HUMAN PROTEINS RESPONSIBLE FOR ER DEGRADATION

Abstract: The invention relates to degradation of proteins via the ubiquitin-proteasome pathway. More particularly, the invention relates to the degradation of proteins in the ER, including cystic fibrosis transmembrane conductance regulator (CFTR) via the ubiquitin-proteasome pathway. The invention provides methods and compositions for inhibiting such degradation and for promoting the maturation of DELTA F508 into a functional CFTR and for understanding the role of loss of CFTR function in CF. The invention resides in the discovery of human proteins responsible for the ubiquitination of DELTA F508, CFTR, and alpha 1-AT.

Description: HUMAN PROTEINS RESPONSIBLE FOR ER DEGRADATION BACKGROUND OF THE INVENTION Field of the invention 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. Summary of the related art 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. Scheffner et al, Cell 75: 495-505 (1993) discloses that ubiquitination of p53 is involved in degradation of this tumor suppressor. The enzymatic pathway for ubiquitination has been reasonably well defined. Jentsch, Annu. Rev. Genet. 26: 179-207 (1992) discloses that ubiquitination requires initial activation of a conserved C-terminal glycine residue by the ubiquitin activating enzyme, El, through formation of ubiquitin adenylate in an ATP-dependent process which liberates PPi, followed by thioester formation at a thiol site in El with release of AMP. 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. Most ubiquitin-conjugating enzymes thus far identified are soluble proteins of the cytosol or of the nucleoplasm. Recent studies in yeast, however, report an integral membrane ubiquitin conjugating enzyme which is associated with endoplasmic reticulum (ER) membrane. Sommer and Jentsch, Nature 365: 176-179 (1993), discloses UBC6, a yeast integral membrane ubiquitin conjugating enzyme, and suggests that this enzyme mediates degradation of proteins at the ER membrane. Biederer et al, EMBO J. 15: 2069-2076 (1996), teaches that polyubiquitination, UBC6, a functional proteasome and UBC7, a soluble ubiquitin-conjugating enzyme, are all required for proteoiysis of key components of the translocation apparatus of the ER membrane. 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. In many cases, the mutant protein undergoes rapid degradation and the disease pathology is triggered by the absence of the protein from its target compartment. In other cases, the mutant protein accumulates in the ER, with resultant toxic effects. Welsh and Smith, Cell 73: 1251-1254 (1993) discloses that cystic fibrosis (CF) is caused by the functional absence of a plasma membrane chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR). Tsui, Trends Genet. 8: 392-398 (1992), teaches that the vast majority of CF cases are due to a deletion of a single phenylalanine ( delta F508, hereafter "ΔF508") from a cytoplasmic portion of CFTR. Ward and Kopito, J. Biol. Chem. 269: 25710-25718 (1994), teaches that ΔF508 is retained and degraded in a pre-Golgi compartment. Yang et al, Proc. Natl. Acad. Sci. USA 90: 9480-9484 (1993), discloses that overexpressed ΔF508 precursors accumulate at ER membranes. Fra and Sitia, Sub-Cell Biochem. 21: 143-168 (1993), suggests that immature ΔF508 molecules fail to fold properly and are degraded by a pathway similar to yeast pathways described for ER degradation. Qu et al, J. Biol. Chem. 27V. 22791-22795 (1996) discloses that mutation in the secretory protein c^-antitrypsin (α 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 «ΓAT, 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 ΔF508, 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. These observations illustrate the need for new approaches for treating diseases mediated by defects in ER processing. Cheng et al, Cell 63: 827-834 (1990) suggests therapeutic treatment for CF by blocking degradation of ΔF508. Unfortunately, such approaches have not been successful. Ward et al, Cell .33: 121-127 (1995), teaches that proteasome inhibitors block the degradation of ΔF508 molecules, but that this leads to accumulation of polyubiquitinated ΔF508 forms which are not processed into a functional post-ER compartment. There is, therefore, a need for methods and compositions for treating human disease associated with proteasome-mediated ER protein degradation, including promoting the maturation of mutant proteins such as ΔF508 and mutant α,-AT. Such methods may reside in preventing the ubiquitination of proteins that are the target of proteasome-mediated ER protein degradation. Ideally, such methods would be specific for such proteins, including ΔF508 or CFTR or c^-AT, rather than preventing ubiquitination of proteins generally. BRIEF SUMMARY OF THE INVENTION 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 ΔF508, CFTR, and o^-AT. In a preferred embodiment, such treatment entails promoting the maturation of ΔF508 into a functional CFTR and provides understanding of the role of loss of CFTR function in cystic fibrosis (CF). In another preferred embodiment, such treatment entails promoting the maturation of mutant c^-AT and/or preventing its accumulation in the ER. Thus, 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 ΔF508, CFTR, and t -AT, than are methods that would prevent ubiquitination of proteins generally. In certain