WO2000050445A1 - Nouveau compose de complexe suppresseur de tumeur de von hippel-lindau et scf ubiquitine ligase - Google Patents

Nouveau compose de complexe suppresseur de tumeur de von hippel-lindau et scf ubiquitine ligase Download PDF

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WO2000050445A1
WO2000050445A1 PCT/US2000/004838 US0004838W WO0050445A1 WO 2000050445 A1 WO2000050445 A1 WO 2000050445A1 US 0004838 W US0004838 W US 0004838W WO 0050445 A1 WO0050445 A1 WO 0050445A1
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protein
complex
rbxl
ring box
cullin
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PCT/US2000/004838
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English (en)
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Joan A. Conaway
Ronald C. Conaway
Takumi Kamura
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Oklahoma Medical Research Foundation
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Priority to US09/914,324 priority Critical patent/US6858709B1/en
Priority to CA002362520A priority patent/CA2362520A1/fr
Priority to JP2000601023A priority patent/JP2002541775A/ja
Priority to AU33787/00A priority patent/AU769012B2/en
Priority to EP00911981A priority patent/EP1155030A4/fr
Publication of WO2000050445A1 publication Critical patent/WO2000050445A1/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
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a bio-affecting and body treating composition and to a biological diagnostic agent.
  • VHL von Hippel-Lindau
  • chromosome 3p25.5 The von Hippel-Lindau (VHL) tumor suppressor gene on chromosome 3p25.5 is mutated in the majority of sporadic clear cell renal carcinomas and in VHL disease, an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors including clear cell renal carcinomas, cerebellar hemangioblastomas and hemangiomas, retinal angiomata, and pheochromocytomas.
  • VHL protein is expressed in most tissues and cell types and appears to perform multiple functions, including general repression of hypoxia-inducible genes (Iliopoulos, et al. 1996. "Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein,” Proc Natl Acad Sci USA 93:10595-10599; Gnarra, et al. 1996.
  • the VHL protein is found in a multiprotein complex that includes the ubiquitin-like Elongin B protein, and Elongin C and the cullin CUL2, which share sequence similarity with the Skpl and Cdc53 components of the Skpl-Cdc53p-F-box protein (SCF) ubiquitin ligase complex, respectively.
  • SCF Skpl-Cdc53p-F-box protein
  • the von Hippel-Lindau tumor-suppressor gene product forms a stable complex with human CUL-2, a member of the Cdc53 family of proteins," Proc Natl Acad Sci USA 94:2156-2161; and Lonergan, et al. 1998.
  • "Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing Elongins B/C and Cul2," Mol Cell Biol 18:732-741).
  • Elongins B and C form a stable subcomplex that interacts with a short BC-box motif in the VHL protein and bridges its interaction with CUL2.
  • VHL mutations found in sporadic clear cell renal carcinomas and in VHL kindreds result in mutation or deletion of the BC-box, disruption of the VHL complex, and deregulation of hypoxia-inducible gene expression, p27 protein stability, and bironectin matrix assembly.
  • Fig. 1A - 1C describe the co-purification of the VHL complex with Rbxl from rat liver cytosol.
  • Fig. 1C depicts a 5-20% SDS-polyacrylamide gel of sample used for peptide sequencing.
  • FIG. 3 A - 3D depict the reconstitution of Rbxl- containing complexes.
  • Fig. 3a demonstrates Rbxl forming complexes with VHL and CUL2 in the presence of Elongin BC. Lysates from Sf21 cells expressing the indicated viruses were immunoprecipitated with anti-FLAG or anti-MYC antibodies, and immunoprecipitated proteins were detected by immunoblotting.
  • Fig. 3B demonstrates independent association of Rbxl with Elongin BC, and VHL. Lysates from Sf21 cells expressing the indicated viruses were immunoprecipitated with anti- HPC4, anti-FLAG, or anti-MYC antibodies, and immunoprecipitated proteins were detected by immunoblotting.
  • Fig. 3C demonstrates in vitro binding of recombinant Rbxl, VHL, and Elongin BC.
  • Proteins expressed in and purified from E. coli were mixed together in the combinations indicated, renatured by dilution and dialysis, and immunoprecipitated with anti-HPC4. Immunoprecipitated proteins were detected by immunoblotting with the indicated antibodies.
  • Fig. 3D demonstrates independent association of Rbxl with Cul l, Cul2, and Cdc53. Lysates from S£21 cells expressing the indicated viruses were immunoprecipitated with anti- MYC antibodies, and immunoprecipitated proteins were detected by immunoblotting.
  • Fig. 4A - 4C depict various activities associated with the presence or absence of Rbxl.
  • Fig. 4A demonstrates that binding of Rbxl protein to endogenous yeast Cdc53 correlates with function.
  • the upper panel shows phenotypes of rbxl A cells expressing wild type or mutant mammalian Rbxl (mRbxl) protein.
  • mRbxl mammalian Rbxl
  • lysates from cells expressing wild type and mutant mammalian MYC-Rbxl proteins in either the rbxl deletion strain (deleted) or in the parental strain MCY453 (wild type) were subjected to immunoprecipitation with anti- MYC antibodies. Immunoprecipitated proteins were detected by immunoblotting with anti- MYC or anti-Cdc53 antibodies.
  • Fig. 4B demonstrates that Sicl protein accumulates in Rbxl -depleted cells.
  • R&c/ ⁇ /pGAL-mrbxl (M4) cells were grown to an OD600 of 1 in galactose-containing medium and then shifted into glucose medium. Cells were harvested after 8 hours of growth in glucose, and cell lysates were analyzed by immunoblotting with anti-MYC and two different anti-Sicl antibodies. Fig. 4C demonstrates morphological changes associated with Rbxl -depletion.
  • Rbx/ ⁇ /pGAL-mrbxl (M4) cells were grown in galactose (gal) or for 8 hours after glucose shift
  • FIG. 5 shows a diagram of a SCF ubiquitin ligase complex and a VHL ubiquitin ligase complex and illustrates the ubiquitin ligases of the present invention.
  • Ring box protein and "Rbxl” as used herein refers to proteins that are components of the von Hippel-Lindau tumor suppressor complex.
