ID NO.138); AA640616 (SEQ ID NO:139); AW079701 (SEQ ID NO.140); AI202368 (SEQ ID NO: 141); N51558 (SEQ ID NO: 142); AA401853 (SEQ ID NO: 143);AW410681 (SEQIDNO:144);N40375 (SEQIDNO:145); AW385667 (SEQ ID NO: 146); N27557 (SEQ ID NO: 147); AW368222 (SEQ ID NO: 148);
  • AW368132 (SEQ ID NO: 149); R60619 (SEQ ID NOT50); D20400 (SEQ ID NOT51); AW517221 (SEQ ID NO.152); AA403126 (SEQ ID NOT53).
  • the present invention is directed to polynucleotides comprising the polynucleotide fragments and full-length polynucleotide (e.g. the coding region) described herein exclusive of one or more of the above recited ESTs.
  • Restriction endonuclease cleavage or shearing by sonication may easily be used to generate fragments of various sizes.
  • the DNA fragments of the present invention can be generated synthetically according to the methods and techniques known and available to those skilled in the art.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode for fragments, analogs or derivatives of the Bridge-1 protein, e.g., polypeptides having biological activity substantially similar to the Bridge-1 protein.
  • Variants may occur naturally, such as isoforms and allelic variants.
  • Non-naturally occurring variants may be produced using any of the mutagenesis techniques known and available to those skilled in the art.
  • Such variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the Bridge-1 protein or fragment thereof. Also especially preferred in this regard are substitution of nucleotides that encode a conservative amino acid substitution. In a preferred embodiment, such variants contain no more than five total substitutions, deletions, and/or additions.
  • nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 80% identical, more preferably at least 85%> identical, more preferably at least 90% identical, and most preferably at least 95%, 96%, 97%, 98%, or 99% identical to: (a) the nucleotide sequence of the cDNA clone in the plasmid pcDN A3 -Bridge-1 deposited with the American Type Tissue Culture (“ATCC”) and assigned accession number 203947; (b) the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1); (c) the nucleotide sequence of the cDNA clone in the plasmid pcDNA3-Bridge-l deposited with the American Type Tissue Culture (“ATCC”) and assigned accession number 203947, which encodes the full-length Bridge-1 protein; (d) the nucleotide sequence of the ORF, i.e.
  • FIG. 1 SEQ ID NO: 1
  • Figure 1 SEQ ID NO: 1
  • Figure 1 a nucleotide sequence complimentary to any of (a)-(d).
  • Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% "identical” can be determined conventionally using known computer algorithms.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence also referred to as a global sequence alignment, can be determined using the F ASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:231-245 (1990).
  • RNA sequence can be compared by converting U's to T's.
  • the result of said global sequence alignment is in percent identity.
  • Preferred parameters used in a F ASTDB alignment of DNA sequences to calculate percent identity are:
  • the present application is directed to such nucleic acid molecules having a nucleotide sequence at least 90%>, 95%), 96%, 97%, 98%,
  • the percent identity is measured by comparing the obtained DNA sequence to that of nucleotides 495-1 162 (i.e., the ORF or coding region) of the nucleotide sequence in FIG. 1 (SEQ ID NO: 1).
  • nucleic acid molecules of the present invention that do not encode a polypeptide having Bridge-1 activity include, wter alia, (X) isolating the Bridge-1 gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the Bridge-1 gene as described in Verma et al, Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting Bridge- 1 mRNA expression in specific tissues, such as placenta tissue .
  • FISH in situ hybridization
  • “Bridge- 1 activity” is intended to mean one or more of the following: protein-protein binding activity, transcription coactivation activity, or transcription activation activity.
  • transcription activation activity is meant positive regulation of gene expression consisting of an increase in the level of transcription and/or translation resulting from interaction with core cellular transcriptional or translational machinery.
  • transcription coactivation activity is meant positive regulation of gene expression consisting of an increase in the level of transcription and/or translation resulting from interaction with other proteins with transcription activation activity.
  • nucleic acid molecules having a nucleotide sequence at least 80%, and preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the above-described nucleic acid molecules which do, in fact, encode a polypeptide having Bridge-1 activity.
  • a polypeptide having a Bridge-1 activity is intended to mean polypeptides exhibiting similar, but not necessarily identical, activity as to the
  • Bridge-1 activity as measured in a particular biological assay.
  • the Bridge-1 protein of the present invention interacts directly with known transcription factors such as PDX-1, E12 and E47.
  • the Bridge-1 protein of the present invention enhances transcription genes modulated by these transcription factors.
  • "a polypeptide having a Bridge-1 protein activity” includes polypeptides that interact with PDX-1, El 2, E47 and other transcription factors or otherwise enhance PDX- 1 or E2A protein dependent transcriptional activation or PDX- 1 or E2A independent transcription activation.
  • nucleic acid molecules having a nucleotide sequence at least 90%>, preferably at least 95%), 96%), 97%, 98%o, 99%) identical to the nucleotide sequence of the above-described nucleic acid molecules will encode "a polypeptide having Bridge-1 activity.”
  • degenerate variants all encode the same polypeptide, this will be clear to the skilled artisan.
  • a reasonable number will also encode a polypeptide having Bridge-1 activity. This is because the skilled artisan is fully aware of possible amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g. , replacing one aliphatic amino acid with a second aliphatic amino acid).
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of Bridge-1 or Bridge-1 fragments.
  • Bridge-1 fragment means a shortened sequence of an amino acid sequence that retains some or all of the the Bridge-1 activity of the full-length sequence, such as a fragment comprising the Bridge-1 PDZ-like domain (amino acids 138-178 of the amino acid sequence shown in FIG.
  • Bridge-1 fragment is also intended to refer to splice-variants and proteolytic fragments of the full-length Bridge-1 amino acid sequence shown in FIG. 1 (SEQ ID NO:2), including the "small form” Bridge-1 (FIG. 17) which migrates at approximately 18 kD in SDS-PAGE Western Blots, is detected with rabbit polyclonal Bridge-1 antisera, and is differentially expressed in cell lines derived from different tissues with preferential expression in pancreatic beta cells.
  • Recombinant constructs may be introduced into host cells using well known techniques such as infection, transduction, transfection, transvection, electroporation and transformation.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • vectors comprising cis-acting control regions to the polynucleotide of interest.
  • Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids.
