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WO2000075670A1 - Interaction de b3-1 avec les facteurs d'echange du gtp du facteur d'adp-rybosylation - Google Patents

Interaction de b3-1 avec les facteurs d'echange du gtp du facteur d'adp-rybosylation Download PDF

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Publication number
WO2000075670A1
WO2000075670A1 PCT/US2000/015910 US0015910W WO0075670A1 WO 2000075670 A1 WO2000075670 A1 WO 2000075670A1 US 0015910 W US0015910 W US 0015910W WO 0075670 A1 WO0075670 A1 WO 0075670A1
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Prior art keywords
polypeptide
arf
binding
gef
binding partner
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PCT/US2000/015910
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English (en)
Inventor
Edith S. Harris
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Icos Corporation
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Priority to AU56030/00A priority Critical patent/AU5603000A/en
Publication of WO2000075670A1 publication Critical patent/WO2000075670A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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

Definitions

  • the present invention relates generally to methods for identifying compounds that modulate interaction between B3-1 and ADP-ribosylation factor GTP exchange factor proteins.
  • ADP-ribosylation factors are members of the Ras family of GTPases that function in vesicle-mediated transport between Golgi cisternae [Schekman and Orci, Science 277:1526-1533 (1996)]. ARFs may also participate in exocytosis, receptor mediated endocytosis and actin cytoskeletal organization [D'Souza-Schorey, et al, Science 267:1175-1178 (1995); Caumont, et al, J. Biol. Chem. 273:1373-1379
  • ARFs are molecular switches that are inactive when bound to GDP and active when bound to GTP. When active, the GTPases bind to, and regulate the activity of, downstream kinases and lipases. For example, ARFs have been reported to directly regulate phospholipase D (PLD) [Brown, et al, Cell 75:1137-1144 (1993);
  • ARF 1 localizes to the Golgi whereas ARF6 localizes to the plasma membrane [D'Souza-Schorey, et al, Science 267:1175-1178 (1995); Teal, et al, J. Biol. Chem. 269:3135-3138 (1994)].
  • Different subcellular sites of ARF localization suggest different specific targeting interactions and functions among family members.
  • ARF activity is induced through an interaction with specific GTP exchange factors (GEFs).
  • GEFs GTP exchange factors
  • ARNO GTP exchange factors
  • a family of four closely related ARF GEFs has recently been described, designated ARNO [Chardin, et al, Nature 354:481-484 (1996)], cytohesin-1 or B2-1 [Dixon, et al, Biochim. Biophys. Ada 7276:321-324 (1993); Kolanus, et al;., Cell 56:233-242 (1996)]
  • ARNO3 [Klarlund, et al, Science 275:1927-1930 (1997), incorporated by reference herein]
  • ARNO4 WO97/39124, published October 23, 1997, incorporated by reference herein] .
  • All four proteins include an amino- terminal coiled-coil structure with limited homology to kinesin, a central Sec7 domain homologous with yeast Sec7 and Gea-1, and a carboxy-terminal pleckstrin homology (PH) domain [Peyroche, et al, Nature 354:479-481 (1996)].
  • the Sec7 domain is sufficient to mediate ARF GTP exchange [Chardin, et al, Nature 354:481-484 (1996)].
  • ARF proteins include one or more N-myristate moieties that are believed to participate in membrane binding [Randazzo, etal, J. Biol. Chem. 270:14809-14815 (1995)].
  • binding of GEF GEF
  • PH domains to a particular type of membrane phosphatidylinositol may be required to both target and regulate ARF activity [Chardin, et al, Nature 354:481-484 (1996); Klarlund, et al, Science 275:1927-1930 (1997)]. It is unclear if these binding mechanisms are sufficient to target ARF-GEF complexes to all downstream effectors of ARF, as other GEF binding proteins may play a role in the localization of ARF GEF complexes.
  • ARF GEF-interacting compounds can provide targets for therapeutic and prophylactic intervention in pathologies which arise from, or are associated with, ARF biological activity.
  • the present invention provides materials and methods to identify binding partners of ADP ribosylation factor GTP exchange factors (ARF GEFs) as well as modulators of ARF GEF biological activity.
  • the invention also provides polynucleotides encoding binding partners of ARF GEFs, expression constructs comprising ARF GEF-encoding polynucleotides, host cells comprising ARF GEF polynucleotides or expression constructs, and methods to produce ARF GEF binding partner polypeptides.
  • B3-1 polypeptides are members of a family of polypeptides designated ARF GEF adaptor polypeptides.
  • a partial B3-1 polypeptide previously characterized is also known as AGA-1.
  • the present invention provides methods for identifying a compound that binds an ADP ribosylation factor GTP exchange factor (ARF GEF) comprising the steps of: a) contacting the ARF GEF with one or more candidate binding partner compounds, and b) identifying the compounds that bind the ARF GEF.
  • ARF GEF is immobilized.
  • the method is performed in a cell.
  • the invention also provides methods for identifying a modulator of binding between B3- 1 and an ADP-ribosylation factor GTP exchange factor (ARF GEF), comprising the steps of: a) measuring binding between a B3-1 polypeptide that binds an ARF GEF and an ARF GEF polypeptide that binds B3-1 , in the presence and absence of a test compound; and b) identifying as a modulator of ARF GEF/B3-1 binding a test compound that decreases or increases binding between the B3- 1 polypeptide and the ARF GEF polypeptide in the presence of the test compound as compared to binding in the absence of the test compound.
  • the ARF GEF is immobilized.
  • the method is performed in a solution assay.
  • the method is performed in a cell.
  • the invention also provides methods for identifying a compound that modulates binding between B3-1 and an ADP-ribosylation factor GTP exchange factor
  • ARF GEF comprising the steps of: a) providing a solid support having immobilized thereon one of a B3-1 polypeptide that interacts with an ARF GEF and an ARF GEF polypeptide that interacts with B3- 1 as an immobilized binding partner; b) contacting the immobilized binding partner with the other of the ARF GEF polypeptide and the B3-1 polypeptide as a non-immobilized binding partner; c) measuring binding between said immobilized binding partner and said non-immobilized binding partner in the presence and absence of a test compound; and d) identifying as a modulating compound one that decreases or increases binding between the immobilized binding partner and said non-immobilized binding partner in the presence of the test compound as compared to in the absence of the test compound.