  • the terms refer to proteins obtained from any eukaryotic species, particularly mammalian species such as bovine, porcine, murine, equine, and human, and from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • the terms encompass polypeptides or proteins having less than the complete amino acid sequence and biologically active variants and gene products.
  • naturally occurring as used herein means an endogenous or exogenous protein isolated and purified from animal tissue or cells.
  • isolated and purified means a protein that is essentially free of association with other proteins or polypeptides, e.g., as a naturally occurring protein that has been separated from cellular and other contaminants by the use of antibodies or other methods or as a purification product of a recombinant host cell culture.
  • biologically active means a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • nucleotide sequence means a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct that has been derived from DNA or RNA isolated at least once in substantially pure form (i.e., free of contaminating endogenous materials) and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods.
  • sequences are preferably provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns that are typically present in eukaryotic genes. Sequences of non-translated DNA may be present 5' or 3' from an open reading frame where the same do not interfere with manipulation or expression of the coding region.
  • nucleic acid molecule as used herein means RNA or DNA, including cDNA, single or double stranded, and linear or covalently closed molecules.
  • a nucleic acid molecule may also be genomic DNA corresponding to the entire gene or a substantial portion thereof or to fragments and derivatives thereof.
  • the nucleotide sequence may correspond to the naturally occurring nucleotide sequence or may contain single or multiple nucleotide substitutions, deletions and/or additions including fragments thereof. All such variations in the nucleic acid molecule retain the ability to encode a biologically active protein when expressed in the appropriate host or a biologically active fragment thereof.
  • the nucleic acid molecule of the present invention may comprise solely the nucleotide sequence encoding a protein or may be part of a larger nucleic acid molecule that comprises the gene for ]extends to the gene for the protein.
  • the non-protein encoding sequences in a larger nucleic acid molecule may include vector, promoter, terminator, enhancer, replication, signal sequences, or non-coding regions of the gene.
  • variant as used herein means a polypeptide substantially homologous to a naturally occurring protein but which has an amino acid sequence different from the naturally occurring protein (human, bovine, ovine, porcine, murine, equine, or other eukaryotic species) because of one or more deletions, insertions, derivations, or substitutions.
  • the variant amino acid sequence preferably is at least 40% identical to a naturally occurring amino acid sequence but is most preferably at least 70% identical.
  • Variants may comprise conservatively substituted sequences wherein a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
  • Conservative substitutions are well known in the art and include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Conventional procedures and methods can be used for making and using such variants. Other such conservative substitutions such as substitutions of entire regions having similar hydrophobicity characteristics are well known. Naturally occurring variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the protein that leave the protein biologically active and capable of performing its biological function.
  • Alternate splicing of mRNA may yield a truncated but biologically active protein. Variations attributable to proteolysis include differences in the N- or C-termini upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids from the protein.
  • nucleic acid or amino acid sequences having sequence variations that do not materially affect the nature of the protein, i.e., the structure and/or biological activity of the protein.
  • nucleic acid sequences the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression and refers primarily to degenerate codons encoding the same amino acid or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
  • amino acid sequences refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
  • percent identity means comparisons among amino acid sequences as defined in the UWGCG sequence analysis program available from the University of Wisconsin. (Devereaux et al, Nucl. Acids Res. 12: 387-397 (1984)).
  • nucleotide sequences of any known and naturally occurring gene will be highly homologous and some will be minimally homologous to the nucleotide sequences of any known and naturally occurring gene.
  • the present invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring Rbxl and all such variations are to be considered as being specifically disclosed.
  • the present invention provides isolated and purified biologically active cullin-interacting
  • Ring box proteins that are a component of the von Hippel-Lindau tumor suppressor complex and of SCF ubiquitin ligase complexes, nucleic acid molecules that encode Ring box proteins, expression vectors having DNA that encode Ring box proteins, host cells that have been transfected or transformed with expression vectors having DNA that encode Ring box proteins, host cells that have been transfected or transformed with expression vectors having DNA that encode Ring box proteins,
  • Ring box proteins methods for producing recombinant Ring box proteins by culruring host cells that have been transfected or transformed with expression vectors having DNA that encode Ring box proteins, isolated and purified recombinant Ring box proteins, complexes and methods that use Ring box proteins to screen for therapeutic agents, methods for diagnosing the predisposition of a patient to certain carcinomas, methods for treating any of several enumerated carcinomas or augmenting metabolically deficient systems in humans and other animals, and methods for evaluating the effectiveness of a therapeutic treatment for Ring box associated carcinomas.
  • the present invention provides a new Ring box protein designated Rbxl.
  • VHL complex from rat liver was first purified by the procedures given below and outlined in Fig. 1 A.
  • the VHL complex was purified from a post-nuclear supernatant prepared from the livers of 360 male Sprague-Dawley rats and fractionated by (NH4) 2 SO 4 precipitation as described previously. (Conaway, et al. 1996. "Purification of RNA polymerase
  • DTT 10% (v/v) glycerol] containing 0.5 MM PMSF, 10 ⁇ g/ml antipain, and 10 ⁇ g/ml leupeptin and was brought to a conductivity equivalent to that of Buffer A containing 100 mM KC1 by a 3 hour dialysis against Buffer A containing 0.5 mM PMSF and dilution with the same buffer.
  • the dialysate was centrifuged for 30 min at 28,000 x g, and the supernatant was gently mixed for -45 minutes with 800 ml of phosphocellulose (Pl l, Whatman) pre-equilibrated in Buffer A containing 100 Mm M KC1 and 0.5 M PMSF.
  • the slurry was then filtered at 500 ml/hr in a
  • Toyopearl DEAE-650M TosoHaas pre-equilibrated in Buffer C [40 mM Tris-HCl (pH 7.9), 0.5 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol] containing 80 mM KCl.
  • Buffer C 40 mM Tris-HCl (pH 7.9), 0.5 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol
  • the slurry was filtered at 150 ml/hr in a 5.0-cm diameter column and then washed at the same flow rate with Buffer C containing 80 mM KCl.
  • the column was eluted stepwise at 250 ml/hr with Buffer A containing 220 mM KCl, and 50 ml fractions were collected.
  • VHL protein which eluted with an apparent native molecular mass between 330 kDa and 200 kDa, were diluted with an equal volume of Buffer E [40 mM Hepes-NaOH (pH 7.9), 0.1 mM
  • Buffer C containing 60 mM KCl.