  • the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, CMV promoters, promoters of retroviral LTRs, and inducible promoters such as tetracycline and IPTG inducible promoters as well as promoters inducible with heavy metals to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as ⁇ . coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, Cos and
  • vectors preferred for use in bacteria include, but are not limited to, pA2, pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Preferred eukaryotic vectors include, but are not limited to, pcDNA-3 (Invitrogen), pM, pVP16 (Clonetech), pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • bacterial promoters suitable for use in the present invention include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al, BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Ausubel, F. M. et al, CURRENTPROTOCOLSINMOLECULARBIOLOGY, (John Wiley and Sons, Inc.) 1994- 1997.
  • Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting elements of DNA, generally about 10 to 300 bp in size, that act to increase transcriptional activity of a promoter in a given host cell-type.
  • Illustrative examples of enhancers include, but are not limited to, the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the SV40 enhancer which is located on the late side of the replication origin at bp 100 to 270
  • the cytomegalovirus early promoter enhancer the polyoma enhancer on the late side of the replication origin
  • adenovirus enhancers adenovirus enhancers.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions.
  • additional amino acids particularly charged amino acids
  • peptide moieties may be added to the polypeptide to facilitate purification.
  • the Bridge-1 protein or fraction thereof can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include, but are not limited to, naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • the polypeptides of the present invention may be post translationally modified (e.g., glycosylated, phosphorylated, famesylated, etc.).
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention further provides an isolated Bridge- 1 polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence as shown in Figure 1 (SEQ ID NO:2), or a fragment thereof.
  • Preferred Bridge-1 fragments will have Bridge-1 activity.
  • Preferred Bridge-1 fragments should at least include amino acid residues 138 to 178 as shown in FIG.l (SEQ ID NO:2) and/or amino acid residues 186-222 as shown in FIG. 1 (SEQ ID NO:2), or amino acid substitutions, additions or deletions thereof that are not significantly detrimental to Bridge-1 activity.
  • Bridge-1 polypeptide fragments may be "free-standing" or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
  • Polypeptide fragments may comprise 9, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more amino acids in length.
  • the present invention is directed to Bridge-1 polypeptides and polypeptide fragments described herein exclusive of one or more of the polypeptides described in SEQ ID NOS: 3-20.
  • an "isolated" polypeptide or protein is intended to mean a polypeptide or protein removed from its native environment, such as recombinantly produced polypeptides and proteins expressed in host cells and native or recombinant polypeptides which have been substantially purified by any suitable technique (e.g. , the single-step purification method disclosed in Smith and Johnson, Gene ⁇ 57/31-40 (1988), which is incorporated by reference herein). Isolated polypeptides or proteins according to the present invention further include such compounds produced synthetically.
  • the inventors have discovered that the full-length Bridge-1 protein is an about 222 amino acid residue protein with a deduced molecular weight of about 24.8 kD and a pi of 6.70.
  • the present invention further includes variations of the Bridge-1 polypeptide which show substantial Bridge- 1 polypeptide activity or which include regions of Bridge-1 protein.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity.
  • polypeptides of the present invention include polypeptides having an amino acid sequence as encoded by the deposited cDNA, an amino acid sequence as shown in FIGURE 1 (SEQ ID NO:2), as well as an amino acid sequence at least 80%> identical, more preferably at least 85%> identical, more preferably at least 90%> identical, and most preferably at least 95%, 96%, 97%, 98%, or 99% identical, to the amino acid sequence encoded by the deposited cDNA, to the amino acid sequence as shown in FIGURE 1 (SEQ ID NO:2), or to the amino acid sequence of a polypeptide fragment described above. Whether two polypeptides have an amino acid sequence that is at least 80%>, 90%o or 95%> identical can be determined using a computer algorithm as described above.
  • nucleic acid molecules and polypeptides of the present invention are useful in screening assays for identifying proteins and protein fragments that bind to Bridge- 1 or a Bridge- 1 fragment, including proteins, protein fragments, biological and chemical compounds and other small molecules that enhance or inhibit Bridge- 1 activity. Accordingly, the nucleic acid molecules and polypeptides of the present invention are useful in assays for identifying drugs capable of enhancing or inhibiting Bridge-1 activity.
  • the full-length Bridge- 1 protein, and Bridge- 1 polypeptide fragments can be used to generate fusion proteins.
  • the Bridge- 1 polypeptide when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the Bridge-1 polypeptide can be used to indirectly detect the second protein by binding to the Bridge-1 protein.
  • the Bridge-1 polypeptides can be used as a targeting molecule once fused to other proteins.
  • Examples of domains that can be fused to Bridge-1 polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
  • Bridge-1 fusion polypeptides may be constructed which include additional N-terminal and/or C-terminal amino acid residues.
  • any N-terminally or C-terminally deleted Bridge-1 polypeptide disclosed herein may be altered by inclusion of additional amino acid residues at the N-terminus to produce a Bridge-1 fusion polypeptide.
  • Bridge-1 fusion polypeptides are contemplated which include additional N-terminal and/or C-terminal amino acid residues fused to a Bridge- 1 polypeptide comprising any combination of N- and C-terminal deletions.
  • fusion proteins may also be engineered to improve characteristics of the Bridge-1 polypeptide.
  • a region of additional amino acids may be added to the N-terminus of the Bridge-1 polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage.
  • peptide moieties may be added to the Bridge- 1 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the Bridge-1 polypeptide.
  • the addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • Bridge-1 polypeptides including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins
  • IgG chimeric polypeptides.
  • fusion proteins facilitate purification and show an increased half-life in vivo.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A394,827; Trauneckeret ⁇ /.,N ⁇ twre 337:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et ⁇ /., J. Biochem.
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
  • EP-A 0232 262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • Bridge-1 polypeptides can be fused to marker sequences, such as a peptide which facilitates purification of Bridge-1.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Another peptide tag useful for purification, the "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein.
  • any of these above fusions can be engineered using the Bridge-1 polynucleotides or the Bridge-1 polypeptides.
  • the inventors have cloned a gene encoding Bridge- 1 and have shown that the full length Bridge-1 protein and Bridge-1 fragments bind other proteins. Further, the inventors have shown that Bridge-1 proteins and protein fragments interact with other transcription factors, including members of the E2A family of transcription factors (which includes the El 2 E47, E2-2, HEB and daughterless transcription factors), PDX-1 and others, to modulate transcription activation.
  • the present invention further provides for screening methods for identifying proteins, protein fragments, biological and chemical compounds, and other small molecules that interact with Bridge-1 or Bridge-1 fragments. Such screening methods are useful for identifying, proteins, protein fragments, biological and chemical compounds and molecules that enhance or inhibit Bridge- 1 activity.
