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WO2000023599A2 - Proteines humaines responsables de la degradation du reticulum endoplasmique (er) - Google Patents

Proteines humaines responsables de la degradation du reticulum endoplasmique (er) Download PDF

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WO2000023599A2
WO2000023599A2 PCT/US1999/024563 US9924563W WO0023599A2 WO 2000023599 A2 WO2000023599 A2 WO 2000023599A2 US 9924563 W US9924563 W US 9924563W WO 0023599 A2 WO0023599 A2 WO 0023599A2
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hsubc15
hsubc14
ubiquitin
protein
nucleic acid
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PCT/US1999/024563
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WO2000023599A3 (fr
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Vincent Chau
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Millennium Pharmaceuticals, Inc.
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Priority to EP99955088A priority Critical patent/EP1123385A2/fr
Priority to JP2000577306A priority patent/JP2002527111A/ja
Priority to CA002348156A priority patent/CA2348156A1/fr
Priority to AU11272/00A priority patent/AU1127200A/en
Publication of WO2000023599A2 publication Critical patent/WO2000023599A2/fr
Publication of WO2000023599A3 publication Critical patent/WO2000023599A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to degradation of proteins via the ubiquitin- proteasome pathway. More particularly, the invention relates to the degradation of proteins at the endoplasmic reticulum, including cystic fibrosis transmembrane conductance regulator (CFTR), via the ubiquitin-proteasome pathway.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Covalent modification of proteins through their conjugation with other proteins is an important biological mechanism for regulating protein metabolism and biological activity.
  • Hershko and Ciechanover, Annu. Rev. Biochem. 61: 761- 807 (1992) discloses conjugation of ubiquitin, one of the most conserved eukaryotic proteins, to other proteins through an enzymatic mechanism, as well as its role in protein degradation.
  • Rock et al, Cell 78: 761-771 (1994) discloses that ubiquitination of protein antigens is required for processing of such antigens.
  • Murray, Cell 81: 149-152 (1995) teaches that ubiquitination of cyclin is involved in cell cycle regulation.
  • Ubiquitin is then transferred to a thiol site in ubiquitin conjugating enzyme, E2, through formation of a thioester bond. Ubiquitin is then transferred to .an epsilon amino group of a lysine residue in the target protein through an amide linkage, usually with the involvement of ubiquitin-protein isopeptide ligase, E3. Hopkin, J. Natl. Inst. Health Res. 9: 36-42 (1997), teaches that target specificity is regulated by the particular combination of E2 and E3 protein, with more than 30 E2 proteins and 10 E3 proteins being known at present.
  • the ER protein degradation system has now been proposed to be associated with human disease.
  • Aridor and Balch, Nature Medicine 5: 745-750 (1999) teaches that many sporadic and inherited diseases arise from point mutations which cause disorders in protein conformation and prevent export of the protein from the ER.
  • the mutant protein undergoes rapid degradation and the disease pathology is triggered by the absence of the protein from its target compartment.
  • the mutant protein accumulates in the ER, with resultant toxic effects.
  • cystic fibrosis is caused by the functional absence of a plasma membrane chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • Qu et al J. Biol. Chem. 27V. 22791-22795 (1996) discloses that mutation in the secretory protein c ⁇ -antitrypsin ( ⁇ j -AT) is associated with adult-onset emphysema and infantile liver disease.
  • the reference further teaches that lung injury is due to a decrease in elastase inhibitory capacity ordinarily provided by « ⁇ AT, whereas liver injury is due to the hepatotoxic effect of the abnormally folded mutant protein, which is retained in the ER.
  • the authors demonstrate that degradation of the mutant a AT protein, like that of ⁇ F508, is mediated by the proteasome.
  • Qu et al. also teaches that c ⁇ -AT associates with the transmembrane molecular chaperone calnexin, and that it is the ubiquitination of calnexin that targets the complex for proteasome-mediated degradation.
  • the invention provides methods and compositions for treating human disease associated with proteasome-mediated ER protein degradation.
  • the invention resides in the discovery of human proteins involved in proteasome- mediated ER protein degradation and responsible for the ubiquitination of ER proteins such as ⁇ F508, CFTR, and o ⁇ -AT.
  • such treatment entails promoting the maturation of ⁇ F508 into a functional CFTR and provides understanding of the role of loss of CFTR function in cystic fibrosis (CF).
  • CF cystic fibrosis
  • such treatment entails promoting the maturation of mutant c ⁇ -AT and/or preventing its accumulation in the ER.
  • the methods and compositions according to the invention are more specific for proteins that are degraded in the ER by the ubiquitin-proteasome pathway, such as ⁇ F508, CFTR, and t -AT, than are methods that would prevent ubiquitination of proteins generally.
  • the invention provides new purified ubiquitin- conjugating enzymes and allelic variants thereof.
  • the new purified ubiquitin-conjugating enzyme is membrane-bound.
  • the primary amino acid sequence of one preferred embodiment of such a membrane-bound ubiquitin-conjugating enzyme (HSUBC14) is shown in Figure 1.
  • the primary amino acid sequence of a second preferred embodiment of such a membrane- bound ubiquitin-conjugating enzyme is shown in Figure 2.
  • the new purified ubiquitin-conjugating enzyme is soluble.
  • the primary amino acid sequence of a preferred embodiment of such a soluble ubiquitin-conjugating enzyme is shown in Figure 3.
  • the invention provides ubiquitin conjugating enzyme expression elements.
  • Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding a ubiquitin conjugating enzyme selected from the group consisting of HSUBC14, HSUBC15, HSUBC18, and dominant negative mutants thereof, isolated or recombinant nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such isolated or recombinant nucleic acid sequences, preferably expression vectors.
  • Such ubiquitin conjugating enzyme expression elements also include, without limitation, isolated or recombinant nucleic acids capable of expressing antisense transcripts targeted against a ubiquitin conjugating enzyme selected from the group consisting of HSUBC14, HSUBC15, and HSUBC18, and vectors comprising such isolated or recombinant nucleic acids, preferably expression vectors.
  • HSUBC14BMs HSUBC14-binding molecules
  • HSUBC15-binding molecules HSUBC15BMs
  • HSUBCl ⁇ -binding molecules HSUBC18BMs
  • the invention provides methods for identifying HSUBC14BMs, HSUBC15BMs, and HSUBC18BMs.
  • One preferred method according to these aspects of the invention comprises screening for HSUBC14BMs, HSUBC15BMs, or HSUBC18BMs by contacting purified HSUBC14, HSUBC15, or HSUBC18 according to the invention with populations of molecules or mixed populations of molecules, and determining the presence of molecules which bind specifically to HSUBC14, HSUBC15, or HSUBC18.
  • Another preferred method according to these aspects of the invention comprises rationally designing molecules to bind HSUBC14, HSUBC15, or HSUBC18 based upon structural information from the purified HSUBC14, HSUBC15, or HSUBC18 provided by the invention, and determining whether such rationally designed molecules bind specifically to HSUBC14, HSUBC15, or HSUBC18.
  • These aspects of the invention include HSUBC14BMs, HSUBC15BMs, and HSUBC18BMs identified by the methods according to the invention.
  • HSUBC14BMs, HSUBC15BMs, and HSUBC18BMs can be used in conventional assays to detect the presence or absence, and/or quantity of HSUBC14, HSUBC15, or HSUBC18, or complexes of HSUBC14, HSUBC15, or HSUBC18 with ubiquitin in a biological sample.
  • the invention provides methods for determining the presence or absence and/or quantity of H UBC14, HSUBC15, HSUBC18, or complexes thereof with ubiquitin in a biological sample.
  • Such methods comprise providing a detectable HSUBC14BM, HSUBC15BM, or HSUBC18BM to a biological sample, allowing the detectable HSUBC14BM, HSUBC15BM, or HSUBC18BM to bind to HSUBC14, HSUBC15, HSUBC18, or complex thereof with ubiquitin, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable HSUBC14BM, HSUBC15BM, or HSUBC18BM with the HSUBC14, HSUBC15, HSUBC18, or complex thereof with ubiquitin.
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding HSUBC14, HSUBC15, or HSUBC18 can also be used in conventional assays to detect the presence or absence of HSUBC14, HSUBC15, or HSUBC18 nucleic acid in a biological sample.
  • the invention provides methods for determining the presence or absence, and/or quantity of, HSUBC14, HSUBC15, or HSUBC18 nucleic acid in a biological sample.
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary and/or specifically homologous to HSUBC14, HSUBC15, or HSUBC18 nucleic acid.
  • the invention provides methods for identifying modulating ligands of HSUBC14, HSUBC15, or HSUBC18.
  • Some HSUBC14BMs, HSUBC15BMs, and HSUBC18BMs are capable of acting as antagonists or agonists of HSUBC14, HSUBC15, or HSUBC18.