  • the non-immobilized binding partner is labeled with a detectable label moiety.
  • the solid support is selected from the group consisting of a chromatographic resin, a bead, plastic
  • the invention also provides purified and isolated B3-1 polypeptides comprising the amino acid sequence set out in SEQ ID NO: 2.
  • Polynucleotides encoding the polypeptide of SEQ ID NO: 2 are also comprehended.
  • a presently preferred polynucleotide encoding the amino acid sequence in SEQ ID NO: 2 comprises the polynucleotide sequence set forth in SEQ ID NO: 1.
  • the invention comprehends polynucleotides including DN A, cDN A, genomic DNA, partially chemically synthesized DNA and wholly chemically synthesized DNA.
  • Antisense polynucleotides which specifically hybridize with the complement of a polynucleotide of the invention are also contemplated.
  • the invention also provides expression constructs comprising a polynucleotide of the invention. Host cells transformed or transfected with a polynucleotide or expression construct of the invention are also contemplated.
  • the invention also provides methods for producing a B3-1 polypeptide comprising the steps of: a) growing a host cell of the invention under conditions appropriate for expression of the B3-1 polypeptide and b) isolating the B3-1 polypeptide from the host cell or medium in which the host cell is grown.
  • the invention further provides antibodies specifically immunoreactive a polypeptide of the invention.
  • the antibody of the invention is a monoclonal antibody.
  • Hybridomas which secrete an antibody of the invention are also provided.
  • the invention further provides anti-idiotype antibodies specifically immunoreactive with the antibody of the invention.
  • the present invention provides methods to identify compounds that bind an ARF GEF comprising the steps of (a) contacting an ARF GEF polypeptide with a candidate binding partner compound, and (b) identifying the compound that binds the ARF GEF polypeptide.
  • the ARF GEF is immobilized.
  • the method is performed in a solution assay.
  • the method is performed in a cell based assay.
  • Cell based assays include methods to screen genomic and cDNA libraries, such as the dihybrid assay as previously described [Fields and Song, Nature 340:245-246 (1989); Fields, Methods: A Companion to Methods in Enzymology 5: 1 16-124 (1993); U.S. Patent 5,283, 173 issued February 1, 1994 to Fields, et al. ] .
  • Modifications and variations on the di-hybrid assay also referred to in the art as "two-hybrid” assay
  • Two-hybrid Two-hybrid
  • Methods of the invention preferably identify components in biological pathways that are mediated by ARF GEF biological activity.
  • the method is carried out in a host cell containing a soluble ARF GEF and a soluble form of its binding partner and wherein decreased or increased binding is quantitated through measurement of a binding- dependent phenotypic change in the host cell, said phenotypic change resulting from a change in expression or a reporter gene product.
  • binding proteins can be identified or developed using isolated or recombinant ARF GEF products, ARF GEF variants, or cells expressing such products. Binding proteins are useful for purifying ARF GEF polypeptides and detection or quantification of ARF GEF products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of ARF GEFs, especially those activities involved in signal transduction.
  • the ARF GEF DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of ARF GEFs.
  • DNA and amino acid sequence information for ARF GEFs also permits identification of binding partner compounds with which an ARF GEF polypeptide or polynucleotide will interact. Identification of binding partner compounds of ARF GEF polypeptides provides potential targets for therapeutic or prophylactic intervention in pathologies associated with ARF GEF biological activity.
  • methods of the invention comprise the steps of (a) contacting an ARF GEF polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the ARF GEF polypeptide.
  • Identification of the compounds that bind the ARF GEF polypeptide can be achieved by isolating the ARF GEF polypeptide/binding partner complex, and separating the ARF GEF polypeptide from the binding partner compound.
  • An additional step of characterizing the physical, biological, and/or biochemical properties of the binding partner compound is also comprehended by the invention.
  • the ARF GEF polypeptide/binding partner complex is isolated using a antibody immunospecific for either the ARF GEF polypeptide or the candidate binding partner compound.
  • the complex is isolated using a second binding partner compound that interacts with either the ARF GEF polypeptide or the candidate binding partner compound.
  • either the ARF GEF polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation
  • methods of the invention to identify binding partner compounds include a step of isolating the ARF GEF polypeptide/binding partner complex through interaction with the label or tag.
  • An exemplary tag of this type is a poly-histidine sequence, generally about six histidine residues, that permits isolation of a compound so labeled using nickel
  • In vitro methods of the invention comprise the steps of (a) contacting an immobilized ARF GEF polypeptide with a candidate binding partner compound and (b) detecting binding of the candidate compound to the ARF GEF polypeptide.
  • the candidate binding partner compound is immobilized and binding of the ARF GEF polypeptide is detected. Immobilization is accomplished using any of the methods well known in the art, including bonding to a support, a bead, or a chromatographic resin, as well as high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
  • Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, as well as other techniques well known and routinely practiced in the art.
  • methods comprise the steps of (a) contacting a ARF GEF polypeptide in a cell with a candidate binding partner compound and (b) detecting binding of the candidate binding partner compound to the ARF GEF polypeptide.
  • a presently preferred method uses the dihybrid assay as previously described elsewhere herein. Modifications and variations on the di-hybrid assay (also referred to in the art as "two-hybrid” assay) have also previously been described [Colas and Brent, TIBTECH 76:355-363 (1998)] and are embraced by the invention.
  • the invention also provides methods for identifying a modulator of binding between an ARF GEF protein and a binding partner compound comprising the steps of: a) measuring binding between an ARF GEF polypeptide and a binding partner compound in the presence and absence of a putative modulator; and b) identifying a modulator as one that decreases or increases binding between the ARF GEF polypeptide and the binding partner compound in the presence of the modulator as compared to binding in the absence of the putative modulator.
  • the ARF GEF binding partner compound is a B3-1 polypeptide.
  • the modulator increases binding between the ARF GEF polypeptide and the binding partner compound.
  • the modulator decreases binding between the ARF GEF polypeptide and the binding partner compound.