  • such methods involve: (a) providing a host cell containing recombinant genes which express a polypeptide comprising a protein ligand binding domain and a polypeptide comprising Bridge- 1 or a Bridge- 1 fragment, or fusion proteins comprising a protein ligand binding domain and a polypeptide comprising Bridge-1 or a Bridge-1 fragment, wherein said Bridge-1 and said Bridge- 1 fragment bind said protein ligand binding domain; (b) administering a candidate polypeptide to said cell; and (c) determining whether said candidate polypeptide reduces either: (1) Bridge-1 or Bridge-1 -fragment-binding to the polypeptide protein binding domain as compared to said binding in the absence of said candidate polypeptide; or (2) Bridge-1 or Bridge-1 -fragment-transcription coactivation activity ; or (3 ) Bridge- 1 or Bridge- 1 fragment transcription activation activity as compared to such activities in the absence of said candidate polypeptide.
  • Bridge-1 fragment capable of binding other proteins are described above.
  • Methods for determining whether a candidate protein enhances or interferes with Bridge-1 or Bridge-1 -fragment binding are known in the art.
  • the effect of a candidate protein on Bridge- 1 or Bridge- 1 -fragment-binding to another protein for example a protein in the E2A transcription factor or PDX-1
  • GST glutathione-S-transferase
  • this determination could be made using the mammalian two-hybrid system (Luo, Y. et al., Biotechniques 22:350-352 (1997).
  • the inventors have identified at least two domains of Bridge- 1 , a PDZ-like domain (amino acids 138- 178 of the amino acid sequence shown in FIG. 1 (SEQ ID NO. 1)
  • polypeptides containing these domains when fused to a DNA-binding domain of a transcriptional activator, are capable of activating transcription.
  • the present invention further provides a screening method for identifying a polypeptide which binds to the Bridge- 1 PDZ- like domain and/or the Bridge-1 carboxyl terminus domain which comprises (a) providing a host cell containing a recombinant gene or genes which express a polypeptide comprising a transcriptional activator DNA-binding domain (DBD) and the Bridge- 1 PDZ-like domain and/or the Bridge- 1 carboxyl terminus domain;
  • DBD transcriptional activator DNA-binding domain
  • the invention further provides for a screening method for identifying polypeptides that bind to the Bridge-1 PDZ-like domain and/or the Bridge-1 carboxyl terminus domain, which comprises: (a) providing a host cell containing a recombinant gene or genes which express a polypeptide comprising a transcriptional activator DNA-binding domain (DBD) and a Bridge-1 PDZ-like domain and/or Bridge-1 carboxyl terminus domain; (b) administering a recombinant gene expressing a candidate polypeptide or a biological or chemical compound or other small molecule to said cell; and (c) determining whether said candidate polypeptide, biological or chemical compound or other small molecule inhibits Bridge-1 activity.
  • a screening method for identifying polypeptides that bind to the Bridge-1 PDZ-like domain and/or the Bridge-1 carboxyl terminus domain which comprises: (a) providing a host cell containing a recombinant gene or genes which express a polypeptide comprising a transcriptional activator DNA
  • transcriptional activator molecules that enhance transcription by RNA polymerase B (II).
  • Transcriptional activators include yeast transcriptional activators, such as GAL4 and GCN4; the herpes simplex activator, VP16; and members of the nuclear receptor family, which includes RAR, RXR, ER, TR, NDR, GR, and AR.
  • Recombinant genes encoding a polypeptide comprising a Bridge-1 PDZ- like domain are described below.
  • Recombinant genes encoding a polypeptide comprising a transcriptional activator DBD are well known in the art.
  • Methods for determining whether a candidate polypeptide enhances or interferes with transcription are well known in the art. For example, the effect of a candidate polypeptide on Bridge- 1 PDZ-like domain activity or Bridge- 1 carboxyl terminus domain activity can be determined using CAT assays as described below and in Gronemeyer et al. (1987) and Bocquel et al, Nucl. Acids Res. (1989).
  • recombinant genes will encode a chimeric polypeptide comprising a transcriptional activator DBD fused to a Bridge- 1 polypeptide comprising the PDZ-like domain.
  • the host cell expressing the recombinant genes will also express a reporter gene. Examples of reporter genes are described above. Bridge-1 Antibodies and Methods of Use
  • Bridge-1 antibodies are also provided by the present invention, as specific for a Bridge-1 protein or a Bridge-1 protein fragment.
  • the term "antibody” is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience,
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and any subclass thereof.
  • a hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region, which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used.
  • Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81 :3273-3277 (1984); European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. patent No. 4,699,880, which is herein entirely incorporated by reference.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein- .
  • antibody is also meant to include both intact immunoglobulin molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciated that Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of a Bridge- 1 according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • an "antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • the antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect a Bridge-1 protein, polypeptide, or fragment, in a sample or to detect presence of cells which express a Bridge-1 of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
  • the antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a Bridge-1 protein, polypeptide, or fragment, of the present invention.
  • In situ detection may be accomplished by removing a histological specimen form a patient, and providing a labeled antibody of the present invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • Assays for a Bridge-1 protein, polypeptide, or fragment, of the present invention typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying a Bridge-1 protein, polypeptide, or fragment, and detecting the antibody by any of a number of techniques well- known in the art.
  • the biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled Bridge-1 -specific antibody.
  • the solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support or carrier may then be detected by conventional means.
  • solid phase support By “solid phase support”, “solid phase carrier”, “solid support”, “solid carrier”, “support” or “carrier” is intended any support or carrier capable of binding antigen or antibodies.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support or carrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of anti-Bridge- 1 antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.
  • Bridge- 1 -specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • This enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a ⁇ counter or a scintillation counter or by autoradiography .
  • an anti-Bridge- 1 antibody with a fluorescent compound.
  • the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence.
  • fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as those of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).
  • fluorescence emitting metals such as those of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).
  • EDTA diethylenetriamine pentaacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • An antibody molecule of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site” or "sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen form the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.
  • a directional INS- 1 cDNA library was constructed in the plasmid vector pJG4-5 using a Stratagene cDNA synthesis kit.
  • the El 2 bait was constructed by RT-PCR amplification of DNA encoding amino acids 521 to 649 of El 2 from total RNA isolated from rat 18 dpc pancreas, followed by cloning into the plasmid pEG202 in frame with an upstream LexA DNA-binding domain.
  • Yeast two-hybrid screening was conducted according to standard methods (Golemis, E.
  • Rat Bridge- 1 cDNA sequence was submitted to Genbank under the accession number AF067728.
  • RNAs were extracted by the guanidinium isothiocyanate method (Davis, L. G., etal, Basic Methods in Molecular, New York, Elsevier( 1986), pp.