  • the method according to these aspects of the invention comprises providing HSUBC14BMs, HSUBC15BMs, or HSUBC18BMs to an assay system for HSUBC14, HSUBC15, or HSUBC18 participation in the ubiquitin-conjugation pathway, and determining whether such HSUBC14BMs, HSUBC15BMs, or HSUBC18BMs interfere with or enhance the ability of HSUBC14, HSUBC15, or HSUBC18 to participate in the ubiquitin- conjugation pathway.
  • the HSUBC14BMs, HSUBC15BMs, or HSUBCl ⁇ BMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure.
  • These aspects of the invention include modulating ligands of HSUBC14, HSUBC15, or HSUBC18 identified by this method according to the invention.
  • the invention provides modulating ligands of HSUBC14, HSUBC15, or HSUBC18.
  • Preferred modulating ligands are HSUBC14BMs, HSUBC15BMs, or HSUBC18BMs which act as antagonists, interfering with the ability of HSUBC14, HSUBC15, or HSUBC18 to participate in the ubiquitin- conjugation pathway.
  • Other preferred modulating ligands are HSUBC14BMs, HSUBC15BMs, or HSUBC18BMs which act as agonists, enhancing the ability of HSUBC14, HSUBC15, or HSUBC18 to participate in the ubiquitin-conjugation pathway.
  • HSUBC18BMs preferably interact with HSUBC14, HSUBC15, or HSUBC18 to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBC14, HSUBC15, or HSUBC18, and/or transfer of ubiquitin to a protein targeted for proteasome-mediated ER protein degradation, such as ⁇ F508, CFTR, or ⁇ r AT.
  • a protein targeted for proteasome-mediated ER protein degradation such as ⁇ F508, CFTR, or ⁇ r AT.
  • the invention provides methods for modulating the conjugation of ubiquitin or its transfer to a target protein, such as ⁇ F508, CFTR, or ⁇ ,-AT.
  • a target protein such as ⁇ F508, CFTR, or ⁇ ,-AT.
  • One preferred embodiment of the method according to these aspects of the invention comprises providing a modulating ligand of HSUBC14, HSUBC15, or HSUBC18, or a recombinant expression unit which expresses HSUBC14,
  • HSUBC15 or HSUBC18, or an antagonist thereof, to a biological system in which ubiquitin is conjugated to a target protein, such as ⁇ F508, CFTR, or ⁇ r AT.
  • a target protein such as ⁇ F508, CFTR, or ⁇ r AT.
  • the invention provides oligonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 1, Figure 2, or Figure 3.
  • Preferred embodiments include hybridization probes and antisense oligonucleotides.
  • the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation.
  • the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ⁇ F508 or CFTR precursors to mature into functional CFTR.
  • the invention provides a method for therapeutically treating emphysema caused by failure of mutant ⁇ AT to be secreted.
  • the invention provides a method for therapeutically treating liver disease caused by an accumulation of mutant c ⁇ -AT in the ER.
  • Preferred embodiments of these methods utilize agents that interfere with HSUBC14, HSUBC15, or HSUBC18 protein function or expression of a gene encoding HSUBC14, HSUBC15, or HSUBC18.
  • Figure 1 shows the nucleotide sequence [SEQ ID NO: 1] and deduced amino acid sequence [SEQ ID NO: 2] for HSUBC14, with the active site cysteine at position 94 underlined.
  • Figure 2 shows the nucleotide sequence [SEQ ID NO: 3] and deduced amino acid deduced amino acid sequence [SEQ ID NO: 4] for HSUBC15, with the active site cysteine at position 91 underlined.
  • Figure 3 shows the nucleotide sequence [SEQ ID NO: 5] and deduced amino acid sequence [SEQ ID NO: 6] for HSUBC18.
  • Figure 4 shows the alignment of the amino acid sequence for HSUBC14 with the yeast protein Ubc6 [SEQ ID NO: 7].
  • Figure 5 shows the alignment of the amino acid sequence for HSUBC15 with HSUBC14, the yeast protein Ubc6 [SEQ ID NO: 7], and the C. elegans protein ced 1022.1 [SEQ ID NO: 8].
  • Figure 6 shows the alignment of the amino acid sequence for HSUBC18 with the yeast protein Ubc7 [SEQ ID NO 9].
  • Figure 7 shows the results of an immunostaining experiment comparing the immunostaining patterns of HSUBC14 (Ubchl4) and calreticulin.
  • Figure 8 shows the results of an immunostaining experiment comparing the immunostaining patterns of HSUBC15 (Ubchl5) and calreticulin.
  • Figure 9 shows the results of an immunostaining experiment comparing the immunostaining patterns of HSUBC18 (Ubchl ⁇ ) and calreticulin.
  • the invention relates to degradation of proteins via the ubiquitin- proteasome pathway. More particularly, the invention relates to the proteasome- mediated ER protein degradation of target proteins, including, but not limited to, cystic fibrosis transmembrane conductance regulator (CFTR) and c ⁇ -antitrypsin ( ⁇ AT), via the ubiquitin-proteasome pathway.
  • target proteins including, but not limited to, cystic fibrosis transmembrane conductance regulator (CFTR) and c ⁇ -antitrypsin ( ⁇ AT)
  • the invention provides methods and compositions for treating human disease associated with proteasome-mediated ER protein degradation.
  • the invention resides in the discovery of human proteins involved in proteasome- mediated ER protein degradation and responsible for the ubiquitination of ER proteins such as ⁇ F508, CFTR, and o j-AT.
  • such treatment entails promoting the maturation of ⁇ F508 into a functional CFTR and provides understanding of the role of loss of CFTR function in cystic fibrosis (CF).
  • CF cystic fibrosis
  • such treatment entails promoting the maturation of mutant r AT and/or preventing its accumulation in the ER.
  • the methods and compositions according to the invention are more specific for proteins that are degraded in the ER by the ubiquitin-proteasome pathway, such as ⁇ F508, CFTR, and c ⁇ -AT, than are methods that would prevent ubiquitination of proteins generally.
  • the novel human proteins of the invention are ubiquitin conjugating enzymes. Certain of the new proteins are membrane-bound, while certain others are soluble proteins. Because there are subtle differences in preferred embodiments of some aspects of the invention relating to the membrane-bound proteins as compared to the soluble proteins, the membrane-bound and soluble proteins are separately described below. In particular, the first ten aspects of the invention described below relate to membrane-bound proteins, while the description of the eleventh to twentieth aspects relates to soluble proteins. Necessarily, there is considerable overlap in the descriptions relating to the two types of proteins, and they are combined in the Summary of the Invention above.
  • the invention provides new purified transmembrane domain-containing ubiquitin-conjugating enzymes and allelic variants thereof.
  • the primary amino acid sequence of one preferred embodiment of the new ubiquitin-conjugating enzyme (HSUBC14) is shown in Figure 1.
  • Amino acid residues 231-249 form a transmembrane domain.
  • the transmembrane domain can be deleted to form a soluble HSUBC14, which is a preferred embodiment of this aspect of the invention.
  • "HSUBC14” refers to both the soluble and transmembrane domain-containing embodiments, unless otherwise specified or evident from context.
  • the full length protein has 39% sequence identity to yeast Ubc6. An alignment of HSUBC14 with yeast Ubc6 is shown in Figure 4.
  • HSUBC15 The primary amino acid sequence of a second preferred embodiment of a new ubiquitin-conjugating enzyme (HSUBC15) is shown in Figure 2.
  • the transmembrane domain can be deleted to form a soluble HSUBC15, which is a preferred embodiment of this aspect of the invention.
  • HSUBC15 refers to both the soluble and transmembrane domain-containing embodiments, unless otherwise specified or evident from context.
  • the full length protein has 22% sequence identity to yeast Ubc6 and 40% sequence identity to a C.
  • HSUBC15 elegans homologue, cedl022.1.
  • An alignment of HSUBC15 with yeast Ubc ⁇ and cedl022.1 is shown in Figure 5.
  • the terms "HSUBC14” and “HSUBC15” are intended to include allelic variants thereof.
  • An "allelic variant”, as used herein, is a protein having at least about 50% amino acid sequence identity, more preferably at least about 75%, even more preferably at least about 85%, still more preferably at least about 95%, , even more preferably at least about 96%, yet even more preferably at least about 97%, more preferably yet at least about 98% and most preferably at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or to a portion or protein conjugate thereof which retains the biological activities of HSUBC14 or HSUBC15, respectively, to (1) form a thioester linkage with ubiquitin, and (2) to transfer ubiquitin to a target protein such as ⁇ F508 or to CFTR, both under conditions as described in the examples below and at a rate at least 10% of that of HSUBC14 or HSUBC15, respectively, preferably at least 25% as fast, more preferably at least 50% as fast, and most preferably at least 75% as fast.
  • such biologically active portion of HSUBC14 comprises an amino acid sequence spanning residue 94 in Figure 1, more preferably comprises at least the amino acid sequence NTRLCLS, yet more preferably comprises at least about 25 additional amino acids of HSUBC14, even more preferably at least about 50 additional amino acids of HSUBC14, still more preferably at least about 75 additional amino acids of HSUBC14, yet even more preferably at least about 100 additional amino acids of HSUBC14, and most preferably at least about 150 additional amino acids of HSUBC14.