  • the column was eluted at 0.8 ml/min with a 40 ml linear gradient from 60 mM to 250 mM KCl in Buffer C, and 0.7 ml fractions were collected.
  • MonoQ column (5- x 50-mm, Pharmacia) pre-equilibrated in Buffer C containing 80 mM KCl.
  • the column was eluted at 0.4 ml/min with a 12 ml linear gradient from 80 mM to 300 mM KCl in Buffer C, and 0.2 ml fractions were collected. Fractions containing the VHL protein eluted between 180 mM and 200 mM KCl.
  • VHL tumor suppressor complex was fractionated by 13% SDS-polyacrylamide gel electrophoresis. Proteins were visualized by staining the gel with Coomassie blue, excised, and subjected to in-gel reduction, S-carboxyamidomethylation, and tryptic digestion. Using 10% of the digestion mixture, peptide sequences were determined in a single run by microcapillary reversed-phase chromatography coupled to the electrospray ionization source of a quadrupole ion trap mass spectrometer (Finnigan LCQ).
  • the ion trap's online data-dependent scans allowed the automatic acquisition of high resolution spectra to determine charge state and exact mass, and tandem mass spectrometry spectra for sequence information.
  • the relative collision energy was 35%, and isolation width was 2.5 Dalton.
  • Searches of the EST database performed using TLASTIN algorithm identified human and mouse ESTs that encoded the peptide sequences NHIMDLCIECQAN, QVCPLDNREWEFQK, WNAVAL and WLK which were determined by ion trap mass spectrometry of the 16 kDa polypeptide that copurified with the VHL complex. The identification was facilitated with the algorithm SEQUEST (Eng, et al. 1994.
  • the amino acid sequence for human and mouse Rbxl is shown in Fig. 2 and in SEQ ID NO:l.
  • the nucleotide sequence for the human Rbxl DNA is shown in nucleotides 7-333 of SEQ ID NO:3 and the nucleotide sequence for the murine Rbxl DNA is shown in nucleotides 18-344 of SEQ ID NO:5, inclusive of the stop codon.
  • Nucleotides 1-6 and 1-17 are 5' untranslated regions respectively and 334-508 and 345-504 are 3' untranslated regions respectively.
  • Rbxl is highly homologous to D. melanogaster ORF 115C2.11, C. elegans ORF ZK287.5, and S. cerevisiae ORF YOL133w.
  • the amino acid sequence for Saccharomyces cerevisiae Rbxl is shown in SEQ ID NO:2 and the nucleotide sequence for the Saccharomyces cerevisiae Rbxl DNA is shown in nucleotides 4-369 of SEQ ID NO:4, inclusive of the stop codon. Comparison of the deduced amino acid sequences demonstrates that the proteins are highly homologous with about an 80 percent identity and that the proteins are substantially the same.
  • Rbxl exhibits significant sequence similarity with Saccharomyces cerevisiae Anaphase-Promoting Complex subunit APC11 (Zachariae, et al. 1998. "Mass spectromerric analysis of the anaphase-promoting complex from yeast: Identification of a subunit related to cullins," Science 279:1216-1219).
  • a DNA sequence may vary from that shown in SEQ ID NO:3 and still encode a Rbxl protein having the amino acid sequence shown in SEQ ID NO:l.
  • Such variant DNA sequences may result from silent mutations, e.g., occurring during PCR amplification, or may be the product of deliberate mutagenesis of a native sequence.
  • the invention therefore, provides equivalent isolated DNA sequences encoding biologically active Rbxl selected from: (a) the coding region of a native Rbxl gene; (b) cDNA comprising the nucleotide sequence presented in SEQ ID NO:3; (c) DNA capable of hybridization to the native Rbxl gene under moderately stringent conditions and which encodes biologically active
  • Isolated and purified recombinant Rbxl is provided according to the present invention by incorporating the corresponding DNA into expression vectors and expressing the DNA in a suitable host cell to produce the protein.
  • Expression Vectors Recombinant expression vectors containing a nucleic acid sequence encoding the protein can be prepared using well known techniques.
  • the expression vectors include a DNA sequence operably linked to suitable transcriptional or translational regulatory nucleotide sequences such as those derived from mammalian, microbial, viral, or insect genes. Examples of regulatory sequences include transcriptional promoters, operators, enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence functionally relates to the DNA sequence for the appropriate protein. Thus, a promoter nucleotide sequence is operably linked to a Rbxl DNA sequence if the promoter nucleotide sequence controls the transcription of the appropriate DNA sequence.
  • the ability to replicate in the desired host cells may additionally be incorporated into the expression vector.
  • sequences encoding appropriate signal peptides that are not naturally associated with Rbxl can be incorporated into expression vectors.
  • a DNA sequence for a signal peptide secretory leader
  • a signal peptide that is functional in the intended host cells enhances extracellular secretion of the appropriate polypeptide.
  • the signal peptide may be cleaved from the polypeptide upon secretion of protein from the cell.
  • Suitable host cells for expression of Rbxl include prokaryotes, yeast, archae, and other eukaryotic cells.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known in the art, e.g., Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985).
  • the vector may be a plasmid vector, a single or double-stranded phage vector, or a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
  • the vectors in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsulated virus by well known techniques for infection and fransduction.
  • Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
  • Cell-free translation systems could also be employed to produce the protein using RNAs derived from the present DNA constructs.
  • Prokaryotes useful as host cells in the present invention include gram negative or gram positive organisms such as E. coli or Bacilli.
  • a polypeptide may include a N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell.
  • the N-terminal Met may be cleaved from the expressed recombinant Rbxl polypeptide.
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include ⁇ -lactamase and the lactose promoter system. Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
  • a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
  • useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
  • an appropriate promoter and a DNA sequence are inserted into the pBR322 vector.
  • vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), pGEMl (Promega Biotec, Madison, Wisconsin., USA), and the pET (Novagen, Madison, Wisconsin, USA) and pRSET (Invitrogen Corporation, Carlsbad, California, USA) series of vectors (Studier, F.W., J Mol. Biol. 219: 37 (1991); Schoepfer, R. Gene 124: 83 (1993)) .
  • Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include T7, (Rosenberg, A.H., Lade, B. N., Chui, D-S., Lin, S-W., Dunn, j. j., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135) , ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al, Nature 275:615, (1978); and Goeddel et al, Nature 281:544, (1979)), tryptophan (tip) promoter system (Goeddel et al, Nucl Acids Res. 8:4057, (1980)), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412 (1982)).
  • Yeasts useful as host cells in the present invention include those from the genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces.
  • Yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
  • ARS autonomously replicating sequence
  • Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3 -phosphoglycerate kinase (Hitzeman et al, J. Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes (Holland et al, Biochem.
  • yeast transformation protocols such as enolase, glyceraldehyde-3-phos ⁇ hate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • suitable vectors and promoters for use in yeast expression are further described in Fleer et al, Gene, 107:285-195 (1991).
  • Other suitable promoters and vectors for yeast and yeast transformation protocols are well known in the art.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by Hinnen et al, Proc Natl Acad Sci USA, 75:1929 (1978).
  • the Hinnen protocol selects for Trp.su ⁇ .+ transformants in a selective medium, wherein the selective medium consists of 0.61% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine, and 20 ⁇ g/ml uracil.
  • Rbxl Mammalian or insect host cell culture systems well known in the art could also be employed to express recombinant Rbxl, e.g., Baculovirus systems for production of heterologous proteins in insect cells (Luckow and Summers, Bio/Technology 6:47 (1988)) or
  • CHO cells for mammalian expression may be used.
  • Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes.
  • Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell, e.g., SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication.
  • Exemplary expression vectors for use in mammalian host cells are well known in the art.
  • Rbxl and other Ring box proteins may, when beneficial, be expressed as a fusion protein that has the Ring box protein attached to a fusion segment.
  • the fusion segment often aids in protein purification, e.g., by permitting the fusion protein to be isolated and purified by affinity chromatography.
  • Fusion proteins can be produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of the protein.
  • Preferred fusion segments include, but are not limited to, glutathione-S-transferase, ⁇ -galactosidase, a poly-histidine segment capable of binding to a divalent metal ion, and maltose binding protein.
  • isolated and purified Rbxl may be produced by the recombinant expression systems described above.
  • the method comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes the protein under conditions sufficient to promote expression of the protein.
  • the protein is then recovered from culture medium or cell extracts, depending upon the expression system employed.
  • procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium.
  • the culture medium first may be concentrated. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium.
  • an anion exchange resin can be employed, e.g., a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media (e.g., silica gel having pendant methyl or other aliphatic groups), ion exchange-HPLC (e.g., silica gel having pendant DEAE or sulfopropyl (SP) groups), or hydrophobic interaction-HPLC (e.g., silica gel having pendant phenyl, butyl, or other hydrophobic groups) can be employed to further purify the protein.
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • ion exchange-HPLC e.g., silica gel having pendant DEAE or sulfopropyl (SP) groups
  • hydrophobic interaction-HPLC e.g., silica gel having pendant phenyl, butyl, or other hydrophobic groups
  • Recombinant protein produced in bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting- out, ion exchange, affinity purification, or size exclusion chromatography steps. Finally, RP- HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • the present invention provides a protein complex useful for screening for potential therapeutic agents that would interfere with or augment Rbxl -dependent stimulation of addition of ubiquitin or a ubiquitin-like protein to any substrate targeted for modification by SCF complexes.
  • the complex contains a cofactor protein and one or more proteins selected from the group consisting of a cullin protein, a substrate recognition protein, and a linker protein. Additional components, e.g., ATP, may be added to the solution or composition containing the complex to facilitate complex formation and utilization.
  • the complex is a ubiquitin ligase protein complex that contains cullin proteins; substrate recognition proteins; linker proteins; and cofactor proteins.
  • the complex preferably has one protein from each group but can have more that one protein from each group if required or preferred.
  • the complex is formed from the proteins but the solution or compositions containing the complex may contain additional components if needed to facilitate complex formation and utilization.
  • solutions or compositions containing the complex can comprise various combinations of (1) Skpl, Elongin C, or other linker protein; (2) ⁇ -TRCP, Cdc4, Grrl, or other F-box substrate recognition protein or VHL or other Elongin C-Binding substrate recognition protein; (3) CULl, Cdc53, CUL2, or other cullin protein; (4) Cdc34, UBC5C, or another E2 ubiquitin conjugating enzyme; (5) phosphorylated or other appropriately modified substrate; (6) El ubiquitin activating enzyme; (7) ATP; and (8) ubiquitin, GST-ubiquitin, GST-Ubiquitin 1 ⁇ , ubiquitin derivatives, or a ubiquitin-like protein such as SUMO, NEDD8, or Rub 1.
  • the complex is a ubiquitin ligase protein complex that contains a cullin protein, a substrate recognition protein, a linker protein, and a cofactor protein.
  • the complex is an isolated and purified ubiquitin ligase protein complex comprising such proteins.
  • the cullin proteins of the present invention are proteins selected from the group consisting of Cdc53, Cullin 1 (CULl), Cullin 2 (CUL2), Cullin 3 (CUL3), Cullin 4A (CUL4A), Cullin 4B (CUL4B), and Cullin 5 (CUL5), preferably CULl or CUL2.
  • the substrate recognition proteins are selected from the group consisting of F-box proteins such as ⁇ -TRCP (HOS), Cdc4, Grrl, and other members of this protein family and VHL and other Elongin C binding proteins, preferably F-box proteins or VHL.
  • the linker proteins are selected from the group consisting of Skpl or Elongin C. Many such cullin, substrate recognition, and linker proteins are known in the art and can be used in the present invention.
  • the cofactor proteins are the Ring box proteins of the present invention, preferably Rbxl.
  • FIG. 5 shows a diagram of SCF and VHL ubiquitin ligase complexes illustrating the ubiquitin ligases of the present invention.
  • the cullin protein is Cdc53/Cull in the SCF complex and CUL2 in the VHL complex
  • the substrate recognition protein is a F-box protein in the SCF complex and the VHL protein in the VHL complex
  • the linker protein is Skpl in the SCF complex and Elongin C in the VHL complex.
  • the VHL complex also contains Elongin B.
  • the cofactor protein Rbxl binds to the other proteins and activates the ubiquitin ligase.