  • the invention provides a method for identifying a compound that modulates binding between an ARF GEF polypeptide and a binding partner compound comprising the steps of: a) immobilizing the ARF GEF polypeptide on a solid support; b) contacting said ARF GEF polypeptide with a labeled binding partner compound in the presence and absence of a putative modulator compound, c) detecting binding between said labeled binding partner compound and said immobilized ARF GEF polypeptide, and d) identifying the modulating compound as one that decrease or increases binding between the ARF GEF polypeptide and the binding partner compound in the presence of the modulator compound as compared to binding in the absence of the putative modulator compound.
  • the ARF GEF binding partner compound is a B3-1 polypeptide.
  • the invention also provides methods wherein the binding partner compound is immobilized and the ARF GEF is detectably labeled.
  • the invention provides methods wherein an ARF GEF polypeptide is immobilized on a solid support which is coated or impregnated with a fluorescent agent, and the binding partner compound is labeled with an agent which excites said fluorescent agent. Interaction between the ARF GEF and the binding partner compound is detected by light emission from said fluorescent agent.
  • the binding partner compound is immobilized and the ARF GEF polypeptide is labeled with a compound that excites the fluorescent agent.
  • the ARF GEF binding partner compound is B3-1.
  • split hybrid assays as described in WO98/13502, published April 2, 1998 and incorporated herein by reference, are contemplated. Variations on the split hybrid assay, described generally in WO 95/20652, published August 3, 1995 and incorporated by reference herein, are also contemplated.
  • Compounds that modulate (i.e., increase, decrease, or block) ARF GEF activity or expression may be identified in a method comprising the steps of (a) incubating a putative modulator with a ARF GEF polypeptide or polynucleotide and (b) determining the effect of the putative modulator on ARF GEF activity or expression.
  • the selectivity of a compound that modulates ARF GEF polypeptide activity or expression can be evaluated by comparing the effects on ARF GEF activity or expression to the effect on other compounds.
  • Cell based methods such as di-hybrid assays to identify DNA encoding a binding compound and split hybrid assays to identify inhibitors of ARF GEF polypeptide interaction with a known binding polypeptide, as well as in vitro methods, including assays wherein an ARF GEF polypeptide, an ARF GEF polynucleotide, or a binding partner is immobilized, and solution assays are contemplated by the invention.
  • Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to an ARF GEF polypeptide or an ARF GEF- encoding nucleic acid, oligonucleotides which specifically bind to an ARF GEF polypeptide or an ARF GEF gene sequence, and other non-peptide compounds (e.g. , isolated or synthetic organic and inorganic molecules) which specifically react with an ARF GEF polypeptide or underlying nucleic acid.
  • Mutant ARF GEF polypeptides which affect the enzymatic activity or cellular localization of the wild-type ARF GEF polypeptides are also contemplated by the invention.
  • Presently preferred targets for the development of selective modulators include, for example: (1) regions of the ARF GEF polypeptide which contact other proteins, (2) regions that localize the ARF GEF polypeptide within a cell, (3) regions of the ARF GEF polypeptide which bind substrate, (4) allosteric binding site(s) of the ARF GEF polypeptide, and (5) phosphorylation site(s) of the ARF GEF polypeptide, as well as other regions of the protein wherein covalent modification regulates biological activity.
  • Still other selective modulators include those that recognize specific ARF GEF encoding and regulatory polynucleotide sequences. Modulators of ARF GEF activity may be therapeutically useful in treatment of diseases and physiological conditions in which ARF GEF activity is known or suspected to be involved.
  • Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator.
  • a modulator is identified in those instances where binding between the ARF GEF polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.
  • a modulator that increases binding between the ARF GEF polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the ARF GEF polypeptide and the binding partner compound is described as an inhibitor.
  • HTS high throughput screening
  • Assays that incorporate robotics, wherein tens to hundreds of thousands of candidate modulator compounds are screened, are particularly preferred.
  • the invention therefore provides high throughput screening assays to identify compounds that interact with or inhibit biological activity (i.e., inhibit enzymatic activity, binding activity, etc.) of a ARF GEF polypeptide.
  • Cell-based HTS systems are contemplated, including melanophore assays to investigate binding interaction, yeast-based assay systems, and mammalian cell expression systems
  • HTS assays are designed to identify "hits” or 'lead compounds” having a desired property, from which modifications can be designed to improve the desired property. Chemical modification of the "hit” or “lead compound” is often based on an identifiable structure/activity relationship between the "hit” and the ARF GEF polypeptide.
  • modulators of ARF GEF activity and in particular HTS assays.
  • libraries used for the identification of small molecule modulators including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.
  • Natural product libraries consist of structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.
  • Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms.
  • Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Cane, et al, Science 252:63-68 ( 1998).
  • Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture.
  • compositions comprising a modulator identified by a methods of the invention.
  • the compositions are pharmaceutical compositions.
  • the pharmaceutical compositions optionally may include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art maybe used.
  • Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
  • the pharmaceutical compositions can be packaged in forms convenient for delivery.
  • the compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the immunogenic composition into the recipient organism and, particularly, when the immunogenic composition is being delivered in unit dose form.
  • the dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
  • compositions may be introduced into the subject to be treated by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drug solutions) or subcutaneous injection (including depot administration for long term release) ; by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • B3-1 modulator product compositions are generally injected in doses ranging from 1 ⁇ g/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 50 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kg/day.
  • the modulator composition may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product.
  • Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration.
  • the optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage.
  • Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.
  • a suitable dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above.
  • Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data.
  • the final dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug's specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
  • the present invention also provides novel purified and isolated human polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the human B3-1 polypeptides.
  • DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences.
  • Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and includes allelic variants of the preferred polynucleotide of the invention. Genomic DNA of the invention is distinguishable from genomic DNAs encoding polypeptides other than B3-1 in that it includes the B3-1 coding region found in B3- 1 cDNA of the invention. Genomic DNA of the invention can be transcribed into
  • RNA, and the resulting RNA transcript may undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcript are removed, or "spliced out.”
  • RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a B3-1 polypeptide are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same DNA sequences but arise from distinct mRNA transcripts.
  • Allelic variants are known in the art to be modified forms of a wild type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are inherently naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).
  • the invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding B3-1, followed by second strand enzymatic synthesis of a complementary strand to provide a double stranded DNA, or cDNA.
  • “Chemically synthesized” as used herein and understood in the art refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. "Wholly chemically synthesized” DNA sequences are therefore produced entirely by chemical means, and “partially chemically synthesized” DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.