  • such biologically active portion of HSUBC15 comprises an amino acid sequence spanning residue 91 in Figure 2, more preferably comprises at least the amino acid sequence KKICLS, yet more preferably comprises at least about 25 additional amino acids of HSUBC15, even more preferably at least about 50 additional amino acids of HSUBC15, still more preferably at least about 75 additional amino acids of HSUBC15, yet even more preferably at least about 100 additional amino acids of HSUBC15, and most preferably at least about 150 additional amino acids of HSUBC15.
  • allelic variants have the biological activity of HSUBC14 or HSUBC15, as discussed above.
  • allelic variants are either rationally designed or naturally occurring allelic variants, i.e., they are expressed in actual individual mammals, most preferably from actual individual humans or mice. Rationally designed allelic variants can be produced according to standard art-recognized procedures (see e.g., international publication W095/18974).
  • Protein as used herein means having less than about 25% by weight, and preferably less than about 10% by weight contamination with other proteins. Such purified proteins may be obtained from natural sources, from recombinant expression, or by chemical synthesis. "Protein”, as used herein and hereinbelow is intended to encompass any polypeptide having at least 10 amino acid residues.
  • the invention provides ubiquitin conjugating enzyme expression elements.
  • Such elements include, without limitation, isolated or recombinant nucleic acid sequences encoding HSUBC14, HSUBC15, or dominant negative mutants thereof, isolated or recombinant nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such isolated or recombinant nucleic acid sequences, preferably expression vectors.
  • Such ubiquitin conjugating enzyme expression elements also include, without limitation isolated or recombinant nucleic acids capable of expressing antisense transcripts targeted against HSUBC14 or HSUBC15 and vectors comprising such isolated or recombinant nucleic acids, preferably expression vectors.
  • amino acid sequence identity and homology are determined using the program Clustal W Version 1.6 to do sequence alignment (Thompson et al, Nucleic Acids Res 22: 4673-4680 (1994)).
  • the program GeneDoc Version 2.2 was used for viewing aligned sequences.
  • a sequence is “specifically homologous” to another sequence if it is sufficiently homologous to specifically hybridize to the exact complement of the sequence.
  • a sequence is “specifically complementary” to another sequence if it is sufficiently homologous to specifically hybridize to the sequence.
  • a sequence "specifically hybridizes" to another sequence if it hybridizes to form Watson-Crick or Hoogsteen base pairs either in the body, or under conditions which approximate physiological conditions with respect to ionic strength, e.g., 140 mM NaCI, 5 mM MgCl 2 .
  • a "recombinant expression element” is a nucleic acid sequence which encodes HSUBC14 or HSUBC15, or a portion encoding at least 15 contiguous amino acids thereof, or a dominant negative mutant thereof, or is capable of expressing an antisense molecule specifically complementary thereto, or a sense molecule specifically homologous thereto, wherein the recombinant expression unit may be in the form of linear DNA or RNA, covalently closed circular DNA or RNA, or as part of a chromosome, provided however that it cannot be the native chromosomal locus for HSUBC14 or HSUBC15.
  • Preferred recombinant expression elements are vectors, which may include an origin of replication and are thus replicatable in one or more cell type. Certain preferred recombinant expression elements are expression vectors, and further comprise at least a promoter and passive terminator, thereby allowing transcription of the recombinant expression element in a bacterial, fungal, plant, insect or mammalian cell.
  • Preferred recombinant expression elements have at least 75% nucleic acid sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99%, and encode a protein or peptide having either HSUBC14 or HSUBC15 biological activity, as described above, or activity as a dominant negative mutant thereof, as further described below.
  • "Dominant negative mutants" are proteins or peptides derived from
  • HSUBC14 or HSUBC15 which inhibit the biological activity of HSUBC14 or HSUBC15, respectively.
  • Preferred dominant negative mutants include variants in which the C at position 94 of HSUBC14 or the C at position 91 of HSUBC15 is substituted, preferably by S.
  • Preferred dominant negative mutants can be derived from HSUBC14 or HSUBC15 and interfere with covalent bond formation between ubiquitin and HSUBC14 or HSUBC15, respectively, or interfere with transfer of ubiquitin from HSUBC14 or HSUBC15 to ⁇ F508, CFTR, or ⁇ AT.
  • Such dominant negative mutants can be prepared by art recognized procedures (see e.g., Townsley et al, Proc. Natl. Acad. Sci.
  • such dominant negative mutant is a protein or peptide having from 50% amino acid sequence identity to about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or to a portion or protein conjugate thereof which inhibits the biological activity of HSUBC14 or HSUBC15 to form a thioester linkage with ubiquitin or transfer ubiquitin to a target protein such as ⁇ F508, CFTR, or o ⁇ -AT, under conditions as described in the following examples.
  • such inhibition is by at least 50%, preferably by at least 75%, more preferably by at least 90% and most preferably by at least 99%.
  • such inhibitory portion comprises an amino acid sequence spanning residue 94 of HSUBC14, as shown in Figure 1, more preferably comprises at least about 25 additional amino acids of HSUBC14, or at least about 50 additional amino acids of HSUBC14, or at least about 75 additional amino acids of HSUBC14, or at least about 100 additional amino acids of HSUBC14, or even at least about 150 additional amino acids of HSUBC14.
  • such inhibitory portion comprises an amino acid sequence spanning residue 91 of HSUBC15, as shown in Figure 2, more preferably comprises at least about 25 additional amino acids of HSUBC15, or at least about 50 additional amino acids of HSUBC15, or at least about 75 additional amino acids of HSUBC15, or at least about 100 additional amino acids of HSUBC15, or even at least about 150 additional amino acids of HSUBC15.
  • spanning residue x means comprising amino acid residues in both the N-terminal and C-terminal directions from residue x, as that residue is shown in Figure 1 or Figure 2.
  • residue x itself may be substituted by one or more amino acids, more preferably from about 1 to about 50 amino acids, or residue x may be absent.
  • the amino acids in the N-terminal and C-terminal directions from residue x are each independently within 20 amino acids of residue x, as shown in Figure 1 or Figure 2, more preferably within 10, even more preferably within 5, and most preferably are immediately adjacent residue x as shown in Figure 1 or Figure 2.
  • ⁇ F508 may be used to refer to either the phenylalanine residue at position 508 of CFTR, or may more usually be used to refer to a mutant CFTR molecule in which such phenylalanine residue is substituted by one or more amino acids, more preferably from about 1 to about 50 amino acids, or in which such phenylalanine residue is deleted.
  • the invention provides methods for identifying HSUBC14BMs or HSUBC15BMs.
  • One preferred method according to this aspect of the invention comprises screening for HSUBC14BMs or HSUBC15BMs by contacting purified HSUBC14 or HSUBC15 according to the invention with populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to HSUBC14 or HSUBC15.
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind HSUBC14 or HSUBC15 based upon structural information from the purified HSUBC14 or HSUBC15 provided by the invention and determining whether such rationally designed molecules bind specifically to HSUBC14 or HSUBC15.
  • soluble HSUBC14 and soluble HSUBC15 can be prepared by removing the respective transmembrane domains, and the soluble proteins are preferably used as targets for HSUBC14BMs or HSUBC15BMs.
  • the terms "HSUBC14" and "HSUBC15” specifically include soluble HSUBC14 and soluble HSUBC15.
  • Molecules that bind specifically to HSUBC14 or HSUBC15 are molecules that bind to HSUBC14 or HSUBC15 with greater affinity than to other unrelated proteins.
  • binding affinity of the molecule for HSUBC14 or HSUBC15 is at least 5-fold greater than its affinity for unrelated proteins, more preferably at least 10-fold greater, still more preferably at least 50-fold greater, and most preferably at least 100-fold greater.
  • This aspect of the invention includes
  • HSUBC14BMs and HSUBC15BMs identified by the methods according to the invention.
  • a"HSUBC14-binding molecule is a molecule or macromolecule which binds under physiological conditions to HSUBC14.
  • a "HSUBC15-binding molecule”, or ⁇ SUBC15BM” is a molecule or macromolecule which binds under physiological conditions to HSUBC15.
  • Binds under physiological conditions means forming a covalent or non-covalent association with an affinity of at least 10 6 M “1 , most preferably at least 10 9 M "1 , either in the body, or under conditions which approximate physiological conditions with respect to ionic strength, e.g., 140 mM NaCI, 5 mM MgCl 2 .
  • a "population of molecules”, as used herein, refers to a plurality of identical molecules.
  • A"mixed population of molecules refers to a plurality of molecules wherein more than one type of molecule is present.
  • a HSUBC14BM or HSUBC15BM according to the invention is a peptide or a peptidomimetic.
  • a "peptide” is a molecule comprised of a linear array of amino acid residues connected to each other in the linear array by peptide bonds.
  • Such peptides according to the invention may include from about three to about 500 amino acids, and may further include secondary, tertiary or quaternary structures, as well as intermolecular associations with other peptides or other non-peptide molecules.