  • the SCF ubiquitin ligase targets such substrate proteins as Sicl, cyclins, ⁇ -catenin, NF- ⁇ B inhibitors such as I ⁇ B ⁇ , and the like
  • VHL ubiquitin ligase complex targets such substrate proteins as hypoxia-inducible factor 1 (HIF1) and hypoxia-inducible factor 2 (HIF2)
  • HIF1 hypoxia-inducible factor 1
  • HIF2 hypoxia-inducible factor 2
  • the present invention also provides methods for screening for potential therapeutic agents that would interfere with or augment Rbxl -dependent stimulation of addition of ubiquitin or a ubiquitin-like protein to any substrate including those targeted for modification by SCF of VHL-containing complexes.
  • the methods require forming a complex in vitro that contains a cofactor protein and one or more proteins selected from the group consisting of a cullin protein, a substrate recognition protein, and a linker protein; adding a test compound to interact with the complex; and determining if the complex remains intact or is disrupted by the compound. If the complex is disrupted, the compound is likely to be a therapeutic agent useful for the treatment of the corresponding disease, e.g., cancer or inflammatory disease.
  • a complex can be formed in vitro between a Cdc53 and Rbxl.
  • a test compound can be added to interact with the complex.
  • SCF ubiquitin ligases control the stability of proteins including but not limited to cyclins and cyclin dependent kinase inhibitors, which regulate the cell cycle, and I ⁇ B, which regulates inflammatory processes. If the complex is disrupted, the compound is a likely candidate for an anti-cancer agent or an anti- inflammatory drug.
  • the complex used in the methods is the ubiquitin ligase protein complex described herein, most preferably an isolated and purified ubiquitin ligase protein complex.
  • Rbxl can be used to screen for agents which augment or inhibit the activity of other Cullin-containing ubiquitin ligases and of the VHL complex controlling the conjugation of ubiquitin or ubiquitin-like proteins to various sets of target proteins.
  • the hypoxia-inducible transcription factor HIFl ⁇ is a likely target for ubiquitination by the VHL complex (Maxwell et al "The tumor suppressor protein VHL targets hypoxi-inducible factors for oxygen-dependent proteolysis" Nature 399:271, 1999).
  • Hypoxia-inducible transcription factors regulate the expression of hypoxia inducible genes including vascular endothelial growth factor, which controls normal vascularization as well as vascularization of tumors.
  • compounds that disrupt or interfere with the function of the VHL complex are likely candidates for anti- cancer drugs or for drugs that promote vascularization.
  • the present invention provides methods for diagnosing the predisposition of a patient to certain carcinomas.
  • the invention is based upon the discovery that the absence of VHL-associated proteins, i.e., Ring box proteins such as Rbxl, from certain patient tissues or body fluids indicates that the patient is predisposed to certain carcinomas.
  • the method comprises collecting a tissue or body fluid sample from a patient, analyzing the tissue or body fluid for the quantity of Ring box protein in the tissue, and predicting the predisposition of the patient to certain carcinomas based upon the amount of Ring box protein in the tissue or body fluid. Specifically, determination of Rbxl protein levels in certain tissues permits specific and early, preferably before metastases occur, detection of carcinomas in the patient.
  • Carcinomas that can be diagnosed using the present method include, but are not limited to, clear cell renal carcinoma.
  • the present invention provides methods for treating any of several enumerated Ring box protein associated carcinomas or augmenting metabolically deficient systems (e.g., fibronectin deposition) in humans and other animals.
  • One method comprises administering a therapeutically effective amount of a compound that enhances or augments in vivo the expression of the target Rbxl gene and enhances the in vivo the expression of the Ring box protein to a patient diagnosed as having a Ring box protein associated carcinoma or cellular deficienciey and having been diagnosed as deficient in Ring box protein, preferably Rbxl.
  • the compound is a nucleobase containing a sequence of the nucleic acid sequence encoding the Ring box protein.
  • the nucleic acid sequence is activated to produce the Ring box protein and increase the amount of the Ring box protein in the cell.
  • Increasing the amount of Ring box protein increases the activity of VHL tumor suppressor activity, ubiquitination, fibronectin deposition, and similar activities.
  • Another method comprises administering a therapeutically effective amount of a Ring box protein to a patient diagnosed as having a Ring box protein associated carcinoma or cellular deficiency and having been diagnosed as deficient in Ring box protein, preferably Rbxl.
  • Carcinomas that can treated using the present method include, but are not limited to, clear cell renal carcinoma.
  • the present invention provides methods for evaluating the effectiveness of a therapeutic treatment for Ring box associated carcinomas.
  • the method comprises collecting a tissue or body fluid sample from a patient suffering from a Ring box associated carcinoma and having been subjected to a therapeutic treatment for such carcinoma, determining the amount of Ring box protein in the tissue or body fluid sample, and comparing the determined amount or Ring box protein to a standard indicative of normal Ring box protein levels.
  • the standard can be averages of Ring box protein levels for normal patients but preferably is the level of Ring box protein for the patient being treated before the treatment began. Elevated levels of Ring box protein compared to the standard indicates that the treatment is effective.
  • Rbxl can be used as a research tool to better understand various complex mechanisms of cell ubiquitination.
  • SCF Cdc4 a standard in vitro Sicl ubiquitination assay that is dependent upon Sicl phosphorylation and the E2 Cdc4 was employed.
  • SCF Cdc4 components were co-expressed in insect cells in the presence or absence of mammalian or yeast Rbxl, and complexes were immunopurified through either MYC-tagged Cdc53(MYC-Cdc53) or FLAG-tagged Skpl (FLAG-Skpl) subunits.
  • Immunopurified SCF Cdc4 complexes were supplemented with phosphorylated Sicl, Cdc4, El ubiquitin activating enzyme, ATP, and GST-Ub 1 ⁇ prior to analysis of Sicl conjugates by immunoblotting.
  • GST-Ub ⁇ forms polyubiquitinated products only poorly, so Sicl conjugates are integrated into a ladder of bands differing by -35 kd, the size of GST-Ub 1 ⁇ .
  • Low but detectable amounts of Sicl-GST- Ub 1 ⁇ conjugates were produced by the SCF Cdc4 complex after a 60 minute reaction.