  • RNAs were electrophoresed on 1%> agarose-formaldehyde gels and blotted onto nylon membranes (GeneScreen, NEN Life Science Products, Boston, MA) prior to probing with 32 P-labeled Bridge-1 cDNA. Membranes were stripped and reprobed with rat ⁇ -actin according to the manufacturer's instructions. Mouse and human endocrine system multiple tissue Northern blots (Clontech Laboratories Inc., Palo Alto, CA) were probed with 32 P-labeled Bridge-1 cDNA using high stringency washing conditions as described by the manufacturer. Plasmid Construction
  • pBSII-Bridge-1 and pcDN A3 -Bridge-1 were constructed by inserting the 1.4 kb Bridge-1 cDNA into an EcoRI site within the multiple cloning region of pBSII or pcDNA3, respectively.
  • the Mammalian Matchmaker Two-Hybrid Assay Kit (Clontech Laboratories Inc., Palo
  • pM and PNP 16 were used to construct plasmids expressing GaW D ⁇ A-binding domain/Bridge- 1, NP16/Bridge-l , GaW D ⁇ A-binding domain/E12, NP16/E12, and GaW D ⁇ A-binding domain/Beta-2 fusion proteins.
  • pM-Bridge-1 and pNP16-Bridge-l were constructed by inserting a blunt-ended 900 bp BstU I/EcoRI fragment of pBSII-Bridge- 1 in frame into a blunt-ended Mlu
  • pM-E12 and pNPl 6-E 12 were constructed by inserting a 2.7 kb blunt-ended ⁇ de I/EcoRI fragment of Pan 2 excised from the vector PARP5 (gift of C. Nelson) in frame into a Sma I site of pM and pNP16 vectors, respectively.
  • pM-Beta-2 was constructed by inserting a 2.4 kB blunt-ended BamHI/cohesive-ended Xba I fragment from pcD ⁇ Al-Beta-2 (gift of J.
  • pcD ⁇ AI-Beta-2 had been previously constructed by inserting a 2.4 kB BamHI/Xho I fragment from pCMN-Beta-2 (gift of F. ⁇ aya and M.J. Tsai) into pcD ⁇ AI that had been digested with BamHI and
  • pcD ⁇ A3-E12 and pcDNA3-E47 were constructed by inserting 2.7 kb Bgl II/EcoRI fragments of Pan 2 from the vector PARP5 or Pan 1 from the vector PARP5P2, respectively, (vectors were gifts of C. Nelson) into pcDNA3 prepared by digestion with BamHI and EcoRI.
  • 5FF 1 CAT was a gift from J. L. Moss.
  • pcDNA3-E12 ⁇ C and pVP16-E12 ⁇ C were constructed by point mutagenesis with insertion of a premature stop codon in pcDNA3 -E 12 and pVP 16-E 12, respectively, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's i n s t r u c t i o n s w i t h t h e o l i g o n u c l e o t i d e s 5'-ACCCGGGCCTGGGTTAGGCCCACAAT-3'(SEQ ID NO: 22) and 5 ' - ATTGTGGGCCTAACCCAGGC-CCGGGT-3 ' (SEQ ID NO: 23).
  • pM-Bridge- 1(1-133) was constructed by digestion of pM-Bridge- 1 with Stu I and Hind III, blunting with Klenow polymerase, and religation. Mutants were verified by sequencing and expression was assessed by Western blotting of transfected cell extracts.
  • HeLa cells American Type Culture Collection, Manassas, VA, and gift from R. Stein
  • BHK-21 C-13 cells
  • RIN1027-B2 cells Philippe, J., J. Clin. Invest. 79:351-358
  • HeLa cells were transfected with 25 ug total DNA by the calcium phosphate precipitation method using the CalPhos Maximizer Transfection Kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer' s instructions.
  • BHK cells or HeLa cells were transfected with 5 ⁇ g total DNA and 5 ⁇ l Lipofectamine as outlined by the manufacturer (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). Cells were harvested 48 hours after transfection.
  • Chloramphenicol acetyltransferase (CAT) assays were conducted with the fluorescent substrate assay kit FAST CAT (Molecular Probes, Eugene, OR) and thin layer chromatography on silica gel plates (Eastman Kodak, Rochester, NY) as previously reported (Staffers, D. A., et al, J. Clin. Invest. 702:232-241 (1998)). Quantitation was performed with a Fluorimager 575 interfaced with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Luciferase assays were conducted as previously described (Lu, M. et al, J. Biol. Chem. 272:28349-28359 (1997)).
  • Rat Bridge- 1 was synthesized in rabbit reticulocyte extracts by coupled in vitro transcription and translation from the plasmid pcDNA3 -Bridge- 1 , using the
  • E12 and E47 were generated using the same procedure with the plasmids pcDNA3-E12 and pcDNA3-E47, respectively. Reactions were conducted with either cold or 35 S-radiolabeled methionine (NEN Life Science Products, Boston, MA). To visualize the incorporation of 35 S-methionine, reactions were subjected to 10% SDS-polyacrylamide electrophoresis, followed by gel incubation in an autoradiography enhancer (Enlightening, NEN Life Science Products, Boston,
  • Extracts from transfected cells were fractionated by SDS-PAGE, electroblotted onto Immobilon-P membranes and incubated with rabbit polyclonal anti-Bridge-1 antisera (1:2000 dilution), rabbit polyclonal anti-GaWDBD antiserum (1 : 1000 dilution) (sc-577, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or rabbit polyclonal anti-El 2 antisera
  • a yeast two-hybrid screening system was developed.
  • a cDNA library derived from the insulinoma cell line, INS-1 (Asfari, M., et al, Endocrinol. 730:167-178 (1992)), was screened using a bait derived from the carboxy terminus of El 2 that included the bHLH DNA-binding and dimerization domains .
  • INS-1 insulinoma cell line
  • INS-1 Asfari, M., et al, Endocrinol. 730:167-178 (1992)
  • Clone #36 encodes a novel 177-amino acid open reading frame with homology to PDZ domains (Yao and Wong, unpublished data) and clone #18 encodes Bridge- 1 , which is the focus of this application. Interactions of these four clones with unrelated baits, including human interleukin receptor (cytoplasmic domain, amino acids 477 to 527) and D. melanogaster bicoid (pRFHM-1) (Golemis, E. A., in Current Protocols in Molecular Biology, F.M. Ausubel, et al, eds., John Wiley and Sons, New York, pp. 13.14.1-13.14.17 (1994)) were tested as negative controls.