  • Such intermolecular associations may be through, without limitation, covalent bonding (e.g., through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, ion-dipole interactions, dipole-dipole interactions, or any combination of the above.
  • such an HSUBC14BM or HSUBC15 comprises a complementarity determining region of an antibody which binds under physiological conditions to a peptide-containing epitope of HSUBC14 or HSUBC15, or a peptidomimetic of such a complementarity determining region.
  • a "complementarity determining region of an antibody” is that portion of an antibody which binds under physiological conditions to an epitope, including any framework regions necessary for such binding, and which is preferably comprised of a subset of amino acid residues encoded by the human heavy chain V, D and J regions, the human light chain V and J regions, and/or combinations thereof.
  • Examples of such preferred embodiments include an antibody, or an antibody derivative, which may more preferably be a monoclonal antibody, a human antibody, a humanized antibody, a single-chain antibody, a chimeric antibody, or an antigen-binding antibody fragment.
  • Reichman et al, Nature 332: 323-327 (1988) discloses a human antibody on which rat hypervariable regions have been grafted. Verhoeyen, et al, Science 239: 1534-1536 (1988) teaches grafting of a mouse antigen binding site onto a human antibody.
  • those skilled in the art are enabled to design and produce peptidomimetics having binding characteristics similar or superior to such complementarity determining region (see e.g., Horwell et al, Bioorg. Med. Chem. 4: 1573 (1996); Liskamp et al, Reel. Trav. Chim. Pays- Bas 1: 113 (1994); Gante et al, Angew. Chem. Int. Ed.
  • compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers.
  • Additional preferred HSUBC14BMs or HSUBC15BMs according to the invention include small molecules, which can be identified using screening or rational design approaches as discussed later herein.
  • HSUBC14BMs and HSUBC15BMs can be used in conventional assays to detect the presence or absence, and/or quantity of HSUBC14 or HSUBC15, or complexes of HSUBC14 or HSUBC15 with ubiquitin, in a biological sample.
  • the invention provides methods for determining the presence or absence and/or quantity of HSUBC14 or HSUBC15 or complex thereof with ubiquitin in a biological sample.
  • Such methods comprise providing a detectable HSUBC14BM or HSUBC15BM to a biological sample, allowing the detectable HSUBC14BM or HSUBC15BM to bind to HSUBC14, HSUBC15, or complex thereof with ubiquitin, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable HSUBC14BM or HSUBC15BM with the HSUBC14, HSUBC15, or complex thereof with ubiquitin.
  • a detectable HSUBC14BM or HSUBC15BM is an HSUBC14BM or HSUBC15BM which can be detected in an assay. Such detection is preferably through the direct or indirect binding of a tag or label on the HSUBC14BM or HSUBC15BM.
  • "Direct or indirect binding" means that the tag or label may be directly connected to the HSUBC14BM or HSUBC15BM by intermolecular association, or may be connected via intermediate molecules to the HSUBC14BM or HSUBC15BM by intermolecular association.
  • Such intermolecular associations may be through, without limitation, covalent bonding (e.g., through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, ion-dipole interactions, dipole-dipole interactions, or any combination of the above.
  • Preferred tags and labels include, without limitation, radioisotopes, heavy metals, fluorescent labels, chemoluminescent labels, enzymes and enzyme substrates.
  • Preferred biological samples include blood, serum, plasma, cells, tissue portions, and cell or tissue extracts.
  • the method according to this aspect of the invention takes the form of a conventional ELISA or RIA.
  • the method employs either direct or indirect immunofluorescence. Additional preferred embodiments utilize in vivo imaging of cells expressing HSUBC14 or HSUBC15 using conventional imaging agents directly or indirectly bound to an HSUBC14BM or HSUBC15BM according to the invention.
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding HSUBC14 or HSUBC15 can also be used in conventional assays to detect the presence or absence of HSUBC14 or HSUBC15 nucleic acid in a biological sample.
  • the invention provides methods for determining the presence or absence and/or quantity of HSUBC14 or HSUBC15 nucleic acid in a biological sample.
  • such assays are nucleic acid hybridization and/or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary and/or specifically homologous to HSUBC14 or HSUBC15 nucleic acid.
  • Particularly preferred embodiments include Northern blotting, dot or slot blotting, and polymerase chain reaction.
  • the invention provides methods for identifying modulating ligands of HSUBC14 or HSUBC15.
  • Some HSUBC14BMs and HSUBC15BMs are capable of acting as antagonists or agonists of HSUBC14 or HSUBC15.
  • the method according to this aspect of the invention comprises providing HSUBC14BMs or HSUBC15BMs to an assay system for HSUBC14 or HSUBC15 participation in the ubiquitin-conjugation pathway, and determining whether such HSUBC14BMs or HSUBC15BMs interfere with or enhance the ability of HSUBC14 or HSUBC15 to participate in the ubiquitin-conjugation pathway.
  • the HSUBC14BMs or HSUBC15BMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure.
  • This aspect of the invention includes modulating ligands of HSUBC14 or HSUBC15 identified by this method according to the invention.
  • the method comprises providing HSUBC14BMs or HSUBC15BMs to an assay system for HSUBC14 or HSUBC15 participation in the ubiquitination of ER proteins, and determining whether such HSUBC14BMs or HSUBC15BMs interfere with or enhance the ability of HSUBC14 or HSUBC15 to participate in the ubiquitination of ER proteins.
  • the ER protein is ⁇ F508, CFTR, or ⁇ r AT.
  • Assessment of ability to interfere with or enhance the ability of HSUBC14 or HSUBC15 to participate in the ubiquitin-conjugation pathway can conveniently be carried out using an in vitro activity system, as later described herein.
  • the cloned gene encoding HSUBC14.or HSUBC15 can be expressed in yeast secGl mutants, thereby allowing them to grow at restrictive temperatures (above 37°C (see Sommer and Jentsch, Nature .365: 176-180 (1993)). Inhibitors can then be identified by their reversal of the ability of cells expressing HSUBC14 or HSUBC15 to grow at restrictive temperatures.
  • interference or enhancement preferably results in a reduction of ubiquitin-conjugation of at least 50%, more preferably at least 90%, and most preferably, at least 99%, or an increase of ubiquitin-conjugation of at least 50%, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 10-fold.
  • ⁇ F508 may be used either to refer to a mutation of the phenylalanine residue at position 508 of CFTR, or may more usually be used to refer to a mutant CFTR molecule in which such phenylalanine residue is substituted by one or more amino acids, more preferably from about 1 to about 50 amino acids, or in which such phenylalanine residue is deleted.
  • the invention provides modulating ligands of
  • HSUBC14 or HSUBC15 Preferred modulating ligands are HSUBC14BMs or HSUBC15BMs which act as antagonists, interfering with the ability of HSUBC14 or HSUBC15 to participate in the ubiquitination of ER proteins, and preferably are capable of interfering with the conjugation of ubiquitin to ⁇ F508, CFTR, or ,- AT.
  • Other preferred modulating ligands are HSUBC14BMs or HSUBC15BMs which act as agonists, enhancing the ability of HSUBC14 or HSUBC15 to participate in the ubiquitination of ER proteins, and preferably are capable of enhancing the conjugation of ubiquitin to ⁇ F508, CFTR, or ⁇ r AT.
  • such HSUBC14BMs or HSUBC15BMs preferably interact with HSUBC14 or HSUBC15 to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBC14 or HSUBC15 and/or inhibit or enhance the transfer of ubiquitin to ⁇ F508, CFTR, or ⁇ r AT.
  • such inhibition or enhancement is specific, i.e., the modulating ligand interferes with or enhances the ability of HSUBC14 or HSUBC15 to participate in the conjugation of ubiquitin to ⁇ F508, CFTR, or c. ⁇ -AT at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect.
  • the concentration of the ligand required for ubiquitin- ⁇ F508, CFTR, or c ⁇ -AT conjugation modulating activity is at least 2-fold lower, more preferably at least 5-fold lower, even more preferably at least 10-fold lower, and most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.
  • the invention provides methods for modulating the conjugation of ubiquitin to HSUBC14 or HSUBC15 or its transfer to a target protein, such as ⁇ F508, CFTR, or c ⁇ -AT.
  • a target protein such as ⁇ F508, CFTR, or c ⁇ -AT.
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of HSUBC14 or HSUBC15 or a recombinant expression unit which expresses HSUBC14 or HSUBC15 or an antagonist thereof to a biological system in which ubiquitin is conjugated to a target protein, preferably an ER protein such as ⁇ F508, CFTR, or ⁇ r AT.
  • biological system includes in vitro cell or tissue extracts, cell cultures, tissue cultures, organ cultures, living plants and animals, including mammals, including without limitation humans and mice.
  • An "antagonist” is a molecule which inhibits the biological activity of HSUBC14 or HSUBC15.
  • the invention provides oligonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 1 or Figure 2.
  • Preferred embodiments include hybridization probes and antisense oligonucleotides .
  • oligonucleotide includes polymers of two or more deoxyribonucleotide, or any modified nucleoside, including 2'- halo-nucleosides, 2'-0-substituted ribonucleosides,-deazanucleosides or any combination thereof.