  • Sicl-GST-Ub 1 ⁇ conjugates In the presence of either yeast or mammalian Rbxl, the accumulation of Sicl-GST-Ub 1 ⁇ conjugates was dramatically increased after 20 minutes, and substantial amounts of higher molecular mass conjugates accumulated after 60 minutes. A large fraction of phosphorylated Sicl was converted to Sicl-GST-Ub 1 ⁇ conjugates when reactions included either the anti-Cdc53 or anti- FLAG-Skpl immune complexes; e.g., >95% or Sicl was converted to conjugates in reactions containing the anti-Skpl immune complex.
  • Rbxl can be used to stimulate ubiquitination of phosphorylated I ⁇ B in the presence of El ubiquitin-activating enzyme, the E2 ubiquitin conjugation enzyme UBC5C, ATP, GST-Ub 1 ⁇ , and SCF ⁇ "TRCP (which contains the F- box protein ⁇ -TRCP).
  • complex of Rbxl and SCF ⁇ TRCP can be purified from lysates of Hi5 or Sf21 insect cells infected with baculoviruses encoding SCF subunits and Rbxl. Complexes can be supplemented with phosphorylated I ⁇ B prepared as described by Winston (Winston, et al. 1999.
  • reaction products can be subject to SDS-PAGE and analyzed by Western blotting using antibodies against I ⁇ B ⁇ . Conjugation of GST-Ub 1 ⁇ to I ⁇ B ⁇ can result in the formation of a ladder of I ⁇ B ⁇ conjugates differing in size by -35 kD, the size of GST-Ub 1 ⁇ . Confirmation that Rbxl can be used as a research tool has been published, e.g. Tan et al, Mol
  • Rbxl was used in assays for ubqitination of HIFl ⁇ (Maxwell et al "The tumor suppressor protein VHL targets hypoxi- inducible factors for oxygen-dependent proteolysis" Nature 399:271, 1999) in the presence of
  • El ubiquitin-activating enzyme the E2 ubiquitin conjugation enzyme UBC5a, ATP, GST-
  • VHL-containing complex is purified from Sf21 insect cells infected with baculoviruses encoding subunits of the VHL complex.
  • VHL complexes are supplemented with lysates from insect cells infected with baculoviruses encoding HIF1 or HIF2 and with
  • Rbxl was used to stimulate modification of the cullin proteins Cdc53 and CUL2 with the ubiquitin-like protein Rubl (also known as NEDD8) in the presence of Rubl -activating enzyme Uba3/Ulal, Rubl -conjugating enzyme Ubcl2, ATP, GST-Rubl, and a complex consisting of Rbxl and either Cdc53 or Cul2 (Kamura et al "The Rbxl subunit of SCF and VHL E3 ubiquitin ligase activates Rubl modification of cullins Cdc53 and Cul2" Genes Dev. 13:2928, 1999).
  • the Cdc53-Rbxl complex is purified from Sf21 cells infected with baculoviruses encoding Cdc53 and Rbxl. Complexes are supplemented with Uba3/Ulal, Ubcl2, ATP, and GST-Rubl. After a period of incubation, reaction products are subjected to SDS-PAGE and analyzed by Western blotting using antibodies that recognize Cdc53. Conjugation of GST-Rubl to Cdc53 results in disappearance of unmodified Cdc53 and appearance of a new, more slowly migrating band corresponding to the Cdc53 -GST-Rubl conjugate.
  • the CUL2-Rbxl complex is purified from Sf21 cells infected with baculoviruses encoding CUL2 and Rbxl. Complexes are supplemented with Uba3/Ulal, Ubcl2, ATP, and GST-Rubl. After a period of incubation, reaction products are subjected to SDS-PAGE and analyzed by Western blotting using antibodies that recognize
  • VHL complex Reconstitution of the VHL complex and its subassemblies in insect cells and with bacterially expressed proteins in vitro shows that Rbxl can interact with VHL, CUL2, and the Elongin BC complex.
  • Rbxl and VHL were subcloned into pBacPAK8 with N-terminal His tags and N-terminal myc and C-terminal FLAG tags, respectively.
  • CULl was introduced in to the same vector with a C-terminal HA tag
  • CUL2 was introduced into pBacPAK-Hisl with N-terminal His and HA tags, and recombinant baculoviruses were generated using the BacPAK baculovirus expression system (Clontech).
  • the baculovirus vectors encoding S. cerevisiae CDC53 (Willems, et al. 1996.
  • Cdc53 targets phosphorylated Gl cyclins for degradation by the ubiquitin proteolytic pathway
  • Cell 86:453-463 and Elongins B and C have been described (Kamura, et al. 1998.
  • the Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat FAMILIES," Genes E>ev 12:3872-3881).
  • Sf21 cells were cultured in Sf-900 II SFM with 5% fetal calf serum at 27°C and coinfected with various combinations of baculoviruses encoding myc-Rbxl, FLAG- VHL, HA-CUL2, HPC4-Elongin B, and HSV-Elongin C.
  • Sf21 cells were collected and lysed by gentle vortexing in ice-cold buffer containing 40 mM Hepes-NaOH, pH 7.9, 150 mM NaCl, 1 mM DTT, 0.5% (v/v) glycerol, 5 ⁇ g/ml leupeptin, 5 ⁇ g/ml antipain, 5 ⁇ g/ml pepstatin A, and 5 ⁇ g/ml aprotinin. Lysates were centrifuged at 10,000 x g for 20 minutes at 4°C. The supernatants were used for immunoprecipitations.
  • Anti-T7 and anti-HSV antibodies were from Novagen.
  • Anti-HA (12CA5) and anti-c-myc (9E10) antibodies were from Boehringer-Mannheim.
  • Anti-FLAG (M2) was from Eastman Kodak.
  • Anti-Elongin C monoclonal antibody was from Transduction Laboratories.
  • Anti-VHL monoclonal antibody (Ig32) was from Pharmingen.
  • Anti-Sicl (yN-19 and yC-19) and anti- Cdc53 (yC-17) antibodies were from Santa Cruz Biotechnology, Inc.
  • Anti-Elongin B rabbit polyclonal antibodies were described previously. (Garrett, et al. 1995.
  • VHL complex all five components of the VHL complex could be coimmunoprecipitated together from Sf21 cell lysates with either anti-FLAG (Lane 5) or anti- myc (Land 9) antibodies.