  • a preferred DNA sequence encoding a human B3-1 polypeptide is set out in SEQ ID NO: 1.
  • the preferred DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ED NO: 1 along with the complementary molecule (the "non-coding strand” or “complement") having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson-Crick base pairing rules for DNA.
  • the complementary molecule the “non-coding strand” or “complement” having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson-Crick base pairing rules for DNA.
  • the invention further embraces species, preferably mammalian, homologs of the human B3-l-encoding DNA.
  • Species homologs in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least
  • Percent sequence "homology" with respect to polynucleotides of the invention is defined herein as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the B3-1 sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • polynucleotide sequence information provided by the invention makes possible large scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
  • Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related B3-1 polypeptides by well known techniques including Southern and or Northern hybridization, and polymerase chain reaction (PCR).
  • related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to B3-1 and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of B3-1.
  • the disclosure of a full length polynucleotide encoding a B3-1 polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotide.
  • the invention therefore provides fragments of B3-1- encoding polynucleotides comprising at least 10 to 20, and preferably at least 15, consecutive nucleotides of a polynucleotide encoding B3-1, however, the invention comprehends fragments of various lengths.
  • fragment polynucleotides of the invention comprise sequences unique to the B3-1 -encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., "specifically") to polynucleotides encoding B3-1, or B3-1 fragments thereof containing the unique sequence.
  • Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full length sequence derived from introns, regulatory regions, and/or other non- translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g. , those made available in public sequence databases.
  • the invention also provides fragment polynucleotides that are conserved in one or more polynucleotides encoding members of the B3-1 family of polypeptides.
  • fragments include sequences characteristic of the family of B3-1 polynucleotides, and are also referred to as "signature sequences.”
  • signature sequences are readily discernable following simple sequence comparison of polynucleotides encoding members of the B3-1 family. Fragments of the invention can be labeled in a manner that permits their detection, including radioactive and non-radioactive labeling.
  • Fragment polynucleotides are particularly useful as probes for detection of full length or other fragment B3-1 polynucleotides.
  • One or more fragment polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding B3-1 , or used to detect variations in a polynucleotide sequence encoding B3-1.
  • Expression constructs such as plasmid and viral DNA vectors incorporating B3-1 sequences are also provided.
  • Expression constructs wherein B3-l-encoding polynucleotides are operatively linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided.
  • Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
  • Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell.
  • Expression constructs are preferably utilized for production of an encoded protein, but may also be utilized to amplify the construct itself when other amplification techniques are impractical.
  • host cells including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention in a manner which permits expression of the encoded B3- 1 polypeptide.
  • Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.
  • Methods for introducing DNA into the host cell well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts.
  • Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, and mammalian cells systems.
  • Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with B3-1.
  • Host cells of the invention are also useful in methods for large scale production of B3-1 polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
  • HPLC high pressure liquid chromatography
  • Still other methods of purification include those wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent.
  • the purified protein can be cleaved to yield the desired protein, or be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
  • Knowledge of B3- 1 -encoding DNA sequences allows for modification of cells to permit, or increase, expression of endogenous B3-1. Cells can be modified (e.g.
  • amplifiable marker DNA e.g. , ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the B3- 1 coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the B3-1 coding sequences in the cells.
  • the DNA sequence information provided by the present invention also makes possible the development through, e.g. homologous recombination or "knock-out” strategies [Capecchi, Science 244:1288-1292 (1989)], of animals that fail to express functional B3- 1 or that express a variant of B3- 1. Such animals are useful as models for studying the in vivo activities of B3-1 and modulators of B3-1.
  • the invention also provides purified and isolated mammalian B3-1 polypeptides encoded by a polynucleotide of the invention.
  • a B3- 1 polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2.
  • Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as maybe needed to confer optimal biological activity on recombinant expression products of the invention.
  • B3-1 polypeptides Glycosylated and non-glycosylated form of B3-1 polypeptides are embraced.
  • the invention also embraces variant (or analog) B3- 1 polypeptides.
  • insertion variants are provided wherein one or more amino acid residues supplement a B3-1 amino acid sequence. Inserted amino acids residues may be located at either or both termini of the protein, or may be positioned within internal regions of the B3-1 amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels. Insertion variants include B3-1 polypeptides wherein one or more amino acid residues are added to a B3-1 acid sequence, or fragment thereof.
  • Variant products of the invention also include mature B3-1 products, i.e. , B3-1 products wherein leader or signal sequences are removed, with additional amino terminal residues.
  • the additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from a specific proteins.
  • B3- 1 products with an additional methionine residue at position -1 (Met " '-B3-1) are contemplated, as are B3-1 products with additional methionine and lysine residues at positions -2 and -1 (Met "2 -Lys " '-B3-1).
  • Met-Lys, Lys residues are particularly useful for enhanced recombinant protein production in bacterial host cell.
  • the invention also embraces B3-1 variants having additional amino acid residues which result from use of specific expression systems.
  • use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide.
  • GST glutathione-S-transferase
  • Insertional variants also include fusion proteins wherein the amino and/or carboxy termini of the B3-1 polypeptide is fused to another polypeptide.
  • fusion proteins are immunogenic polypeptides such as B3-1, proteins with long circulating half life, such as immunoglobulin constant regions, marker proteins (e.g., fluorescent, others?) and proteins or polypeptide that facilitate purification of the desired B3-1 polypeptide.
  • the invention provides deletion variants wherein one or more amino acid residues in a B3-1 polypeptide are removed. Deletions can be effected at one or both termini of the B3-1 polypeptide, or with removal of one or more residues within the B3-1 amino acid sequence. Deletion variants, therefore, include all fragments of a B3-1 polypeptide.
  • the invention also embraces polypeptide fragments of the sequence set out in SEQ ID NO: 2 wherein the fragments maintain biological or immunological properties of aB3-l polypeptide. Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO: 2 are comprehended by the invention. Preferred polypeptide fragments display antigenic properties unique to or specific for the
  • Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
  • the invention provides substitution variants of B3- 1 polypeptides.
  • Substitution variants include those polypeptides wherein one or more amino acid residues of a B3-1 polypeptide are removed and replaced with alternative residues.
  • the substitutions are conservative in nature, however, the invention embraces substitutions that ore also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables A or B below.
  • Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A (from
  • conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77] as set out in Table B, below.
  • the invention also provides derivatives of B3-1 polypeptides.
  • Derivatives include B3-1 polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.
  • the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.
  • Derivatives of the invention may be prepared to increase circulating half-life of a B3-1 polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.
  • the invention further embraces B3-1 products covalently modified or derivatized, e.g., B3-1, to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Particularly preferred are B3-1 products covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the B3-1 products, or randomly attached to one or more side chains of the polypeptide.
  • antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) and other binding proteins specific for B3-1 products or fragments thereof.
  • Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published June 20, 1993, which is incorporated herein by reference in its entirety.
  • Antibody fragments, including Fab, Fab ' , F(ab ' ) 2 , and F v are also provided by the invention.
  • variable regions of the antibodies of the invention recognize and bind B3- 1 polypeptides exclusively (i. e. , able to distinguish B3-1 polypeptides from the family of B3-1 polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELIS A techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art.
  • Antibodies that recognize and bind fragments of the B3-1 polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, B3-1 polypeptides.
  • antibodies of the invention that recognize B3-1 fragments are those which can distinguish B3-1 polypeptides from the family of B3-1 polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
  • Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of B3- 1 ), diagnostic purposes to detect or quantitate B3-
  • Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.
  • a kit of the invention also includes a control antigen for which the antibody is immunospecific.
  • anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding B3-1.
  • Full length and fragment anti-sense polynucleotides are provided.
  • fragment antisense molecules of the invention include (i) those which specifically recognize and hybridize to B3-1 RNA (as determined by sequence comparison of DNA encoding B3-1 to DNA encoding other known molecules) as well as (ii) those which recognize and hybridize to RNA encoding variants of the B3-1 family of proteins.
  • Antisense polynucleotides that hybridize to RNA encoding other members of the B3-1 family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of B3-1 by those cells expressing B3-1 mRNA.
  • Antisense nucleic acids preferably 10 to 20 base pair oligonucleotides capable of specifically binding to B3-1 expression control sequences or B3- 1 RNA are introduced into cells (e.g. , by a viral vector or colloidal dispersion system such as a liposome).
  • the antisense nucleic acid binds to the B3- 1 target nucleotide sequence in the cell and prevents transcription or translation of the target sequence.
  • Phosphorothioate and methylphosphate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.
  • the antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5 ' end.
  • the invention further comprehends methods to modulate B3- 1 expression through use of ribozymes.
  • Ribozyme technology can be utilized to inhibit translation of B3-1 mRNA in a sequence specific manner through (i) the hybridization of a complementary RNA to a target mRNA and (ii) cleavage of the hybridized mRNA through nuclease activity inherent to the complementary strand.
  • Ribozymes can identified by empirical methods but more preferably are specifically designed based on accessible sites on the target mRNA (Bramlage, etal, Trends Biotechnol. 76:434-438 (1998).
  • Ribozymes can specifically modulate expression of B3- 1 when designed to be complementary to regions unique to a polynucleotide encoding B3- 1. "Specifically modulate” therefore is intended to mean that ribozymes of the invention recognizes only a polynucleotide encoding B3-1. Similarly, ribozymes can be designed to modulate expression of all or some of the B3-1 family of proteins. Ribozymes of this type are designed to recognize polynucleotide sequences conserved in all or some of the polynucleotides which encode the family of proteins.
  • the invention further embraces methods to modulate transcription of B3-1 through use of oligonucleotide-directed triplet helix formation.
  • triplet helix formation is accomplished using sequence specific oligonucleotides which hybridize to double stranded DNA in the major groove as defined in the Watson-Crick model.
  • Hybridization of a sequence specific oligonucleotide can thereafter modulate activity of DNA-binding proteins, including, for example, transcription factors and polymerases.
  • Preferred target sequences for hybridization include promoter and enhancer regions to permit transcriptional regulation of B3-1 expression.
  • triplet helix formation techniques of the invention also embrace use of peptide nucleic acids as described in Corey, Trends Biotechnol. 75:224-229 (1997). Oligonucleotides which are capable of triplet helix formation are also useful for site-specific covalent modification of target DNA sequences. Oligonucleotides useful for covalent modification are coupled to various DNA damaging agents as described in Lavrovsky, et al. [supra]. Mutations in the B3- 1 gene that result in loss of normal function of the
  • B3-1 gene product and underlie B3-1 -related human disease states.
  • the invention comprehends gene therapy to restore B3-1 activity would thus be indicated in treating those disease states (for example, various forms of cancer described herein).
  • Delivery of a functional B3-1 gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g. , adenovirus, adeno- associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998).
  • Example 1 addresses identification of polynucleotides in a human B cell cDNA library that encode proteins which interact with IRP3.
  • Example 2 describes verification of the 5 ' end of the clones identified in Example 1.
  • Example 3 describes specificity of interaction between
  • Example 4 relates to the identification of regions of IRP3 required for the interaction with B3-1.
  • Example 5 describes sequences of B3-1 required for the interaction with IRP3.
  • Example 6 addresses further evidence of a direct interaction of B3-1 and IRP3.
  • Example 7 characterizes expression of B3-1 in various tissue and cell types by Northern blot analysis.
  • GAL4 DNA-binding and transcription-activating domains are encoded on separate plasmids as portions of fusion proteins. Expression of the fusion proteins and interaction of the expression products results in association of GAL4 DNA binding and transactivating domains and ultimate expression of the ⁇ -galactosidase reporter gene under transcriptional control of the GAL4 promoter.
  • a "bait-encoding" pASl plasmid was constructed that contained sequences encoding the GAL4-binding domain, a selection gene (trp), a hemagglutinin (HA) epitope tag, and DNA sequences encoding cytohesin
  • the cytohesin I-encoding sequences were amplified by PCR using the primers B2- 1 5 ' S (SEQ ID NO: 3) and B2-1 3 ' S (SEQ ID NO: 4) and a cytohesin I subclone as template.
  • the subclone was originally amplified from a spleen cDNA library [Liu, et al, Biochim. Biophys. Ada 1132:75-78 (1992)].
  • Reaction conditions included an initial incubation at 94°C for four minutes, followed by thirty cycles of: 94°C for one minute, 50°C for one minute, and 72°C for two minutes.