  • such oligonucleotides have from about 10 to about 100 nucleosides, more preferably from about 15-50, and most preferably from about 15 to 35.
  • Such monomers may be coupled to each other by any of the numerous known internucleoside linkages.
  • these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof.
  • oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
  • the term "2'-0-substituted" means substitution of the 2' position of the pentose moiety with a halogen (preferably CI, Br, or F), or an O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a 2'- H group. Certain embodiments of such oligonucleotides are useful in hybridization assays. Other embodiments are useful as antisense
  • the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation.
  • the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ⁇ F508 or CFTR precursors to mature into functional CFTR. Slowing the rate of ubiquitination of ⁇ F508 allows it to mature into functional CFTR. Thus, interference with HSUBC14 or HSUBC15 function or expression should allow maturation into functional CFTR.
  • the invention provides a method for therapeutically treating emphysema caused by failure of mutant ⁇ j-AT to be secreted. Slowing the rate of ubiquitination of calnexin should slow the rate of degradation of the c ⁇ -AT that is associated to it, thereby allowing it to be secreted.
  • these embodiments utilize agents that interfere with HSUBC14 or HSUBC15 protein function or expression of a gene encoding HSUBC14 or HSUBC15.
  • Preferred agents that interfere with HSUBC14 or HSUBC15 protein function include modulating ligands of HSUBC14 or HSUBC15, respectively, preferably modulating ligands of HSUBC14 or HSUBC15 which act as antagonists of HSUBC14 or HSUBC15.
  • Preferred agents that interfere with the expression of a gene encoding HSUBC14 or HSUBC15 include antisense nucleic acids or antisense oligonucleotides specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the invention provides a method for therapeutically treating liver disease caused by accumulation of mutant ⁇ AT in the ER. Qu et al. teaches that there is a lag in ER degradation of mutant c ⁇ -AT in hosts susceptible to the development of liver disease. Enhancing the rate of ubiquitination of calnexin should increase the rate of degradation of the ⁇ r AT associated with it. Preferably, these embodiments utilize agents that enhance HSUBC14 or
  • Preferred agents that enhance HSUBC14 or HSUBC15 protein function include modulating ligands of HSUBC14 or HSUBC15, respectively, preferably modulating ligands of HSUBC14 or HSUBC15 which act as agonists of HSUBC14 or HSUBC15.
  • the invention provides a new purified soluble ubiquitin-conjugating enzyme and allelic variants thereof.
  • the primary amino acid sequence of a preferred embodiment of the new ubiquitin-conjugating enzyme (HSUBC18) is shown in Figure 3.
  • the protein has 62% sequence identity to yeast Ubc7.
  • An alignment of HsUbcl ⁇ with yeast Ubc7 is shown in Figure 6.
  • HSUBC18 is intended to include allelic variants thereof.
  • An "allelic variant”, as used herein, is a protein having at least about 75%, more preferably at least about 85%, still more preferably at least about 95%, even more preferably at least about 96%, yet even more preferably at least about 97%, more preferably yet at least about 98% and most preferably at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, or to a portion or protein conjugate thereof which retains the biological activities of HSUBC18 to (1) form a thioester linkage with ubiquitin, and (2) to transfer ubiquitin to a target protein such as ⁇ F508 or to CFTR, both under conditions as described in the examples below and at a rate at least 10% of that of HSUBC18, preferably at least 25% as fast, more preferably at least 50% as fast, and most preferably at least 75% as fast.
  • such biologically active portion comprises an amino acid sequence spanning residue 89 in Figure 3, more preferably comprises at least about 25 additional amino acids of HSUBC18, even more preferably at least about 50 additional amino acids of HSUBC18, still more preferably at least about 75 additional amino acids of HSUBC18, yet even more preferably at least about 100 additional amino acids of HSUBC18, and most preferably at least about 150 additional amino acids of HSUBC18.
  • "Spanning residue 89" is intended to mean comprising amino acid residues in both the N- terminal and the C-terminal directions from residue 89, as that residue is indicated in Figure 3.
  • such residues in the N-terminal and C-terminal directions are immediately adjacent residue 89.
  • allelic variants have the biological activity of HSUBC18, as discussed above.
  • allelic variants are either rationally designed or naturally occurring allelic variants, i.e., they are expressed in actual individual mammals, most preferably from actual individual humans or mice.
  • Rationally designed allelic variants can be produced according to standard art-recognized procedures (see e.g., international publication W095/ 18974).
  • Purified as used herein means having less than about 25% by weight, and preferably less than about 10% by weight contamination with other proteins. Such purified proteins may be obtained from natural sources, from recombinant expression, or by chemical synthesis.
  • Protein as used herein and hereinbelow is intended to encompass any polypeptide having at least 10 amino acid residues.
  • the invention provides HSUBC18 expression elements.
  • HSUBC18 expression elements include, without limitation, isolated or recombinant nucleic acid sequences encoding HSUBC18 or dominant negative mutants thereof, or capable of expressing antisense transcripts thereof or nucleic acid sequences specifically homologous or specifically complementary thereto, and vectors comprising any such recombinant expression elements, preferably expression vectors.
  • a sequence is "specifically homologous” to another sequence if it is sufficiently homologous to specifically hybridize to the exact complement of the sequence.
  • a sequence is “specifically complementary” to another sequence if it is sufficiently homologous to specifically hybridize to the sequence.
  • a sequence "specifically hybridizes" to another sequence if it hybridizes to form Watson- Crick or Hoogsteen base pairs either in the body, or under conditions which approximate physiological conditions with respect to ionic strength, e.g., 140 mM NaCI, 5 mM MgCl 2 .
  • such specific hybridization is maintained under stringent conditions, e.g., 0.2X SSC at 68 °C.
  • a “recombinant expression element” is a nucleic acid sequence which encodes HSUBC18, or a portion encoding at least 15 contiguous amino acids thereof or encoding a dominant negative mutant thereof, a nucleic acid sequence specifically homologous or specifically complementary thereto, or a nucleic acid capable of expressing an antisense molecule specifically complementary thereto or a sense molecule specifically homologous thereto, wherein the recombinant expression unit may be in the form of linear DNA or RNA, covalently closed circular DNA or RNA, or as part of a chromosome, provided however that it cannot be the native chromosomal locus for HSUBC18.
  • Preferred recombinant expression elements are vectors, which may include an origin of replication and are thus replicatable in one or more cell type. Certain preferred recombinant expression elements are expression vectors, and further comprise at least a promoter and passive terminator, thereby allowing transcription of the recombinant expression element in a bacterial, fungal, plant, insect or mammalian cell. Preferred recombinant expression elements have at least 75% nucleic acid sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 5, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99%, and encode a protein or peptide having either HSUBC18 biological activity, as described above, or activity as a dominant negative mutant thereof, as further described below.
  • Dominant negative mutants are proteins or peptides derived from HSUBC18 which inhibit the biological activity of HSUBC18.
  • Preferred dominant negative mutants include variants in which the C at position 89 of HSUBC18 is substituted, preferably by S.
  • Preferred dominant negative mutants can be derived from HSUBC18 and interfere with covalent bond formation between ubiquitin and HSUBC18 or transfer of ubiquitin from HSUBC18 to a target protein such as ⁇ F508, CFTR, or ⁇ j-AT.
  • Such dominant negative mutants can be prepared by art recognized procedures (see e.g., Townsley et al, Proc. Natl. Acad. Sci. USA 94: 2362-2367 (1997)).
  • such dominant negative mutant is a protein or peptide having from 75% amino acid sequence identity to about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 5, or to a portion or protein conjugate thereof which inhibits the biological activity of HSUBC18 to form a thioester linkage with ubiquitin or transfer ubiquitin to a protein target such as ⁇ F508, CFTR, or a AT, under conditions as described in the following examples by at least 50%, preferably by at least 75%, more preferably by at least 90% and most preferably by at least 99%.
  • such inhibitory portion comprises an amino acid sequence spanning residue 89, more preferably comprises at least about 25 additional amino acids of HSUBC18, or at least about 50 additional amino acids of HSUBC18, or at least about 75 additional amino acids of HSUBC18, or at least about 100 additional amino acids of HSUBC18, or even at least about 150 additional amino acids of HSUBC18.
  • spanning residue 89 means comprising amino acid residues in both the N-terminal and C-terminal directions from residue 89, as that residue is shown in Figure 3.
  • residue 89 itself may be substituted by one or more amino acids, more preferably from about 1 to about 50 amino acids, or residue 89 may be absent.
  • amino acids in the N-terminal and C-terminal directions from residue 89 are each independently within 20 amino acids of residue 89, as shown in Figure 3, more preferably within 10, even more preferably within 5, and most preferably are immediately adjacent residue 89 as shown in Figure 3.
  • ⁇ F508 may be used to refer to either the phenylalanine residue at position 508 of CFTR, or may more usually be used to refer to a mutant CFTR molecule in which such phenylalanine residue is substituted by one or more amino acids, more preferably from about 1 to about 50 amino acids, or in which such phenylalanine residue is deleted.