  • Rbxl, VHL, and the Elongin CB complex could be coimmunoprecipitated from Sf21 cell lysates with either anti-FLAG (Lane 6) or anti-myc (Lane 10) antibodies in the absence of CUL2; and Rbxl, CUL2, and Elongin BC complex could be coimmuoprecipitated from Sf21 cell lysates with anti-myc antibody in the absence of VHL (Lane 11).
  • Rbxl could also be coimmunoprecipitated with anti- myc antibody with either VHL, CUL2, or the Elongin BC complex from lysates of Sf21 cells coinfected with either myc-Rbxl and FLAG- VHL, myc-Rbxl and HA-CUL2, or myc-Rbxl, HPC4-Elongin B, and HSV-Elongin C. Consistent with these results, as shown in Fig.
  • Rbxl could be coimmunoprecipitated with VHL-Elongin BC and Elongin BC subassemblies reconstituted in vitro with bacterially expressed FLAG- VHL, HPC4-elongin B, and HSV- Elongin C.
  • CUL2 is a member of the multiprotein Cullin family (Kipreos, et al. 1996. "Cul-1 is required for cell cycle exit in C. elegans and identifies a novel gene family," Cell 85:829-839), which also includes the SCF component CULl and its S. cerevisiae homolog Cdc53. Because Rbxl interacted with CUL2 as shown in Fig.
  • Rbxl binds both human CULl and S. cerevisiae Cdc53.
  • Myc-Rbxl and human HA-CULl could be coimmunoprecipitated with anti- myc antibodies from lysates of Sf21 cells coinfected with baculoviruses encoding myc-Rbxl and HA-CULl.
  • myc-Rbxl and Cdc53 could be coimmunoprecipitated with anti- myc antibodies form lysates of Sf21 cells coinfected with baculoviruses encoding myc-Rbxl and Cdc53.
  • Example 6 Effect of Rbxl on Function of SCF cdc4 Ubiquitin Ligase
  • Cdc53/CUL1 is a component of the recently described SCF ubiquitin ligase complex, which catalyzes ubiquitination of a diverse collection of proteins with critical roles in cell cycle, transcription, and development.
  • Cdc53 is a scaffold protein for multiple Cdc34/Skpl/F-box protein complex that regulate cell division and methionine biosynthesis in yeast," Genes Dev 12:692-705; Bai, et al. 1996.
  • SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box," Cell 86:263-274; Patton, et al. 1998.
  • SCF complexes include Cdc53/CUL1, Skpl, and one of the variety of F-box proteins, which recruit substrates to the SCF for ubiquitination.
  • Cdc53/CUL1, Skpl and one of the variety of F-box proteins, which recruit substrates to the SCF for ubiquitination.
  • the results demonstrate that Rbxl can assemble into complexes containing CULl and the additional SCF component Skpl, since Myc-Rbxl could be coimmunoprecipitated with anti-T7 antibodies from lysates of Sf21 cells coinfected with viruses encoding myc-Rbxl, T7-tagged Skpl (T7-Skpl), and HA-CULl.
  • the WD40 repeat protein Cdc4, which recruits the cdk inhibitor Sicl for ubiquitination by the SCF Cdc4 complex, and the leucine-rich repeat protein Grrl, which recruits Gl cyclin Cln2 for ubiquitination by the SCF Grrl complex, are among several F-box proteins found in yeast. Having shown that Rbxl interacts in cells not only with CUL2 and the VHL complex but also with the SCF components Cdc53/CUL1 (Fig. 3 A) and Skpl, mutant yeast strains lacking the chromosomal RBXl gene were generated in order to test the possibility that Rbxl might affect functions of SCF Cdc4 and SCF Grrl .
  • a S. cerevisiae strain lacking the Rbxl gene was constructed as follows.
  • the Rbxl gene was disrupted in MCY453 (Mata/MAThis3)-200/his3)-200 canlR canlR cyh2Rcyh2R ura3/ura3 Ieu2/leu2 trpl/trpl Iys2/lys2) by replacing the complete Rbxl ORF (YOL133w) with the HIS3 gene (Baudin, et al. 1993. "A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae, " Nucleic Acids Res 21:3329-3330).
  • the Rbxl deletion strain was also rescued by expression of either myc-tagged mammalian Rbxl (mRbxl) or a mutant mRbxl (M4), in which putative ring finger cysteine 53 and cysteine 56 were replaced with serines.
  • the rbxl deletion strain was not rescued, however, by expression of a mutant mRbxl (M3), in which putative ring finger cysteine 42 and cysteine 45 were replaced by serines.
  • myc-tagged mRbxl coimmunoprecipitated with the endogenous yeast Cdc53 protein, suggesting that it associates with Cdc53 in cells (Fig. 4A).
  • yeast Rbxl (yRbxl) is a subunit and activator of the SCF Cdc4 ubiquitin ligase and that mammalian Rbxl (mRbxl) can substitute for its yeast counterpart in reconstitution of the active SCF Cdc4 complex.
  • the SCF C c4 complex is required for ubiquitination and targeted degradation of a variety of cell cycle regulatory proteins including Sicl, whose degradation is essential for the Gl/S transition.
  • the SCF Grrl complex is required for ubiquitination and targeted degradation of the Gl cyclin Cln2.
  • myc-tagged wild type or M4 mutant mRbxl were expressed in the rbxl deletion strain on a high copy number plasmid under control of the GAL 1,10 promoter.
  • Rbxl is a general subunit of SCF ubiquitin- ligases demonstrates (1) that
  • Rbxl interacts with subunits of Cdc53/CUL1 containing SCF ubiquitin-ligase complexes, (2) that the abilities of Rbxl mutants to bind the Cdc53 subunit of SCF ubiquitin ligase correlate with their abilities to prevent cell cycle arrest in a yeast strain lacking chromosomal Rbxl, and (3) that depletion of Rbxl from yeast interferes with the function of SCF c c4 and SCF Grrl .
  • Rbxl is also a component of the CUL2-containing VHL complex
  • Rbxl can now be applied in delineating the function of the VHL complex and perhaps other Cullin complexes as possible ubiquitin ligases and ubiquitin-like ligases for as yet unidentified sets of target proteins. Since the multiprotein VHL complex has been shown to have roles in cell cycle regulation through its control of the levels of the cdk inhibitor p27, in repression of hypoxia- inducible genes, and in assembly of the extracellular fibronectin matrix, Rbxl can be applied in the reconstitution of VHL complexes to determine therapeutics that cause the dissociation of the VHL complex.