  • the amplification product was purified, digested, and subcloned into vector p AS 1 to give expression vector cytohesin I/pASl.
  • the resulting product was sequenced to rule out PCR-derived errors.
  • a yeast strain, aY190 was transformed with cytohesin I/pASl by standard methods and grown in trp " selective media to mid-log phase. Cells were harvested and lysed in buffer containing 100 mM Tris, pH 6.8, 2% SDS, 10% glycerol,
  • a "target" expression plasmid was constructed with vector pACT modified to contain sequences encoding a second selection gene (leu) and DNA encoding the GAL4 transcription-activation domain II fused to a B cell cDNA library. Lymphocyte cDNA library sequences were inserted at an Xh ⁇ l site of the vector. Ayl90 cells previously transformed with cytohesin I/pASl were transformed with the pACT- lymphocyte library DNA by standard methods and grown under Leu " -Trp " -His " selective media also including 3-amino triazole. Resulting colonies were tested for ⁇ -galactosidase ( ⁇ -gal) activity using the blue/white selection method well known in the art. Several hundred ⁇ -gal positive colonies were obtained and sequence analysis of the B cell cDNA- derived pACT inserts from 176 positives was carried out. BLASTN and BLASTP
  • L06633 is also known as B3-1.
  • clone 111 included the longest insert, as judged by 5' start of the insert, and the shortest insert was found in clone 184, encoding amino acids corresponding to residues 174 through 359 of clone 111.
  • Clone 184 contained four of the five heptad repeats found in the leucine zipper region of B3-1.
  • the third discrepancy was that nucleotides 847 and 848 of clone 111 were AC and in B3-1, the corresponding nucleotides were CA. This difference resulted in an amino acid change at position 278 in clone 111 from a glutamine at the corresponding position in B3-1 to a threonine.
  • the fourth discrepancy spanned nucleotides 1031 through 1038 of clone 111 which were AGCAACTC, but were GAGTTGCT in B3-1.
  • a BLASTN search of the NCBI GenBank® EST database using the entire known sequence of LO6633 identified two ESTs which had essentially perfect homology to the 5 ' end of the LO6633.
  • AA380876 sequences were identical in the overlapping 52 nucleotides except for unidentified bases.
  • a methionine codon was identified beginning 14 nucleotides 5 ' of the first nucleotide of LO6633.
  • An in- frame stop codon was also identified 24 nucleotides 5 ' to the methionine codon indicating that the methionine codon probably represented the beginning of the open reading frame.
  • B3-1 cl 11 isolated from the dihybrid assay contained a sequence 5 ' of the start of LO6633 which was identical to the T28954 and AA380876 EST sequences through the starting methionine but did not include the 5 ' stop codon.
  • the two-hybrid assay was used to test the specificity of B3-1 with family members of cytohesin I. "Bait" plasmid sequences were first generated by PCR encoding
  • ARNO, ARNO-E156A (a mutant of ARNO wherein glutamate at position 156 was changed to alanine), ARNO4, and ARNO3 (also known as Grb) which were cloned into to plasmid pASl discussed above.
  • DNA encoding the ARNO-E156A mutant was
  • IRP 1 5 ' S and IRP 1 3 ' S oligonucleotide primers were used with an ARNO and an ARNO-
  • IRP 1 3 ' S CCCGTCGACTCAGGGCTGCTCCTGCTTCTTC SEQ ID NO: 8
  • Reaction conditions included an initial incubation at 94°C for two minutes, followed by thirty cycles of: 94°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds and 72°C for one minute.
  • the PCR products were purified, digested, cloned into pASl, and sequenced to rule out PCR derived errors.
  • the correct constructs were transformed into the aY190 yeast strains and expression was examined as described above. Western blots indicated that the IRPl bait fusion proteins were expressed at detectable levels. DNA encoding other bait proteins ARNO4 and ARNO3 were generated also by PCR.
  • IRP2 5 ' S and IRP2 3'S primers were used in PCR reactions with an ARNO4 template [Klarlund, et al, Science 275:1927-1930 (1997)] and primers IRP4 1EE and IRP43 ' S were used in PCR reactions with an ARNO3 template [WO97/39124, published October 27,1998)].
  • IRP25'S CATATCCCGGGGATGGACCTGTGCCACCCAG SEQ ID NO: 9
  • IRP23'S CCCGTCGACTCACTGCTTGCTGGCAATCTTC
  • IRP41EE ATAGAATTCGATATCCATGGATGAAGACGGCGGCGGC
  • PCR was carried out under conditions as described in Example 1.
  • the ARNO4 PCR product was digested with Smal and Sail and subcloned into pAS 1.
  • the ARNO3 PCR product was digested with EcoRV and Sail and subcloned into pAS 1. Clones were sequenced to rule out PCR-derived errors.
  • nucleotide PCR error In generating the ARNO3 bait-encoding sequence, a single nucleotide PCR error was detected and the error resulted in an amino acid change in the encoded protein sequence.
  • the nucleotide at position 906 was altered from C to a G which caused amino acid residue 302 to be changed from an asparagine to a lysine.
  • the ARNO-, ARNO- ⁇ 156A-, ARNO4- and ARNO3-encoding bait plasmids were then used in cotransformations of aY 190 cells with various target plasmids encoding B3-1 proteins (clones 184 and 111) identified as described in Example 1. Both clone 184 and clone 111 interacted with all of the proteins encoded by the bait plasmids.
  • the two-hybrid assay was used to identify regions of cytohesin I responsible for the interaction with the B3-1 clones.
  • Two bait-encoding plasmids were constructed; one encoding the cytohesin I pleckstrin homology (PH) domain and the other encoding the cytohesin I Sec7 domain.
  • DNA encoding the cytohesin- 1 PH domain was amplified by PCR using primers IRP3-770S (SEQ ID NO: 14) and IRP3-3 S (SEQ ID NO: 8) and DNA encoding the Sec7 domain was amplified using primers IRP3-5 ' S (SEQ ID NO: 7) and IRP3-760S (SEQ ID NO: 13).
  • the cytohesin I Sec7 bait plasmid also encoded the cytohesin I kinesin-like domain. Reaction conditions were as described in Example 3.