aspects, the invention provides new purified ubiquitin- conjugating enzymes and allelic variants thereof. In some embodiments, 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 (HSUBC15) is shown in Figure 2. In some other embodiments, the new purified ubiquitin-conjugating enzyme is soluble. The primary amino acid sequence of a preferred embodiment of such a soluble ubiquitin-conjugating enzyme (HSUBC18) is shown in Figure 3. In other aspects, 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. The purified protein and its structural information provided herein enables the preparation of HSUBC14-binding molecules (HSUBC14BMs), HSUBC15-binding molecules (HSUBC15BMs), and HSUBClδ-binding molecules (HSUBC18BMs), molecules that bind to HSUBC14, HSUBC15, or HSUBC18, respectively. Thus, in some other aspects, 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. Thus, in other aspects, 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. Thus, in other aspects, the invention provides methods for determining the presence or absence, and/or quantity of, HSUBC14, HSUBC15, or HSUBC18 nucleic acid in a biological sample. In preferred embodiments, 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. In a sixth aspect, 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. Thus, 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 HSUBClδBMs 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. In other aspects, 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. In certain embodiments, such HSUBC14BMs, HSUBC15BMs, or 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 ΔF508, CFTR, or α r AT. In yet other aspects, the invention provides methods for modulating the conjugation of ubiquitin or its transfer to a target protein, such as ΔF508, CFTR, or α,-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 ΔF508, CFTR, or α r AT. In still yet other aspects, 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. In still yet other aspects, the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation. In certain preferred embodiments, the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ΔF508 or CFTR precursors to mature into functional CFTR. In certain other preferred embodiments, the invention provides a method for therapeutically treating emphysema caused by failure of mutant α^AT to be secreted. In yet other preferred embodiments, 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. BRIEF DESCRIPTION OF THE FIGURES 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 (Ubchlδ) and calreticulin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 (α AT), via the ubiquitin-proteasome pathway. 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 ΔF508, CFTR, and o j-AT. In a preferred embodiment, such treatment entails promoting the maturation of ΔF508 into a functional CFTR and provides understanding of the role of loss of CFTR function in cystic fibrosis (CF). In another preferred embodiment, such treatment entails promoting the maturation of mutant r AT and/or preventing its accumulation in the ER. Thus, 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 ΔF508, 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. In a first aspect, 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. The active site cysteine, C94, forms thioester bonds with ubiquitin. 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. Accordingly, for purposes 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. The primary amino acid sequence of a second preferred embodiment of a new ubiquitin-conjugating enzyme (HSUBC15) is shown in Figure 2. The active site cysteine, C91, forms thioester bonds with ubiquitin. Amino acid residues 288-303 form a transmembrane domain. The transmembrane domain can be deleted to form a soluble HSUBC15, which is a preferred embodiment of this aspect of the invention. Accordingly, for purposes 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. elegans homologue, cedl022.1. An alignment of HSUBC15 with yeast Ubcό and cedl022.1 is shown in Figure 5. For purposes of the invention, 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 ΔF508 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. Preferably, 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. Preferably, 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. The term "spanning residue x" is intended to mean comprising residue x and amino acid residues in both the N-terminal and the C-terminal directions from residue x, as that residue is indicated in Figure 1 or Figure 2. Preferably, such residues in the N-terminal and C-terminal directions are immediately adjacent residue x. Such allelic variants have the biological activity of HSUBC14 or HSUBC15, as discussed above. In alternative preferred embodiments, such 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. In a second aspect, 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. For purposes of the invention, 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)). For viewing aligned sequences, the program GeneDoc Version 2.2 was used. 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 . Preferably, such specific hybridization is maintained under stringent conditions, e.