  • human interleukin receptor cytoplasmic domain, amino acids 477 to 52
  • pRFHM-1 D. melanogaster bicoid
  • Clone #18 contained a cDNA insert of 934 bp. Because Northern blot analysis of INS-1 RNAs revealed two larger Bridge-1 transcripts of 1.3 and 1.0 kb (FIG. 3a), a 14 dpc rat pancreatic cDNA library was screened, using a 30-mer oligonucleotide from the clone #18 sequence. A single clone (Bridge-1) with an insert of 1.4 kb was isolated (FIG. 1). DNA sequence analysis revealed an open reading frame of 222 amino acids. The start codon for this open reading frame lies within the yeast two-hybrid clone #18.
  • IL-R human interleukin-1 receptor
  • D. melanogaster bicoid D. melanogaster bicoid
  • rat E12 fusion proteins by semi-quantitative yeast two-hybrid interaction assay.
  • Strength of interaction was measured by growth on leucine dropout plates to follow LEU reporter gene activity and by the intensity of blue color on X-gal plates to assess lacZ reporter gene activity, after 30°C incubation for 72 hours.
  • - denotes no growth on leucine dropout plates and white colonies on X-gal plates.
  • ++ indicates growth on leucine dropout plates and intense blue colonies on X-gal plates.
  • the Bridge-1 sequence of 222 amino acids predicts a protein with a molecular mass of 24.8 kD and a pi of 6.70.
  • RNA polymerase promoter a single radiolabeled protein was produced that migrated at approximately 28-29 kD (FIG. 3d).
  • Bridge-1 is highly conserved across species, including C. elegans, and S. cerevisiae (FIG. 2A).
  • Bridge-1 cDNA is homologous to murine and human expressed sequence tag sequences from a variety of embryonic and adult tissues as well as to a human sequence with the designation proteasomal modulator subunit p27 (Genbank ace. no. AB003177).
  • Rat Bridge-1 and human p27 are highly homologous, with 70 percent identity (156 of 222 amino acids) and 82 percent similarity at the protein level. The two sequences diverge at the carboxy termini of the proteins. Comparison of the first
  • Bridge- 1 protein sequence revealed a 41 amino acid segment of Bridge- 1 , extending from amino acids 138 to 178, that is homologous to protein-protein interaction domains of the PDZ type within several other proteins (FIG. 2B). Homologies with the aligned proteins within this segment range from 27 to 54 percent identity and 46 to 77 percent similarity.
  • This segment of homologous sequence in Bridge- 1 is shorter than prototypical PDZ domains of approximately 80 to 90 amino acids that form 5 or 6 beta sheets and two alpha helices by crystal structure analysis (Cabral, J. H. M., etal, Nature 3352:649-652 (1996); Doyle, D. A. et al, Cell 55:1067-1076 (1996)).
  • the high degree of sequence similarity in this region indicates that Bridge-1 contains a PDZ-like domain.
  • PDZ domains from the proteins PSD-95, DLG- 1 , and ZO- 1 demonstrates that the 41 amino acid segment identified within Bridge-1 corresponds to 3 beta sheets ( ⁇ C- ⁇ E) and 2 alpha helices ( ⁇ A and B) of PDZ domains (FIG. 2C).
  • the distance between beta sheets B and C varies among PDZ domains within different proteins (Cabral, J. H. M., et al, Nature 3352:649-652 (1996); Doyle, D. A. et al, Cell 55:1067-1076 (1996)), raising the possibility that additional upstream sequences within Bridge-1 may contribute to forming a complete PDZ domain.
  • Bridge-1 sequence is less similar to the amino-terminal portions of typical PDZ domains.
  • Bridge- 1 like ZO- 1 , lacks the conserved sequence GLGF, between beta sheets A and B, that has been identified as the substrate binding site within the third PDZ domain of PSD-95 (Doyle, D. A. et al, Cell 55: 1067-1076 (1996)). Although secondary structure predictions suggest that multiple alpha helices and beta sheets may form within the Bridge- 1 protein, they do not predict a secondary structure pattern that resembles the crystal structures of PDZ domains within PSD-95 or DLG-1 (Cabral, J. H. M, et al, Nature 3352:649-652 (1996); Doyle, O. A. et al, Cell 55:1067-1076 (1996)).
  • Bridge-1 cDNA was used as a probe for Northern analysis of RNAs from cells and tissues derived from rodents and humans (FIGs. 3A, 3B, and 3C). Two transcripts of approximately 1.0 kb and 1.3 - 1.4 kb in size are consistently noted in RNA from rodent tissues (FIGs. 3A and 3B), however only a single transcript of approximately 1.0 kb in size is observed in human tissues (FIG. 3C).
  • Bridge-1 RNA is highly expressed in a variety of cell lines derived from pancreatic islets, including the insulinoma line INS-1 (Asfari, M., et al, Endocrinol.
  • Bridge- 1 expression in RINl 027-B2 cells by immunocytochemistry revealed that Bridge-1 is predominantly located in the nucleus, although lower levels of cytoplasmic staining are observed in some cells (FIG. 4A). Bridge-1 expression did not appear to localize to the cytoplasmic projections extended by the RIN 1027-B2 cells growing in culture. In embryonic day 19 mouse pancreas, nuclear
  • Bridge-1 immunostaining was prominent within the cells of pancreatic islets, ductal cells, and in a few scattered nuclei of pancreatic exocrine cells (FIG. 4D). A lower level of cytoplasmic Bridge-1 staining was seen in islets but not in the exocrine pancreas. In the adult murine pancreas, Bridge-1 is expressed in pancreatic ⁇ -cells, as demonstrated by coexpression with insulin (FIGs. 5 A, 5B).
  • fusion protein constructs were transiently transfected into HeLa cells and tested for their ability to activate a GaWCAT reporter (FIG. 7). Fusion protein constructs were generated with the GaWDNA-binding domain or the activation domain of the VP 16 protein from the herpes simplex virus. Empty vectors or the VP16/E12 fusion protein alone have little activity in this system. The GaW DNA-binding domain Bridge- 1 fusion construct has a slight but detectable level of activation of the reporter. However, in the presence of both the NP16/E12 and the GaW D ⁇ A-binding domain/Bridge- 1 fusion constructs, an 18-fold increase in CAT activity is seen.
  • Beta-2 NeuroD is known to directly interact with E2A proteins via heterodimerization of bHLH domains (Mutoh, H., et al, Proc. Natl Acad. Sci. USA 94:3560-3564 (1997); Naya, F. , et al, Genes Dev. 9: 1009-1019 (1995)).