  • Rbxl is a component of the SCF Cdc4 complex, functioning as a common SCF subunit and participating in regulation of ubiquitination by SCF complexes containing additional F-box proteins, including MET30 and ⁇ -TRCP, which direct ubiquitination of such target proteins as Swel and the transcriptional regulators I ⁇ B and ⁇ -catenin.
  • Rbxl can be applied in determining therapeutics capable of regulating ubiquitination via other SCF complexes.
  • the present invention relates to a component in VHL tumor suppressor activity found in the majority of sporadic clear cell renal carcinomas as well as autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors including clear cell renal carcinomas, cerebellar hemangioblastomas and hemangiomas, retinal angiomata, and pheochromocytomas; general repression of hypoxia-inducible genes; and regulation of p27 protein stability and fibronectin matrix assembly.
  • Ring box proteins described act as a cellular marker useful (1) in detecting a possible predisposition to certain carcinomas, (2) as molecular targets for treating those carcinomas therapeutically, (3) as a target for inhibition by drugs that manipulate the growth of cells, and (4) as a research tool to better understand the various complex mechanisms of cell ubiquitination, binding of certain activator proteins, fibronectin deposition and other aspects of the cellular division process.

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Abstract

L'invention concerne Rbx1, une protéine à doigt RING-H2 conservée au cours de l'évolution, interagissant avec Cullin. Rbx1 de mammifère à été découverte en tant que composant du complexe VHL contenant CUL2. Un homologue de Rbx1, obtenu à partir de S. cerevisiae a également été identifié en tant que sous-unité et activateur de SCFCdc4 ubiquitine ligase contenant Cdc53, nécessaire à l'ubiquitination de l'inhibiteur Sic1 de cdk ainsi qu'à la transition du cycle cellulaire G1/S des levures, assurant une liaison entre le complexe suppresseur de tumeur VHL multiprotéique et l'ubiquitination cellulaire. La protéine Rbx1 agit en tant que marqueur cellulaire, utile (1) afin de détecter une éventuelle prédisposition à certains carcinomes, (2) en tant que cible moléculaire en vue de traiter ces carcinomes de manière thérapeutique, (3) en tant que cible pour l'inhibition par des médicaments agissant sur la croissance cellulaire, et (4) en tant qu'outil de recherche permettant de mieux comprendre les différents mécanismes complexes de l'ubiquitinaion cellulaire, la liaison de certaines protéines activatrices, le dépôt de fibronectine et d'autres aspects du processus de division cellulaire.
PCT/US2000/004838 1999-02-26 2000-02-25 Nouveau compose de complexe suppresseur de tumeur de von hippel-lindau et scf ubiquitine ligase WO2000050445A1 (fr)

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US7625732B2 (en) 1999-03-31 2009-12-01 The University Of North Carolina At Chapel Hill Isolated DNA encoding cullin regulators ROC1 and ROC2, isolated proteins encoded by the same, and methods utilizing the same
US7078203B1 (en) 1999-03-31 2006-07-18 The University Of North Carolina At Chapel Hill Isolated DNA encoding cullin regulators ROC1 and ROC2, isolated proteins encoded by the same, and methods utilizing the same
EP1988165A1 (fr) * 1999-03-31 2008-11-05 The University of North Carolina at Chapel Hill ADN isolé codant pour le ROC1 régulant la culline, protéines isolées codées par celui-ci et leurs procédés d'utilisation
US6740495B1 (en) 2000-04-03 2004-05-25 Rigel Pharmaceuticals, Inc. Ubiquitin ligase assay
US6737244B2 (en) 2000-04-03 2004-05-18 Rigel Pharmaceuticals, Inc. Ubiquitin ligase assay
US7022493B2 (en) 2000-04-03 2006-04-04 Rigel Pharmaceuticals, Inc. Ubiquitin conjugation assays
US7524642B2 (en) 2000-04-03 2009-04-28 Rigel Pharmaceuticals, Inc. Assays for identifying ubiquitin agents and for identifying agents that modify the activity of ubiquitin agents
US7781182B2 (en) 2000-04-03 2010-08-24 Rigel Pharmaceuticals, Inc. Ubiquitin ligase assay
EP1456647A4 (fr) * 2001-11-19 2006-10-18 Proteologics Inc Procede pour identifier et valider des cibles de medicament potentielles
EP1456647A2 (fr) * 2001-11-19 2004-09-15 Proteologics, Inc. Procede pour identifier et valider des cibles de medicament potentielles
RU2781452C2 (ru) * 2012-01-12 2022-10-12 Йейл Юниверсити Соединения и способы усиления деградации белков-мишеней и других полипептидов с помощью e3 убиквитин лигазы
WO2013106643A3 (fr) * 2012-01-12 2013-09-06 Yale University Composés et procédés pour la dégradation améliorée de protéines cibles et d'autres polypeptides par une e3 ubiquitine ligase
US10730862B2 (en) 2012-01-12 2020-08-04 Yale University Compounds and methods for the enhanced degradation of targeted proteins and other polypeptides by an E3 ubiquitin ligase
US9993514B2 (en) 2013-07-03 2018-06-12 Glaxosmithkline Intellectual Property Development Limited Compounds
US9988376B2 (en) 2013-07-03 2018-06-05 Glaxosmithkline Intellectual Property Development Limited Benzothiophene derivatives as estrogen receptor inhibitors
US10071164B2 (en) 2014-08-11 2018-09-11 Yale University Estrogen-related receptor alpha based protac compounds and associated methods of use
US10730870B2 (en) 2015-03-18 2020-08-04 Arvinas Operations, Inc. Compounds and methods for the enhanced degradation of targeted proteins
US11512083B2 (en) 2015-03-18 2022-11-29 Arvinas Operations, Inc. Compounds and methods for the enhanced degradation of targeted proteins
US9938264B2 (en) 2015-11-02 2018-04-10 Yale University Proteolysis targeting chimera compounds and methods of preparing and using same

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EP1155030A4 (fr) 2005-12-14
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AU3378700A (en) 2000-09-14
EP1155030A1 (fr) 2001-11-21
AU769012B2 (en) 2004-01-15

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