  • IRP3 760S CCCGTCGACTCAGAGGTCATTCCCGTCGTC SEQ ID NO: 13
  • IRP3 770S CATATCCCGGGGACTTTCTTCAATCCAGAC SEQ ID NO: 14
  • PCR products were cloned into pAS 1 and verified by sequence analysis. Constructs identified to have the correct sequences were transformed into aY190 yeast strains and expression was examined by Western analysis as described in Example 1.
  • Cells transformed with plasmid encoding the cytohesin I PH domain were designated IRP3PH/aYl 90 and cells transformed with plasmid encoding the cytohesin I Sec7 region were designated CytohesinSec7/aYl 90.
  • Plasmids encoding clone 111 or clone 184 were co-transformed into aY190 cells and ⁇ -gal assays carried out, also as described. Both clones 111 and 184 were found to interact with the Sec7 domain of cytohesin I, but neither clone interacted with the PH domain.
  • Sec7 bait plasmid included the amino half of the protein with the kinesin-like coiled-coil domain in addition to the Sec7 domain, another bait plasmid was generated which separated the two domains in order to examine which, if either, of the two domains alone participate in interaction with B3-1. Site directed mutagenesis
  • I sequence encoding the Sec7 domain was carried out using primers IRP3KS (SEQ ID NO: 15) and IRP3KA S (SEQ ID NO: 16) under conditions including an initial incubation at 95 ° C for thirty seconds, followed by sixteen cycles of denaturation at: 95 ° C for thirty seconds, annealing at 45 ° C for one minute, and extension at 58 ° C for twelve minutes.
  • IRP3KAS ATT AAATTTTTTCCTTC AC ATGGCT ACCTGTTT
  • Resulting clones were sequenced to confirm the presence of the mutation and to rule out PCR-derived errors .
  • the resulting bait plasmid was found to encode only the kinesin-like coiled-coil region of cytohesin I and was designated CytohesinK.
  • Plasmid CytohesinK was cotransformed into aY190 cells with either (i) the parental library vector including no additional library sequences, (ii) a library plasmid identified in an unrelated dihybrid assay including DNA that did not encode a cytohesin I-interacting protein (an irrelevant clone), (iii) plasmid encoding clone 111, full length B3-1, or (iv) the plasmid encoding the leucine zipper region of B3-1, B3-1 D4 (see Example 5). Transformants were grown on selective media and the standard filter ⁇ -gal assay was performed.
  • B3-1 has previously been shown to include two significant structural domains and one or both of these domains were identified in the clones isolated in
  • the amino terminal PDZ domain corresponds to amino acids 77 through 166 clone 111 and a leucine zipper motif, with five heptad repeats, is found in clone 111 at amino acids 167 through 202.
  • PDZ domains are 80-100 residue domains that have been reported to function in the organization and localization of signalizing complexes to various membrane sites [Ponting, et al, Bioessays 79:469-479 (1997)].
  • the PDZ domain is presumed not to be required for the interaction with cytohesin I since several clones identified by two-hybrid screening in Example 1 (for example, clone 184) did not include the PDZ region.
  • the leucine zipper region may be required for interaction with cytohesin I since all of the clones identified in Example 1 contained at least four of the five heptad repeats of this region.
  • the leucine zipper domain is the region of clone 111 that interacted with cytohesin I
  • another plasmid was constructed which encoded a truncated clone 111 including amino acid residues 153 through 208.
  • the truncated protein was designed to contain the complete leucine zipper region as well as additional sequence both 5 ' and 3 ' to that region.
  • Primers B31/458E and B31/624X were used in PCR under conditions described in Example 1 with clone 111 as template to amplify the underlying DNA.
  • the amplification product was purified, digested, and subcloned into pACT2.
  • the insert was also sequenced to rule out PCR-derived errors.
  • the resulting subclone was referred to as Clone 111 D4.
  • Yeast aY190 cells were cotransformed with plasmids encoding B3-1 D4 and cytohesin I by standard methods, the host cells were plated on selection media plates (SC-LeuTrp), and a standard filter ⁇ -gal assay was performed.
  • the PDZ domain may contribute to localizing GEF containing ARF complexes in certain cell types.
  • DNA encoding B3-1 as a fusion protein with the enhanced green fluorescent protein (EGFP) was transfected into cells and immunoprecipitation carried out.
  • EGFP enhanced green fluorescent protein
  • the EGFP coding sequence was excised from the vector pEGFP-Cl (Clontech) by digestion with Nhel and BamRl, and the resulting 800 bp fragment was ligated into pCEP4 (Invitrogen) previously digested with the same two enzymes. The sequence of the resulting clone was confirmed and the plasmid designated EGFP/pCEP4.
  • PCR was carried out to generate Xhol and H dHI restriction sites at the 5 ' and 3 ' ends, respectively, of a B3-1 clone.
  • B3-1 -encoding DNA was amplified using clone 111 as template and the primers B31.GFP.5.X and B31.GFP.3.H3 as set out in SEQ ID NOs: 19 and 20.
  • Reaction conditions included an initial incubation at 94°C for 6 minutes followed by 30 cycles of denaturation at 94°C for one minute, annealing at 55°C for one minute, and extension at 72°C for one minute.
  • the amplification product was digested wit Xhol and HmdIII, purified, and ligated into EGFP/pCEP4 previously digested with Xhol and Hz ⁇ di ⁇ .
  • the resulting plasmid designated B31/GFP/pCEP4/l l, was sequenced to eliminate the possibility of errors resulting from PCR.
  • EGFP/pCEP4 fusion constructs were generated with B3- 1 DNA truncations encoding discrete B3-1 protein domains.
  • a DNA truncation encoding the amino terminal domain B3-1 protein sequence through amino acid 165 (a region that does not include the leucine zipper domain) was generated in addition to a DNA truncation encoding the carboxy terminal region of B3-1 , starting at residue 166 (the beginning of the leucine zipper domain) and terminating at residue 359, the end of the protein.
  • the amino terminal-encoding DNA was generated by PCR using primer B31.GFP.5.X (SEQ ID NO: 19) and B31.GFP.PD. ⁇ 3 (SEQ ID NO: 21) and the carboxy terminal-encoding DNA was generated by PCR using primers B31.GFP.3.H3 (SEQ ID NO: 20) and B31.GFP.LZ.X (SEQ ID NO: 22).