  • Beta-2/NeuroD and El 2 interactions were assessed (FIG. 8).
  • Example 7 Bridge-1 Utilizes its PDZ-like Domain to Interact with E12
  • Bridge-1 deletion mutants were constructed as GaW DNA-binding domain fusion proteins for analysis of interaction with the VP16/E12 fusion protein construct in mammalian two-hybrid studies.
  • Bridge-1 fusion constructs containing the PDZ domain interacted efficiently with the full length E 12 fusion protein while Bridge-1 constructs without the PDZ-like domain were weak interactors relative to full length Bridge-1 (FIG. 18).
  • PDZ-like domain (amino acids 120-222) demonstrated a potent interaction with El 2.
  • Bridge-1 requires an intact PDZ-like domain in order to interact with El 2.
  • the PDZ domain within Bridge-1 has some atypical structural features, it functions as an acceptor site for carboxy-terminal residues of interacting proteins analogous to more typical PDZ-like domains (Saras, J., and C.-H. Heldin, Trends in Biochem. Sci. 27:455-458 (1996)).
  • a mutant (E12 ⁇ C) was generated to prematurely truncate El 2 by removing the terminal 9 amino acids but retaining the two activation domains and the basic helix-loop-helix domain (FIG. 10 A). With this mutation, E 12 no longer terminates in a hydrophobic residue.
  • the E12 ⁇ C mutant retains the ability to interact with Beta-2/NeuroD.
  • the VP16/E12 ⁇ C fusion construct interaction with the GaW DNA-binding domain/Beta-2 fusion construct is not impaired relative to the VP16/E12 construct interaction with the GaW DNA-binding domain/Beta-2 construct.
  • Endogenous Bridge-1 inactivation impairs insulin promoter activity in INS-1 cells.
  • an antisense Bridge- 1 cDN A construct AS-Bridge-l-pcDNA3 was employed in transient transfections of the INS-1 rat insulinoma cell line.
  • a promoter-reporter construct consisting of -410 to +47 rat insulin I promoter sequences, -410INS-LUC (Lu, M. et al., J. Biol. Chem. 272:28349-28359 (1997)), was utilized to assess the effect of expression of the antisense Bridge-1 construct.
  • Expression of the AS-Bridge-l-pcDNA3 construct decreased insulin promoter activity by 45%) (FIG.24).
  • Bridge-1 signaling is operative during the development of the pancreas.
  • Bridge- 1 expression during early pancreas development was demonstrated by three independent methods.
  • the Bridge-1 expression pattern closely mimics that of PDX-1.
  • the homeodomain transcription factor PDX-1 is implicated in the pathogenesis of forms of pancreatic agenesis and diabetes mellitus in mice and humans.
  • Bridge-1 interacts with PDX-1
  • Bridge- 1 was tested with several other fusion protein constructs for interaction. Bridge- 1 interacted with a fragment of E12 and did not interact with the protein bicoid or the interleukin-1 receptor in this system, as indicated in the first manuscript.
  • FIG. 14b The functional consequences of the Bridge-1 /PDX-1 interaction are currently under investigation, but Bridge- 1 modulation of PDX- 1 function is likely to be directly relevant to PDX-1 regulation of pancreas development, PDX-1 regulation of insulin gene transcription (known to occur in synergy with El 2, an additional target of Bridge- 1 coactivation and interaction), and PDX- 1 regulation of differentiated pancreatic beta cell function.
  • Example 13 Bridge-1 transactivation utilizes its PDZ-like domain and carboxy-terminal sequences
  • PDZ-like domain (retaining amino acids 1-73, for example), lack Bridge-1 transactivation activity. Interestingly, a series of point mutants were made within the 41 amino acid domain of Bridge-1 identified as the PDZ-like domain. These mutants were designed to disrupt conserved residues within the PDZ-like domain. Seven distinct point mutations of conserved residues within the PDZ-like domain disrupt Bridge-1 transactivation (five examples are shown in FIG. 15).
  • Bridge- 1 The utilization of the PDZ-like domain for Bridge- 1 transactivation activity indicates that targeting inhibition of this domain with small molecule inhibitors will alter Bridge-1 activity. Further, the observation that different structural requirements specify transactivation versus interaction activities indicates that it is possible to design small molecule inhibitors that specifically target distinct activities of the Bridge- 1 protein. Since Bridge- 1 is a key regulator of insulin gene transcription, such inhibitors should modulate insulin gene transcription and be of therapeutic relevance to the disease diabetes mellitus.
  • Example 14 Bridge-1 function is modulated by p300
  • Bridge-1 The ability of Bridge-1 to interact with coactivators with histone acetyl transferase activity that interact with basal transcriptional machinery, such as CBP and p300, was tested.
  • the Bridge-1/GaW DNA binding domain fusion protein construct transactivting a GaWCAT reporter was utilized as our test system in transient transfections in which we added increasing amounts of p300.
  • the addition of p300 increased Bridge-1 transactivation activity, as much as 25-fold (FIG. 16).
  • Bridge- 1 mRNAs of different sizes are routinely observed, indicating that multiple Bridge-1 transcripts are made, possibly resulting in different forms of Bridge-1 protein.
  • human homologues of Bridge-1 have different carboxy-terminal sequences, indicating that alternate splicing of the carboxyl terminus occurs.
  • this splice junction occurs out of frame and hence entirely changes the carboxy-terminal Bridge- 1 amino acid sequence .
  • This splicing occurs at a position near the site of the carboxy-terminal deletion mutation (1-186) that disrupts Bridge-1 transactivation activity. Since we have implicated the carboxyl terminus of Bridge- 1 as essential for transactivation activity, changing its amino acid sequence by alternate splicing mechanisms will affect transactivation activity. The implication of this finding is that small molecules that target distinct forms of carboxy-terminal Bridge-1 sequences have different biological effects.
  • Bridge-1 In addition to the evidence for different Bridge-1 transcripts, multiple forms of Bridge-1 were detected with rabbit polyclonal antisera on Western blots of cellular extracts.
  • the full length in vitro translated Bridge-1 protein migrates at approximately 28-29 kilodaltons on Western blots.
  • a 28-29 kilodalton protein On Western blots of cellular extracts, a 28-29 kilodalton protein is detected, but often a protein of approximately 18 kilodaltons in size is also detected (FIG. 17).
  • the ratio of this "small form" of Bridge-1 relative to the full length protein is high for multiple insulinoma cell lines, intermediate for the hamster fibroblast cell line BHK, and low for the human cell line HeLa.
  • Bridge-1 A novel protein that interacts with El 2 by yeast two-hybrid analysis has been identified.