  • the invention provides methods for identifying HSUBCl ⁇ BMs.
  • One preferred method according to this aspect of the invention comprises screening for HSUBC18BMs by contacting purified HSUBC18 according to the invention and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to HSUBC18.
  • Another preferred method according to this aspect of the invention comprises rationally designing molecules to bind HSUBC18 based upon structural information from the purified HSUBC18 provided by the invention and determining whether such rationally designed molecules bind specifically to HSUBC18.
  • Molecules that bind specifically to HSUBC18 are molecules that bind to HSUBC18 with greater affinity than to other unrelated proteins.
  • binding affinity of the molecule for HSUBC18 is at least 5-fold greater than its affinity for unrelated proteins, more preferably at least 10-fold greater, still more preferably at least 50-fold greater, and most preferably at least 100-fold greater.
  • This aspect of the invention includes HSUBC18BMs identified by the methods according to the invention.
  • a"HSUBC18-binding molecule is a molecule or macromolecule which binds under physiological conditions to
  • HSUBC18 Binds under physiological conditions means forming a covalent or non-covalent association with an affinity of at least 10 6 M " ⁇ most preferably at least 10 9 M “1 , either in the body, or under conditions which approximate physiological conditions with respect to ionic strength, e.g., 140 mM NaCI, 5 mM MgCl 2 .
  • A"mixed population of molecules refers to a plurality of molecules wherein more than one type of molecule is present.
  • a HSUBC18BM according to the invention is a peptide or a peptidomimetic.
  • a "peptide” is a molecule comprised of a linear array of amino acid residues connected to each other in the linear array by peptide bonds.
  • Such peptides according to the invention may include from about three to about 500 amino acids, and may further include secondary, tertiary or quaternary structures, as well as intermolecular associations with other peptides or other non-peptide molecules.
  • Such intermolecular associations may be through, without limitation, covalent bonding (e.g., through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, ion-dipole interactions, dipole-dipole interactions, or any combination of the above.
  • such an HSUBC18BM comprises a complementarity determining region of an antibody which binds under physiological conditions to a peptide-containing epitope of HSUBC18, or a peptidomimetic of such a complementarity determining region.
  • a "complementarity determining region of an antibody” is that portion of an antibody which binds under physiological conditions to an epitope, including any framework regions necessary for such binding, and which is preferably comprised of a subset of amino acid residues encoded by the human heavy chain V, D and J regions, the human light chain V and J regions, and/or combinations thereof.
  • Examples of such preferred embodiments include an antibody, or an antibody derivative, which may more preferably be a monoclonal antibody, a human antibody, a humanized antibody, a single-chain antibody, a chimeric antibody, or an antigen-binding antibody fragment.
  • Reichman et al, Nature 332: 323-327 (1988) discloses a human antibody on which rat hypervariable regions have been grafted. Verhoeyen, et al, Science 239: 1534-1536 (1988) teaches grafting of a mouse antigen binding site onto a human antibody.
  • compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers.
  • Additional preferred HSUBCl ⁇ BMs according to the invention include small molecules, which can be identified using screening or rational design approaches as discussed later herein.
  • HSUBC18BMs can be used in conventional assays to detect the presence or absence, and/or quantity of HSUBC18, or HSUBCl ⁇ /ubiquitin complex in a biological sample.
  • the invention provides methods for determining the presence or absence and/or quantity of HSUBC18 or HSUBC18/ubiquitin complex in a biological sample.
  • Such methods comprise providing a detectable HSUBCl ⁇ BM to a biological sample, allowing the detectable HSUBC18BM to bind to HSUBC18 or HSUBC18/ubiquitin complex, if any is present in the biological sample, and detecting the presence or absence and/or quantity of a complex of the detectable HSUBC18BM and HSUBC18, or HSUBCl ⁇ /ubiquitin complex.
  • a detectable HSUBCl ⁇ BM is an HSUBCl ⁇ BM which can be detected in an assay. Such detection is preferably through the direct or indirect binding of a tag or label on the HSUBCl ⁇ BM.
  • Direct or indirect binding means that the tag or label may be directly connected to the HSUBCl ⁇ BM by intermolecular association, or may be connected via intermediate molecules to the HSUBCl ⁇ BM by intermolecular association.
  • intermolecular associations may be through, without limitation, covalent bonding (e.g., through disulfide linkages), or through chelation, electrostatic interactions, hydrophobic interactions, hydrogen bonding, ion-dipole interactions, dipole-dipole interactions, or any combination of the above.
  • Preferred tags and labels include, without limitation, radioisotopes, heavy metals, fluorescent labels, chemoluminescent labels, enzymes and enzyme substrates.
  • Preferred biological samples include blood, serum, plasma, cells, tissue portions, and cell or tissue extracts.
  • the method according to this aspect of the invention takes the form of a conventional ELISA or RIA.
  • the method employs either direct or indirect immunofluorescence.
  • Additional preferred embodiments utilize in vivo imaging of cells expressing HSUBC18 using conventional imaging agents directly or indirectly bound to an HSUBC18BM according to the invention.
  • Nucleic acid sequences specifically complementary to and/or specifically homologous to nucleic acid sequences encoding HSUBC18 can also be used in conventional assays to detect the presence or absence of HSUBC18 nucleic acid in a biological sample.
  • the invention provides methods for determining the presence or absence and/or quantity of HSUBC18 nucleic acid in a biological sample.
  • such assays are nucleic acid hybridization and/ or amplification assays, such assays comprising providing to the biological sample a nucleic acid sequence which is specifically complementary and /or specifically homologous to HSUBC18 nucleic acid.
  • Particularly preferred embodiments include Northern blotting, dot or slot blotting, and polymerase chain reaction.
  • the invention provides methods for identifying modulating ligands of HSUBC18.
  • Some HSUBC18BMs are capable of acting as antagonists or agonists of HSUBCl ⁇ .
  • the method according to this aspect of the invention comprises providing HSUBCl ⁇ BMs to an assay system for HSUBCl ⁇ participation in the ubiquitin-conjugation pathway, and determining whether such HSUBCl ⁇ BMs interfere with or enhance the ability of HSUBCl ⁇ to participate in the ubiquitin-conjugation pathway.
  • the HSUBCl ⁇ BMs are preferably provided as a population of molecules (most preferably rationally designed molecules), or as a mixed population of molecules, as for example in a screening procedure.
  • This aspect of the invention includes modulating ligands of HSUBC18 identified by this method according to the invention.
  • the method comprises providing HSUBCl ⁇ BMs to an assay system for HSUBCl ⁇ participation in the ubiquitination of ER proteins, and determining whether such HSUBCl ⁇ BMs interfere with or enhance the ability of HSUBCl ⁇ to participate in the ubiquitination of ER proteins.
  • the ER protein is ⁇ F50 ⁇ , CFTR, or ⁇ AT.
  • HSUBCl ⁇ Assessment of ability to interfere with or enhance the ability of HSUBCl ⁇ to participate in the conjugation of ubiquitin to ⁇ F50 ⁇ , CFTR, or c ⁇ -AT can conveniently be carried out using an in vitro activity system, as later described herein.
  • the cloned gene encoding HSUBC18 can be expressed in yeast sec ⁇ l mutants, thereby allowing them to grow at restrictive temperatures (above 37°C (see Sommer and Jentsch, Nature 365: 176-180 (1993)). Inhibitors can then be identified by their reversal of the ability of cells expressing HSUBCl ⁇ to grow at restrictive temperatures.
  • interference or enhancement preferably results in a reduction of ubiquitin- ⁇ F50 ⁇ , CFTR, or - AT conjugation of at least 50%, more preferably at least 90%, and most preferably, at least 99%, or an increase of ubiquitin- ⁇ F50 ⁇ , CFTR, or ⁇ r AT conjugation of at least 50%, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 10-fold.
  • the invention provides modulating ligands of HSUBCl ⁇ .
  • Preferred modulating ligands are HSUBCl ⁇ BMs which act as antagonists, interfering with the ability of HSUBCl ⁇ to participate in the ubiquitination of ER proteins and preferably are capable of interfering with the conjugation of ubiquitin to ⁇ F50 ⁇ , CFTR, or ⁇ r AT.
  • Other preferred modulating ligands are HSUBC18BMs which act as agonists, enhancing the ability of
  • HSUBC18 to participate in the ubiquitination of ER proteins and preferably are capable of enhancing the conjugation of ubiquitin to ⁇ F50 ⁇ , CFTR, or ⁇ AT.
  • HSUBCl ⁇ BMs preferably interact with HSUBCl ⁇ to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBCl ⁇ and/or transfer of ubiquitin to a protein targeted for proteasome- mediated ER protein degradation, such as ⁇ F50 ⁇ , CFTR, or ⁇ j-AT.
  • such inhibition or enhancement is specific, i.e., the modulating ligand interferes with or enhances the ability of HSUBCl ⁇ to participate in the conjugation of ubiquitin to an ER protein such as ⁇ F50 ⁇ , CFTR, or o ⁇ -AT at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect.