£., 0.2X SSC at 68°C 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 ΔF508, CFTR, or α 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)). Preferably, 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 ΔF508, CFTR, or o^-AT, under conditions as described in the following examples. Preferably, 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%. In certain preferred embodiments, 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. In certain other preferred embodiments, 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. For purposes of this aspect of the invention, the term "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. Preferably, 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. Preferably 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. As used herein, ΔF508 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 purified protein and its structural information provided herein enables the preparation of HSUBC14BMs and HSUBC15BMs, molecules that bind to HSUBC14 or HSUBC15, respectively. Thus, in a third aspect, 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. In either case, 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. Thus, for purposes of this aspect of the invention, 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. Preferably, 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. As used herein, a"HSUBC14-binding molecule", or "HSUBC14BM", is a molecule or macromolecule which binds under physiological conditions to HSUBC14. A "HSUBC15-binding molecule", or ΗSUBC15BM", 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. In certain preferred embodiments, a HSUBC14BM or HSUBC15BM according to the invention is a peptide or a peptidomimetic. For purposes of the invention, 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. In certain preferred embodiments, 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. For purposes of the invention, 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. Those skilled in the art are enabled to make any such antibody derivatives using standard art-recognized techniques. For example, Jones et al, Nature 321: 522-525 (1986) discloses replacing the CDRs of a human antibody with those from a mouse antibody. Marx, Science 229: 455- 456 (1985) discusses chimeric antibodies having mouse variable regions and human constant regions. Rodwell, Nature 342: 99-100 (1989) discusses lower molecular weight recognition elements derived from antibody CDR information. Clackson, Br. J. Rheumatol. 3052: 36-39 (1991) discusses genetically engineered monoclonal antibodies, including Fv fragment derivatives, single chain antibodies, fusion proteins chimeric antibodies and humanized rodent antibodies. 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. In addition, 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. Engl. 33: 1699 (1994); Seebach et al, Helv. Chim. Acta 79: 913 (1996)). Accordingly, all such antibody derivatives and peptidomimetics thereof are contemplated to be within the scope of the present invention. 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. Thus, in a fourth aspect, 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. In certain preferred embodiments, the method according to this aspect of the invention takes the form of a conventional ELISA or RIA. In another preferred embodiment, 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. Thus, in a fifth aspect, the invention provides methods for determining the presence or absence and/or quantity of HSUBC14 or HSUBC15 nucleic acid in a biological sample. In preferred embodiments, 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. In a sixth aspect, 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. Thus, 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. In preferred embodiments of this aspect of 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. More preferably, the ER protein is ΔF508, CFTR, or α 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. Alternatively, 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. In either case, such 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. As used herein, ΔF508 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. In a seventh aspect, 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 ΔF508, 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 ΔF508, CFTR, or α r AT. In certain embodiments, 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 ΔF508, CFTR, or α r AT. Preferably, 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 ΔF508, CFTR, or c.ι-AT at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect. Preferably, the concentration of the ligand required for ubiquitin- ΔF508, 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. In an eighth aspect, the invention provides methods for modulating the conjugation of ubiquitin to HSUBC14 or HSUBC15 or its transfer to a target protein, such as ΔF508, 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 ΔF508, CFTR, or α r AT. The term "biological system", as used herein, 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. In a ninth aspect, 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 . For purposes of the invention, the term 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. Preferably, 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. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term 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. For purposes of the invention 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 oligonucleotides for use in animal model or human therapeutic settings. In a tenth aspect, the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation. In certain preferred embodiments, the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ΔF508 or CFTR precursors to mature into functional CFTR. Slowing the rate of ubiquitination of ΔF508 allows it to mature into functional CFTR. Thus, interference with HSUBC14 or HSUBC15 function or expression should allow maturation into functional CFTR. In certain other preferred embodiments, the invention provides a method for therapeutically treating emphysema caused by failure of mutant αj-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. Preferably, 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. In other preferred embodiments, the invention provides a method for therapeutically treating liver disease caused by accumulation of mutant α 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 α r AT associated with it. Preferably, these embodiments utilize agents that enhance HSUBC14 or HSUBC15 protein function or expression of a gene encoding HSUBC14 or HSUBC15. 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. In an eleventh aspect, 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 active site cysteine, C89, forms thioester bonds with ubiquitin. The protein has 62% sequence identity to yeast Ubc7. An alignment of HsUbclδ with yeast Ubc7 is shown in Figure 6. For purposes of the invention, the term "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 ΔF508 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. Preferably, 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. Preferably, such residues in the N-terminal and C-terminal directions are immediately adjacent residue 89. Such allelic variants have the biological activity of HSUBC18, as discussed above. In alternative preferred embodiments, such 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. In a twelfth aspect, the invention provides HSUBC18 expression elements. Such 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 . Preferably, 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 ΔF508, CFTR, or αj-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)). Preferably, 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 ΔF508, 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%. Preferably, 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. For purposes of this aspect of the invention, the term "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. Preferably, 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. Preferably the 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. As used herein, ΔF508 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 purified protein and its structural information provided herein enables the preparation of HSUBC18 binding molecules, HSUBC18BMs. Thus, in a thirteenth aspect, the invention provides methods for identifying HSUBClδBMs. 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. Preferably, 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. As used herein, a"HSUBC18-binding molecule", or "HSUBC18BM", 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 "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. In certain preferred embodiments, a HSUBC18BM according to the invention is a peptide or a peptidomimetic. For purposes of the invention, 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. In certain preferred embodiments, 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. For purposes of the invention, 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. Those skilled in the art are enabled to make any such antibody derivatives using standard art-recognized techniques. For example, Jones et al, Nature 321: 522-525 (1986) discloses replacing the CDRs of a human antibody with those from a mouse antibody. Marx, Science 229: 455- 456 (1985) discusses chimeric antibodies having mouse variable regions and human constant regions. Rodwell, Nature 342: 99-100 (1989) discusses lower molecular weight recognition elements derived from antibody CDR information. Clackson, Br. J. Rheumatol. 3052: 36-39 (1991) discusses genetically engineered monoclonal antibodies, including Fv fragment derivatives, single chain antibodies, fusion proteins chimeric antibodies and humanized rodent antibodies. 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. In addition, 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. Engl. 33: 1699 (1994); Seebach et al, Helv. Chim. Acta 79: 913 (1996)). Accordingly, all such antibody derivatives and peptidomimetics thereof are contemplated to be within the scope of the present invention. Compositions according to the invention may further include physiologically acceptable diluents, stabilizing agents, localizing agents or buffers. Additional preferred HSUBClδBMs 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 HSUBClδ/ubiquitin complex in a biological sample. Thus, in a fourteenth aspect, 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 HSUBClδBM 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 HSUBClδ/ubiquitin complex. A detectable HSUBClδBM is an HSUBClδBM which can be detected in an assay. Such detection is preferably through the direct or indirect binding of a tag or label on the HSUBClδBM. "Direct or indirect binding" means that