  • the protein is designated Bridge-1 to reflect its probable role as a coactivator that functions via protein-protein interactions. Consistent with this concept of protein-protein interactions is the localization within Bridge-1 of a truncated PDZ-like domain that is required for its interaction with E12. Whereas most characterized PDZ domain-containing proteins have been localized to membrane or cytoplasmic compartments, a small number of nuclear proteins with
  • the PDZ-like domain within Bridge-1 may be a subtype that functions within the nucleus, as it is similar to PDZ domains within the nuclear protein SIP- 1.
  • SIP- 1 interacts with the testis determining factor SRY and has an identical sequence to proteins designated TKA- 1 (tyrosine kinase activator- 1, Genbank ace. no. Z50150) and E3KARP (NH3 kinase A regulatory protein (Zhuang, Y., et al, Cell 79:875-884 (1994))).
  • Bridge- 1 Other proteins with PDZ domains homologous to Bridge- 1 include a Tax-binding protein (Zhuang, Y., et al, Cell 79:875-884 (1994)), and human proteins of unknown function with leucine zipper or LIM domain motifs usually found in transcription factors (FIG. 2C).
  • the PDZ-like domain within Bridge-1 also shares homology with cytoplasmic proteins, including a regulator of renal Na+/H+ exchange, proteases of the DEGS type and the tight junction protein ZO-1.
  • the PDZ-like domain of Bridge-1 has some atypical structural features.
  • the stretch of PDZ homology within Bridge-1 is 41 amino acids, approximately half the size of typical PDZ domains (Cabral, J. H. M., et al, Nature 3352:649-
  • Bridge-1 may contain a distinct type of PDZ-like domain with different structural determinants.
  • E12 and E47 interact with other basic helix-loop-helix proteins through heterodimerization via helix-loop-helix domains (Murre, C, et al, Biochim. Biophys. Acta 7275:129-135 (1994)).
  • the yeast two-hybrid bait used to clone Bridge- 1 was derived from carboxy terminal E 12 sequences, including the bHLH domain and the carboxy terminus (FIG. 10A). Although the Bridge-1 protein sequence includes regions of hydrophobicity, homologies with basic helix-loop-helix proteins were not observed.
  • the El 2 carboxy terminus is important both for interaction with Bridge-1 and for Bridge- 1 coactivation of El 2.
  • Bridge-1 coactivation of El 2-mediated transactivation of the insulin promoter enhancer sequence FarFlat was impaired by approximately 65%o in the absence of the nine carboxy-terminal amino acids of El 2.
  • These data support a model in which interaction of the carboxy-terminal domain of El 2 with the PDZ-like domain of Bridge-1 results in increased transcriptional activation of El 2 targets.
  • This Bridge-1 -El 2 interaction model is distinct from typical heterodimerization models thought to be employed by most
  • Bridge-1 may function both as an E2A coactivator and in some additional capacity as a regulator of E2A degradation.
  • E2A null mice have abnormalities of lymphocyte development (Bain, G., etal, Cell 79:885- 892 (1994); Mutoh, H., et al, Proc. Natl. Acad. Sci. USA 94:3560-3564 (1997)), and mice nullizygous for beta-2/neuroD, the pancreas-specific dimerization partner of E2A, demonstrate abnormalities in pancreas development (Naya, F. J., et al, Genes Development 11 :2323-2334 (1997)), proteins that modulate the transactivation functions of E2A are candidate developmental regulators. Bridge-1 functions as a strong activator in the context of its fusion to the
  • GaW DNA-binding domain The difference in the activity of this construct in the two cell types tested indicates that this transactivational activity is a regulated function. Possible explanations for these differences in activity include different post-translational modifications of Bridge- 1 protein that alter its conformation and function, or differences in expression patterns of protein-binding partners for
  • the transactivation data are consistent with either an intrinsic transactivation domain within Bridge-1 or a recruitment function that attracts additional transactivating proteins.
  • Bridge-1 is important in the pancreas in regulating the insulin and other islet genes.
  • a high level of Bridge-1 expression in pancreatic islet cell lines was observed compared to a cell line derived from exocrine pancreas, as well as expression predominantly in the islets in sections of mouse pancreas.
  • Bridge-1 is expressed in the insulin-producing ⁇ -cells of murine islets.
  • the nuclear localization of Bridge-1 is consistent with its function as a coactivator.
  • E2A regulates the promoter activities of islet-specific genes, including the insulin and glucagon genes, via binding to E boxes within the promoters of these genes (Cordier-Bussat, M., et ⁇ /., Mol. Cell. Biol.
  • E boxes in the promoter are partially responsible for glucose-responsive transcription (German, M. S., and J. Wang, Mol. Cell. Biol. 74:4067-4075 (1994)).
  • Previous investigators have demonstrated that E2A proteins heterodimerize with the tissue-specific bHLH transcription factor Beta-2/NeuroD in binding to the E boxes of the insulin promoter (Naya, F. J., et al, Genes Dev. 9: 1009-1019 (1995)).
  • These heterodimers work in synergy with homeodomain proteins, including PDX- 1 , to activate the insulin promoter via minienhancers such as FarFlat (German, M. S.
  • Bridge-1 enhanced the activation of this minienhancer by either E 12 or E47.
  • the lack of DNA-binding activity of Bridge- 1 on FarFlat oligonucleotides is consistent with a role for Bridge-1 in this context as a coactivator rather than a direct transactivator.
  • the absence of any obvious DNA-binding domain within the Bridge-1 sequence supports this model. It is possible that, with other target DNA sequences, or under other conditions, Bridge-1 might have a cryptic DNA-binding function.
  • the Bridge-1 coactivation of E12-mediated FarFlat transactivation is largely dependent on an intact carboxy terminus within El 2, indicating that Bridge-1 /El 2 interaction mediates the observed coactivation.
  • the coactivator p300 is proposed to stimulate insulin gene transcription via direct interactions with E47 and Beta-2/NeuroD (Qiu, Y., et al, Mol. Cell Biol 75:2957-2964 (1998)).
  • the present data show that Bridge-1 coactivation of E12 is occurs through a mechanism independent of Beta-2/NeuroD.
  • polypeptides, biological or chemical compounds or other small molecules that enhance or inhibit Bridge- 1 activity are used to treat diabetes or other developmental disorders by modifying the transcription activation of target genes including the insulin gene.