  • the concentration of the ligand required for ubiquitin- ⁇ F50 ⁇ , CFTR, or ⁇ AT conjugation modulating activity is at least 2-fold lower, more preferably at least 5-fold lower, even more preferably at least 10-fold lower, and most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.
  • the invention provides methods for modulating the conjugation of ubiquitin to HSUBCl ⁇ or its transfer to a target protein, preferably an ER protein such as ⁇ F508, CFTR, or ⁇ r AT.
  • a target protein preferably an ER protein such as ⁇ F508, CFTR, or ⁇ r AT.
  • One preferred embodiment of the method according to this aspect of the invention comprises providing a modulating ligand of HSUBC18 or a recombinant expression unit which expresses HSUBCl ⁇ or an antagonist thereof to a biological system in which ubiquitin is conjugated to a target protein, such as ⁇ F50 ⁇ , CFTR, or ⁇ AT.
  • biological system includes in vitro cell or tissue extracts, cell cultures, tissue cultures, organ cultures, living plants and animals, including mammals, including without limitation humans and mice.
  • An "antagonist” is a molecule which inhibits the biological activity of HSUBCl ⁇ .
  • the invention provides oligonucleotides that are specifically complementary to a portion of a nucleotide sequence shown in Figure 3. Preferred embodiments include hybridization probes and antisense oligonucleotides .
  • oligonucleotide includes polymers of two or more deoxyribonucleotide, or any modified nucleoside, including 2'- halo-nucleosides, 2'-0-substituted ribonucleosides, deazanucleosides or any combination thereof.
  • such oligonucleotides have from about 10 to about 100 nucleosides, more preferably from about 15-50, and most preferably from about 15 to 35.
  • Such monomers may be coupled to each other by any of the numerous known internucleoside linkages.
  • these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof.
  • oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
  • the term "2'-0-substituted" means substitution of the 2' position of the pentose moiety with a halogen (preferably CI, Br, or F), or an O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or a halo group, but not with a I'll group. Certain embodiments of such oligonucleotides are useful in hybridization assays. Other embodiments are useful as antisense oligonucleo
  • the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation.
  • the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ⁇ F50 ⁇ or CFTR precursors to mature into functional CFTR. Slowing the rate of ubiquitination of ⁇ F50 ⁇ allows it to mature into functional CFTR. Thus, interference with HSUBC14 or HSUBC15 function or expression should allow maturation into functional CFTR.
  • the invention provides a method for therapeutically treating emphysema caused by failure of mutant c ⁇ -AT to be secreted.
  • Slowing the rate of ubiquitination of calnexin should slow the rate of degradation of the ⁇ j-AT that is associated to it, thereby allowing it to be secreted.
  • these embodiments utilize agents that interfere with HSUBC l ⁇ protein function or expression of a gene encoding HSUBCl ⁇ .
  • Preferred agents that interfere with HSUBCl ⁇ protein function include modulating ligands of HSUBCl ⁇ , preferably modulating ligands of HSUBC18 which act as antagonists of HSUBC18.
  • Preferred agents that interfere with the expression of a gene encoding HSUBCl ⁇ include antisense nucleic acids or antisense oligonucleotides specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 5.
  • the invention provides a method for therapeutically treating liver disease caused by accumulation of mutant ⁇ AT in the ER.
  • Qu et al. teaches that there is a lag in ER degradation of mutant ⁇ r-AT in hosts susceptible to the development of liver disease.
  • Enhancing the rate of ubiquitination of calnexin should increase the rate of degradation of the c ⁇ -AT associated with it.
  • these embodiments utilize agents that enhance HSUBCl ⁇ protein function or expression of a gene encoding HSUBCl ⁇ .
  • Preferred agents that enhance HSUBCl ⁇ protein function include modulating ligands of HSUBC18, preferably modulating ligands of HSUBC18 which act as agonists of
  • Example 1 Identification and Cloning of Human HSUBC14 Gene
  • the human EST database was searched using the first 120 amino acids of the S, cerevisiae Ubc6 gene product as a query sequence.
  • the EST clone GenBank accession # W90647 was found to contain a nucleic acid sequence encoding the amino acid sequence MITPNGRXKCNTRLCLSITDFHPDTWNP. This sequence contains 24 amino acids which are identical to the yeast Ubc6 sequence flanking the active site cysteine. This clone was used to search for further EST clones.
  • the second PCR was carried out with the primers GGGAATTCCCATATGAGCAGCACCAGCAGTAAG (forward, initiator methionine codon underlined) and CCCAAGCTTTCACTCCTGCGCGATGCTCCTCAG (reverse, reverse compliment of stop codon underlined).
  • the second PCR product was digested with Ndel and Hindlll and ligated with the large fragment of Ndel/Hindl ⁇ -digested pT7-7 to yield the plasmid pT7-7-HSUBC14.
  • the insert was sequenced by standard procedures. The nucleotide sequence and deduced amino acid sequence are shown in Figure 1.
  • the encoded full-length protein has 259 amino acids, and shares 39% amino acid sequence identity and 55% sequence homology with yeast Ubc6. This homology suggests that HSUBC14 plays a role in human ER proteasome-mediated protein degradation, analogous to the role played by Ubc6 in yeast. An alignment of HSUBC14 with yeast Ubc6 is shown in Figure 4.
  • HSUBC14 HeLa cells transfected with the pFLAG-CMV-2-HSUBC14 plasmid described above were subjected to immunostaining using a mouse anti-FLAG antibody in conjunction with a rabbit anti-calreticulin antibody (Affinity Bioreagents, Inc.).
  • the mouse anti-FLAG antibody was detected with a secondary goat anti-mouse antibody conjugated to OG4 ⁇ (Molecular Probes).
  • the rabbit anti-calreticulin antibody was detected with a goat secondary anti-rabbit antibody conjugated to RedX (Molecular Probes).
  • Calreticulin is a resident protein of the endoplasmic reticulum and is present in all cells.
  • the immunostaining pattern of calreticulin is perinuclear, with reticular staining extending to the cell periphery.
  • the immunostaining pattern of the anti-FLAG in the transfected cells was largely coincident with the calreticulin pattern, indicating that HSUBC14 was mainly localized to the endoplasmic reticulum (see Figure 7).
  • the anti-FLAG immunostaining pattern detected localization of HSUBC14 at the nuclear rim, which was not distinctly stained by the anti-calreticulin antibody, and is therefore a specific characteristic of HSUBC14 localization.
  • a mutant of HSUBC14 was constructed by replacing the active site cysteine with a serine using standard site-specific mutagenesis.
  • the immunostaining pattern of the mutant was determined by the same methods described for the wild-type and was found to be identical to that of wild-type HSUBC14.
  • Thioester bond formation of HSUBC14 with ubiquitin Components are incubated in a reaction buffer containing 25 mM Hepes (pH7.0), 10 mM Mg 2+ and 1 mM ATP for 5 minutes at 30 °C. The reaction is stopped by addition of SDS sample loading buffer. Each sample is divided into two aliquots, to one of which was added DTT to a final concentration of 10 mM. The DTT-containing sample is heated in a 95 °C bath for two minutes. Samples are separated on 10% SDS-Tricine PAGE, followed by transfer to nitrocellulose filters. Filters are stained using conventional Western blot staining procedures with anti-FLAG antibodies.
  • HSUBC14 is expected to migrate as both a 29 kDa band and a 35 kDa band in the presence of ubiquitin and ATP, and the presence of the 35 kDa band is expected to be reversible by DTT. Reaction No. Proteins 1 El + ubiquitin
  • the human EST database was searched using GenBank accession #
  • CON2 sequence was translated, and the process was repeated until an initiator methionine and a stop codon were identified.
  • the coding sequence of HSUBC15 was obtained by nested PCR on a human leukocyte cDNA library.
  • HSUBC15 contains 31 ⁇ amino acids and has a predicted molecular mass of 35,l ⁇ l Da.
  • the full-length protein shares 22% amino acid sequence identity with yeast Ubc6 and 40% amino acid sequence identity with C. elegans cedl022.1. This homology suggests that HSUBC15 may play a role in human ER proteasome- mediated protein degradation analogous to the role played by Ubc in yeast.
  • An alignment of HSUBC15 with yeast Ubc6 and cedl022.1 is shown in Figure 5.
  • HSUBC15 in HeLa cells was carried out by transfecting HeLa cells with pFLAG-CMV-2 expression vector (Kodak, IBI) in which the human Ubcl5 cDNA was inserted at the Clal and Kpnl site. This insertion generates a protein sequence in which the N-terminus of HSUBC15 is extended by the amino acid sequence MDYKDDDDKLAAANSS. Expression of the protein was confirmed by Western blot using anti-FLAG antibodies that recognize the sequence DYKDDDDK. When cell extract is centrifuged to separate the soluble and particulate fractions, anti-FLAG immunoreactivity on Western blot is detected in the particulate fraction, as expected for a membrane-anchored ubiquitin conjugating enzyme. Deletion of the transmembrane domain allows expression of soluble protein.