the tag or label may be directly connected to the HSUBClδBM by intermolecular association, or may be connected via intermediate molecules to the HSUBClδBM 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. In certain preferred embodiments, the method according to this aspect of the invention takes the form of a conventional ELISA or RIA. In another preferred embodiment, 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. Thus, in a fifteenth aspect, the invention provides methods for determining the presence or absence and/or quantity of HSUBC18 nucleic acid in a biological sample. In preferred embodiments, 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. In a sixteenth aspect, the invention provides methods for identifying modulating ligands of HSUBC18. Some HSUBC18BMs are capable of acting as antagonists or agonists of HSUBClδ. Thus, the method according to this aspect of the invention comprises providing HSUBClδBMs to an assay system for HSUBClδ participation in the ubiquitin-conjugation pathway, and determining whether such HSUBClδBMs interfere with or enhance the ability of HSUBClδ to participate in the ubiquitin-conjugation pathway. The HSUBClδBMs 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. In preferred embodiments of this aspect of the invention, the method comprises providing HSUBClδBMs to an assay system for HSUBClδ participation in the ubiquitination of ER proteins, and determining whether such HSUBClδBMs interfere with or enhance the ability of HSUBClδ to participate in the ubiquitination of ER proteins. More preferably, the ER protein is ΔF50δ, CFTR, or α AT. Assessment of ability to interfere with or enhance the ability of HSUBClδ to participate in the conjugation of ubiquitin to ΔF50δ, CFTR, or c^-AT can conveniently be carried out using an in vitro activity system, as later described herein. Alternatively, the cloned gene encoding HSUBC18 can be expressed in yeast secβl 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 HSUBClδ to grow at restrictive temperatures. In either case, such interference or enhancement preferably results in a reduction of ubiquitin- ΔF50δ, CFTR, or - AT conjugation of at least 50%, more preferably at least 90%, and most preferably, at least 99%, or an increase of ubiquitin- ΔF50δ, CFTR, or α r AT conjugation of at least 50%, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 10-fold. In a seventeenth aspect, the invention provides modulating ligands of HSUBClδ. Preferred modulating ligands are HSUBClδBMs which act as antagonists, interfering with the ability of HSUBClδ to participate in the ubiquitination of ER proteins and preferably are capable of interfering with the conjugation of ubiquitin to ΔF50δ, CFTR, or α 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 ΔF50δ, CFTR, or α AT. In certain embodiments, such HSUBClδBMs preferably interact with HSUBClδ to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBClδ and/or transfer of ubiquitin to a protein targeted for proteasome- mediated ER protein degradation, such as ΔF50δ, CFTR, or αj-AT. Preferably, such inhibition or enhancement is specific, i.e., the modulating ligand interferes with or enhances the ability of HSUBClδ to participate in the conjugation of ubiquitin to an ER protein such as ΔF50δ, CFTR, or o^-AT at a concentration that is lower than the concentration of the ligand required to produce another, unrelated biological effect. Preferably, the concentration of the ligand required for ubiquitin- ΔF50δ, CFTR, or α 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. In an eighteenth aspect, the invention provides methods for modulating the conjugation of ubiquitin to HSUBClδ or its transfer to a target protein, preferably an ER protein such as ΔF508, CFTR, or α 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 HSUBClδ or an antagonist thereof to a biological system in which ubiquitin is conjugated to a target protein, such as ΔF50δ, CFTR, or α AT. The term "biological system", as used herein, 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 HSUBClδ. In a nineteenth aspect, 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 . For purposes of the invention, the term 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. Preferably, 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. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term 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. For purposes of the invention 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 oligonucleotides for use in animal model or human therapeutic settings. In a twentieth aspect, the invention provides a method for therapeutically treating diseases associated with proteasome-mediated ER protein degradation. In certain preferred embodiments, the invention provides a method for therapeutically treating cystic fibrosis caused by failure of ΔF50δ or CFTR precursors to mature into functional CFTR. Slowing the rate of ubiquitination of ΔF50δ allows it to mature into functional CFTR. Thus, interference with HSUBC14 or HSUBC15 function or expression should allow maturation into functional CFTR. In certain other preferred embodiments, 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 αj-AT that is associated to it, thereby allowing it to be secreted. Preferably, these embodiments utilize agents that interfere with HSUBC lδ protein function or expression