  • the B3- 1/GFP plasmid encoding full-length B3- 1 , the plasmids encoding the two B3-1 truncations, and a EGFP/pCEP4 control were separately electroporated into a lymphoblastoid cell line JY8 (which is a JY cell line stably transfected to express the IL- 8 receptor). Electroporation was carried out as follows.
  • the individual B3-l/pCEP4 constructs were transfected into JY8 by electroporation (capacitance 960 ⁇ F and voltage 250 V). Briefly, 10 7 cells were collected by centrifugation and resuspended in 0.5 ml of D-PBS, after which 30 ⁇ g of plasmid DNA was added to separate aliquots of the cells. The cells were incubated on ice for ten minutes, electroporated, and allowed to recover on ice for 10 minutes. The cells were transferred to approximately 5 ml of RPMI media and cultured at 37°C in 5% CO 2 . After 48 hours, the cells were collected by centrifugation and resuspended in fresh media with 0.5 mg/ml of hygromycin B (Calbiochem).
  • B3-1/EGFP fusion protein was first immunoprecipitated using a GFP polyclonal antibody (Clontech). Approximately 50 x 10 6 EGFP/B3-1 or EGFP transfectants were collected by centrifugation and washed three times in D-PBS.
  • Cells were lysed in buffer containing 1% CHAPS with 0.01 mg/ml each of Soybean Trypsin Inhibitor (SBTI), aprotinin and leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 0.2 M Na 3 VO 4 on ice for 30 minutes and centrifuged for ten minutes. The supernatant was pre-cleared by incubation with 15 ⁇ l of Protein A agarose for one hour at 4°C with rotation. Each sample was divided into two tubes prior to immunoprecipitation.
  • SBTI Soybean Trypsin Inhibitor
  • AEBSF 4-(2-aminoethyl)benzenesulfonyl fluoride
  • the monoclonal antibody 200A was used to detect the presence of all ARNO family proteins. ARNO was specifically detected using monoclonal antibody 233G and cytohesin I was detected using monoclonal antibody 200B.
  • MOPC21 was used as an IgGl isotype matched control.
  • the 50 kDa band was detected with 200A, 200B and 233G antibodies but not with the MOPC21 control.
  • a DNA fragment encompassing the entire B3-1 coding region was used as a hybridization probe and prepared as follows.
  • the DNA fragment was amplified by PCR using B3 lcl 11 DNA as a template and primers B31.5Nde (SEQ ID NO: 23) and B31.3Xho (SEQ ID NO: 24).
  • Reaction conditions included an initial incubation at 94°C for six minutes followed by 30 cycles of denaturation at 94°C for one minute, annealing at 55°C for one minute, and extension at 72°C for one minute.
  • the amplification product was purified, digested with NJel and Xhol, and purified a second time on a 1% agarose gel.
  • a 240 ng aliquot of the fragment was labeled with 32 P using a Random Primed Labeling Kit (Boehringer Mannheim) with unincorporated nucleotides removed using a Centrisep column.
  • RNA blots (all from Clontech) were prehybridized in ExpressHyb hybridization solution (Clontech) at 68°C for one hour, after which the prehybridization solution was removed and replaced with fresh Express Hybridization solution containing labeled probe. Hybridization was carried out at 68°C for 1.5 hours. The filters were first washed with 2X SSC at room temperature followed by a final wash at 50°C in 0.2X SSC
  • the 4.3 kb transcript may represent an alternatively processed B3- 1 mRNA or a transcript encoded by an unidentified B3-1 -related gene.

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Abstract

L'invention concerne des méthodes permettant d'identifier des partenaires de liaison de B3-1 et des modulateurs de B3-1 se liant à des protéines du facteur d'échange du GTP du facteur d'ADP-ribosylation.
PCT/US2000/015910 1999-06-09 2000-06-09 Interaction de b3-1 avec les facteurs d'echange du gtp du facteur d'adp-rybosylation WO2000075670A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56030/00A AU5603000A (en) 1999-06-09 2000-06-09 Interaction of b3-1 with adp-ribosylation factor gtp exchange factors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13834999P 1999-06-09 1999-06-09
US60/138,349 1999-06-09

Publications (1)

Publication Number Publication Date
WO2000075670A1 true WO2000075670A1 (fr) 2000-12-14

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PCT/US2000/015910 WO2000075670A1 (fr) 1999-06-09 2000-06-09 Interaction de b3-1 avec les facteurs d'echange du gtp du facteur d'adp-rybosylation

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AU (1) AU5603000A (fr)
WO (1) WO2000075670A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039124A1 (fr) * 1996-04-15 1997-10-23 Icos Corporation Modulateurs cytoplasmiques de la regulation/signalisation de l'integrine
WO1998015629A1 (fr) * 1996-10-07 1998-04-16 University Of Massachusetts Proteines de liaison pour phosphoïnositides, grp1 ou recepteur general 1 pour phosphoïnositide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039124A1 (fr) * 1996-04-15 1997-10-23 Icos Corporation Modulateurs cytoplasmiques de la regulation/signalisation de l'integrine
WO1998015629A1 (fr) * 1996-10-07 1998-04-16 University Of Massachusetts Proteines de liaison pour phosphoïnositides, grp1 ou recepteur general 1 pour phosphoïnositide

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL - EMHUM1 10 June 1998 (1998-06-10), HEUFLER C: "Dentritic cell derived cytohesin binding protein", XP002152465 *
DATABASE EMBL - TREMBL 1 August 1998 (1998-08-01), HEUFLER C: "Cytohesin binding protein HE", XP002152464 *
DATABASE EMBL - TREMBL 1 November 1996 (1996-11-01), DIXON B ET AL.: "Transcription factor", XP002152466 *
DIXON, B. ET AL.: "Cloning a cDNA from human NK/T cells which codes for an unusual leucine zipper containing protein", BIOCHIMICA BIOPHYSICA ACTA, vol. 1216, no. 2, 1993, pages 321 - 324, XP000961540 *
MIKAKO TSUCHIYA ET AL: "Molecular identification of ADP-ribosylation factor mRNAs and their expression in mammalian cells", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 266, no. 5, 15 February 1991 (1991-02-15), pages 2772 - 2777, XP002117028, ISSN: 0021-9258 *

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AU5603000A (en) 2000-12-28

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