  • HSUBC15 HeLa cells transfected with the pFLAG-CMV-2-HSUBC15 plasmid described above were subjected to immunostaining using a mouse anti-FLAG antibody in conjunction with a rabbit anti-calreticulin antibody (Affinity Bioreagents, Inc.).
  • the mouse anti-FLAG antibody was detected with a secondary goat anti-mouse antibody conjugated to OG4 ⁇ 8 (Molecular Probes).
  • the rabbit anti-calreticulin antibody was detected with a goat secondary anti-rabbit antibody conjugated to RedX (Molecular Probes).
  • Calreticulin is a resident protein of the endoplasmic reticulum and is present in all cells.
  • the immunostaining pattern of calreticulin is perinuclear, with reticular staining extending to the cell periphery.
  • the immunostaining pattern of the anti-FLAG in the transfected cells was coincident with the calreticulin pattern, indicating that HSUBC15 was mainly localized to the endoplasmic reticulum (see Figure ⁇ ).
  • the anti-FLAG immunostaining pattern did not detect localization of HSUBC15 at the nuclear rim, which is a specific characteristic of HSUBC14 localization.
  • a mutant of HSUBC15 was constructed by replacing the active site cysteine with a serine using standard site-specific mutagenesis.
  • the immunostaining pattern of the mutant was determined by the same methods described for the wild-type and was found to be identical to that of wild-type HSUBC15.
  • Components are incubated in a reaction buffer containing 25 mM Hepes (pH 7.0), 10 mM Mg 2+ and 1 mM ATP for 5 minutes at 30°C.
  • the reaction is stopped by addition of SDS sample loading buffer.
  • Each sample is divided into two aliquots, to one of which was added DTT to a final concentration of 10 mM.
  • the DTT-containing sample is heated in a 95 °C bath for two minutes. Samples are separated on 10% SDS-Tricine PAGE, followed by transfer to nitrocellulose filters. Filters are stained using conventional Western blot staining procedures with anti-FLAG antibodies.
  • HSUBC15 is expected to migrate as both a 35 kDa band and a 41 kDa band in the presence of ubiquitin and ATP, and the presence of the 41 kDa band is expected to be reversible by DTT. Reaction No. Proteins
  • the active site cysteine of a cloned HSUBC14 or HSUBC15 is replaced by a serine using standard site-specific mutagenesis.
  • the mutant protein is expressed in bacteria and purified.
  • the ability of the mutant protein to form a stable oxygen ester with ubiquitin is established as described in Example 3 above, except that the bond formation is not labile in DTT.
  • Dominant negative mutant activity is then established by introducing the mutant protein in increasing concentrations in an assay as described in Example 3 above and demonstrating dose-dependent inhibition of ubiquitin/HSUBC14 or ubiquitin/HSUBC15 complex formation.
  • Example ⁇ Reversal of ⁇ F508 phenotype HEK cells are transfected with an expression vector expressing ⁇ F508 mutant CFTR using standard procedures (see Ward et al, Cell ⁇ 3: 121-127 (1995)). Transfected cells are established as a cell line and transfected with an expression vector expressing the dominant negative mutant prepared according to Example 4 or Example 9. In these transfectants, ⁇ F508 protein is not expected to form the 7 kDa ladder characteristic of polyubiquitination.
  • HSUBCl ⁇ The human EST database was searched using the yeast Ubc7 active site sequence as the initial query sequence in the homology search.
  • the EST clone H ⁇ 5522 was discovered to contain a similar active site sequence and was used as the query sequence for a further search. Cloning was achieved using nested PCR.
  • the first PCR utilized the primers AGGCGAGGTCGCTCGGCGCA (forward) and GCGCCTGTGCGAGGCCAGGT (reverse).
  • the second PCR used the primers GGGAATTCCATATGGCGGGGACC (forward) and CCC AAGCTTTCACAGTCCCAGAGACTT (reverse).
  • the PCR product was inserted into the plasmid pT7 at the Ndel and Hindlll sites to generate plasmid pT7-7-HsUBCl ⁇ .
  • the plasmid insert was sequenced and the nucleotide sequence and deduced amino acid sequence are shown in Figure 3.
  • the encoded full- length protein has 165 amino acids (l ⁇ .565 kDa) and shares 62% amino acid sequence identity and 75% homology with yeast Ubc7. This high level of homology predicts that HSUBCl ⁇ plays a role in human ER proteasome protein degradation analogous to the role played by Ubc7 in yeast.
  • An alignment of HSUBCl ⁇ with the yeast protein Ubc7 is shown in Figure 6.
  • HSUBCl ⁇ Expression of HSUBC18 in HeLa cells was carried out by transfecting HeLa cells with pFLAG-CMV-2 expression vector (Kodak, IBI) in which the human Ubcl8 cDNA was inserted at the Clal and Kpnl site. This insertion generates a protein sequence in which the N-terminus of HSUBC18 was extended by the amino acid sequence MDYKDDDDKLAAANSS. Expression of the protein was confirmed by Western blot using anti-FLAG antibodies that recognize the sequence DYKDDDDK.
  • HSUBCl ⁇ human immunostaining
  • HeLa cells transfected with the pFLAG-CMV-2-HSUBC15 plasmid described above were subjected to immunostaining using a mouse anti-FLAG antibody in conjunction with a rabbit anti-calreticulin antibody (Affinity Bioreagents, Inc.).
  • the mouse anti-FLAG antibody was detected with a secondary goat anti-mouse antibody conjugated to OG4 ⁇ (Molecular Probes).
  • the immunostaining pattern of the anti-FLAG in the transfected cells indicated that HSUBC18 was concentrated in the nucleus but was detected throughout the cytoplasm as well.
  • the HSUBCl ⁇ gene construct prepared according to Example 9, was subcloned into the Ndel and Hindlll sites of a modified pGEX-2TK plasmit (Pharmacia) so as to express a fusion protein of glutathione-S-transferase (GST) and HSUBCl ⁇ .
  • the pGEX-HSUBCl ⁇ plasmid was transformed into the E. coli strain BL21(DE3) (Novagen). Expression of the GST-HSUBCl ⁇ fusion was induced by the addition of 1 mM IPTG. An S100 fraction of bacterial cells expressing the GST-HSUBCl ⁇ fusion was incubated with glutathione-Sepharose resin (Pharmacia).
  • the resin was washed with PBS buffer containing 0.1% TritonX-100 and 0.25 M KC1, and the GST-HSUBC18 fusion eluted with PBS buffer containing 5 mM glutathione.
  • the eluate was incubated with biotinylated thrombin, which cleaves the GST-HSUBCl ⁇ fusion to generate GST and HSUBCl ⁇ .
  • the sample was dialyzed to remove glutathione.
  • the biotinylated thrombin was removed by incubation with streptavidin agarose (Pierce) and the GST was removed by incubation with glutathione-Sepharose.
  • Example 12 Thioester bond formation with ubiquitin Proteins (as indicated below) were incubated in a reaction buffer containing 25 mM Hepes (pH7.0), 10 mM Mg 2+ and 1 mM ATP for 5 minutes at 30°C. The reaction was stopped by addition of SDS sample loading buffer. Each sample was divided into two aliquots, to one of which was added DTT to a final concentration of 10 mM. The DTT-containing sample was heated in a 95 °C bath for two minutes. Samples were separated on 10% SDS-Tricine PAGE, followed by silver staining. HSUBCl ⁇ migrates at a slower rate in the presence of ubiquitin and ATP, and that effect is reversible by DTT. Reaction No. Proteins
  • Example 13 Preparation of dominant negative mutants of HSUBC18
  • the mutant protein was expressed in bacteria and purified.
  • the ability of the mutant protein to form a stable oxygen ester with ubiquitin was established as described in Example 12 above, except that the bond formation was not labile in DTT.
  • Dominant negative mutant activity was then established by introducing the mutant protein in increasing concentrations in an assay as described in Example 12 above and demonstrating dose-dependent inhibition of ubiquitin/HSUBC18 complex formation.

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Abstract

L'invention concerne la dégradation de protéines par la voie ubiquitine-protéasome. Plus particulièrement, l'invention concerne la dégradation de protéines dans l'ER, y compris le régulateur de perméabilité transmembranaire de la fibrose kystique (CFTR) par la voie ubiquitine-protéasome. L'invention concerne également des méthodes et compositions d'inhibition de cette dégradation et d'activation de la maturation de ΔF508 dans un CFTR fonctionnel, ainsi que de compréhension du rôle de la perte de fonction du CFTR dans la fibrose kystique. L'invention concerne enfin la découverte de protéines responsables du transfert d'ubiquitine dans ΔF508, CFTR et α1-AT.
PCT/US1999/024563 1998-10-21 1999-10-21 Proteines humaines responsables de la degradation du reticulum endoplasmique (er) WO2000023599A2 (fr)

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CA002348156A CA2348156A1 (fr) 1998-10-21 1999-10-21 Proteines humaines responsables de la degradation du reticulum endoplasmique (er)
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