  • an animal may be treated by introducing into the animal one or more of the isolated nucleic acid molecules of the invention comprising a polynucleotide encoding a mammalian Bridge-1 polypeptide or a fragment thereof, particularly a polynucleotide that is 90% or 95%) identical to the Bridge-1 nucleotide sequence or a Bridge-1 fragment nucleotide sequence, such as a fragment encoding the PDZ-like domain and/or the carboxyl terminus domain of Bridge-1.
  • This approach known generically as
  • “gene therapy ' is designed to increase the level of mammalian Bridge-1 gene expression in the cells affected by or causing the disorder (such as cancer or tumor cells) and thereby to cure, delay or prevent the development of, or induce remission of, the disorder by restoring cell cycle checkpoint control to the affected cells.
  • Analogous gene therapy approaches have proven effective or to have promise in the treatment of certain mammalian diseases such as cystic fibrosis (Drumm, M.L. et al, Cell (52:1227-1233 (1990); Gregory, R.J. et al, Nature 347:358-363 (1990); Rich, D.P. et al, Nature 347:358-363 (1990)), Gaucher's disease (Sorge, J.
  • one or more isolated nucleic acid molecules of the invention comprising a polynucleotide having a nucleotide sequence at least 90% or at least 95%) identical to the Bridge-1 sequence or a Bridge-1 fragment, such as a fragment encoding the PDZ-like domain, is introduced into or administered to the animal that is suffering from or predisposed to the disorder.
  • Such isolated nucleic acid molecules may be incorporated into a vector or virion suitable for introducing the nucleic acid molecules into the cells or tissues of the animal to be treated, to form a transfection vector.
  • Suitable vectors or virions for this purpose include those derived from retroviruses, adenoviruses and adeno- associated viruses.
  • nucleic acid molecules of the invention may be complexed into a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or with viral components (e.g., viral capsid proteins).
  • a virus e.g., an adenovirus or an adeno-associated virus
  • viral components e.g., viral capsid proteins
  • vectors comprising the isolated polynucleotides of the present invention are directly introduced into the cells or tissues of the affected animal, preferably by injection, inhalation, ingestion or introduction into a mucous membrane via solution; such an approach is generally referred to as "in vivo" gene therapy.
  • cells, tissues or organs may be removed from the affected animal and placed into culture according to methods that are well- known to one of ordinary skill in the art; the vectors comprising the mammalian Bridge- 1 polynucleotides may then be introduced into these cells or tissues by any of the methods described generally above for introducing isolated polynucleotides into a cell or tissue, including viral infection or transfection, and, after a sufficient amount of time to allow incorporation of the Bridge- 1 polynucleotides, the cells or tissues may then be re-inserted into the affected animal. Since the introduction of the mammalian Bridge- 1 gene is performed outside of the body of the affected animal, this approach is generally referred to as "ex vivo" gene therapy.
  • the isolated mammalian Bridge- 1 polynucleotides of the invention may alternatively be operatively linked to a regulatory DNA sequence, which may be a mammalian Bridge-1 promoter or an enhancer, or a heterologous regulatory DNA sequence such as a promoter or enhancer derived from a different gene, cell or organism, to form a genetic construct as described above.
  • a regulatory DNA sequence which may be a mammalian Bridge-1 promoter or an enhancer, or a heterologous regulatory DNA sequence such as a promoter or enhancer derived from a different gene, cell or organism, to form a genetic construct as described above.
  • This genetic construct may then be inserted into a vector, which is then directly introduced into the affected animal in an in vivo gene therapy approach, e.g., by intratumoral administration (i.e., introduction of the nucleic acid molecule or vector directly into a tumor in an animal, for example by injection), or into the cells or tissues of the affected animal in an ex vivo approach.
  • the genetic construct of the invention may be introduced into the cells or tissues of the animal, either in vivo or ex vivo, in a molecular conjugate with a virus (e.g., an adenovirus or an adeno-associated virus) or viral components (e.g. , viral capsid proteins; see WO 93/07283).
  • a virus e.g., an adenovirus or an adeno-associated virus
  • viral components e.g. , viral capsid proteins; see WO 93/07283

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Abstract

La présente invention concerne un facteur de transcription «Bridge-1», et plus particulièrement des molécules d'acide nucléique isolées codant pour «Bridge-1». Cette invention concerne également des procédés de recombinaison permettant de produire des polypeptides «Bridge-1», ainsi que des anticorps «Bridge-1». Cette invention concerne enfin des méthodes de criblage destinées à identifier des polypeptides qui se lient à «Bridge-1» ou à un fragment de celui-ci, par exemple un fragment contenant un domaine de liaison de type PDZ de «Bridge-1».
PCT/US2000/010616 1999-04-22 2000-04-21 «bridge-1», un facteur de transcription WO2000065052A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2006007377A2 (fr) * 2004-06-16 2006-01-19 The General Hospital Corporation Methodes de depistage des troubles a mediation bridge-1, notamment du diabete de type ii
US8278268B2 (en) 2001-04-04 2012-10-02 Ortho-Mcneil Pharmaceutical, Inc. Combination therapy comprising glucose reabsorption inhibitors and PPAR modulators
EP3740213A4 (fr) * 2018-01-19 2021-10-27 Baker Heart and Diabetes Institute Inhibiteurs de psmd9 pour le traitement d'une dysrégulation des lipides hépatiques

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8278268B2 (en) 2001-04-04 2012-10-02 Ortho-Mcneil Pharmaceutical, Inc. Combination therapy comprising glucose reabsorption inhibitors and PPAR modulators
WO2006007377A2 (fr) * 2004-06-16 2006-01-19 The General Hospital Corporation Methodes de depistage des troubles a mediation bridge-1, notamment du diabete de type ii
WO2006007375A2 (fr) * 2004-06-16 2006-01-19 The General Hospital Corporation Utilisation de bridge-1 et activateurs et inhibiteurs de ceux-ci utilises dans le traitement du diabete insulino-dependant
WO2006007377A3 (fr) * 2004-06-16 2006-08-17 Gen Hospital Corp Methodes de depistage des troubles a mediation bridge-1, notamment du diabete de type ii
WO2006007375A3 (fr) * 2004-06-16 2006-08-24 Gen Hospital Corp Utilisation de bridge-1 et activateurs et inhibiteurs de ceux-ci utilises dans le traitement du diabete insulino-dependant
US7875275B2 (en) * 2004-06-16 2011-01-25 The General Hospital Corporation Use of Bridge-1 and activators and inhibitors thereof in the treatment of insulin deficiency and diabetes
EP3740213A4 (fr) * 2018-01-19 2021-10-27 Baker Heart and Diabetes Institute Inhibiteurs de psmd9 pour le traitement d'une dysrégulation des lipides hépatiques

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