of a gene encoding HSUBClδ. Preferred agents that interfere with HSUBClδ protein function include modulating ligands of HSUBClδ, preferably modulating ligands of HSUBC18 which act as antagonists of HSUBC18. Preferred agents that interfere with the expression of a gene encoding HSUBClδ include antisense nucleic acids or antisense oligonucleotides specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 5. In other preferred embodiments, the invention provides a method for therapeutically treating liver disease caused by accumulation of mutant α^AT in the ER. Qu et al. teaches that there is a lag in ER degradation of mutant αr-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. Preferably, these embodiments utilize agents that enhance HSUBClδ protein function or expression of a gene encoding HSUBClδ. Preferred agents that enhance HSUBClδ protein function include modulating ligands of HSUBC18, preferably modulating ligands of HSUBC18 which act as agonists of 3δ HSUBClδ. The patents and publications cited hereinabove and hereinbelow reflect the knowledge in the art and are hereby incorporated by reference in entirety, whether or not specifically so stated. Any inconsistency between these patents and publications and the present disclosure shall be resolved in favor of the present disclosure. The following examples are intended to further illustrate certain particularly preferred embodiments of the invention and are not intended to limit the scope of the invention. Searches of the human EST database utilized the program BLAST (Altschul et al., Nucleic Acids Res 25: 3389-3402 (1997)). Searches for transmembrane helices used the program Antheprot V.3.0 Gilbert Deleague, Institute de Biologie et Chemie des Proteines 69 367 Lyon cdex 07, France. 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 search led to the construction of a contiguous consensus sequence from overlapping clones which predicts a gene to encode a protein having 259 amino acids, with a predicted molecular mass of 28,δ97 Da. The contiguous nucleotide sequence was obtained using nested PCR on a human leukocyte cDNA library. The first PCR used primers having the sequence GAGAGATGAGCAGCACCAGC (forward) and CTCACTCCTGCGCGATGCTC (reverse). 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/Hindlϋ-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. Example 2 Expression and localization of HSUBC14 Expression of Ubcl4 in HeLa cells was carried out by transfecting HeLa cells with pFLAG-CMV-2 expression vector (Kodak, IBI) in which the human Ubcl4 cDNA was inserted at the Clal and Kpnl site. This insertion generates a protein sequence in which the N-terminus of Ubcl4 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. When cell extract was centrifuged to separate the soluble and particulate fractions, anti-FLAG immunoreactivity on Western blot was detected only in the particulate fraction, as expected for a membrane-anchored ubiquitin conjugating enzyme. To determine the intracellular localization of 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 OG4δδ (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). In addition, 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. Example 3 Thioester bond formation of HSUBC14 with ubiquitin Components (as indicated below) 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 2 particulate fraction (from Example 2) + ubiquitin 3 particulate fraction (from Example 2) + ubiquitin + El Example 4 Identification and Cloning of Human HSUBC15 Gene The human EST database was searched using GenBank accession # AA4δδδ73 as the starting query to obtain other human EST clones that contain overlapping nucleic acid sequences. Sequences of the identified EST clones were aligned using the program CLUSTALW (Thompson et al, Nucleic Acids Res. 22:4673-46δ0 (1994)) or the program SeqMan (DNASTAR). These analyses generated a contiguous sequence, as well as a consensus sequence (CON1). Homology search of CON1 led to the identification of a putative C. elegans Ubc gene (cedl022.1 ce02575). Another homology search was conducted using 10-20% of the 5' and 3' sequences of CON1 as the query sequence, the new EST clones were assembled and used to construct a second consensus sequence (CON2). The 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. In the first PCR reaction, the primers CAGCGACCCACCATGGAGACC (forward) and GAAGCAGTTGAGTCACAGCTC (reverse) were used. The primers CCATCGATGGAGACCCGCTACAAC (forward) and CGCGGATCCTTATAACTCAAAGTC (reverse) were used in the second PCR reaction. The product was ligated to pCR2.1 and sequenced. The experimentally obtained nucleotide and deduced protein sequences are shown in Figure 2. HSUBC15 contains 31δ amino acids and has a predicted molecular mass of 35,lδl 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. Example 5 Expression and localization of HSUBC15 Expression of 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. To determine the intracellular localization of 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 OG4δ8 (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 δ). 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. Example 6 Thioester bond formation of HSUBC15 with ubiquitin Components (as indicated below) 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 1 El + ubiquitin 2 particulate fraction (from Example 5) + ubiquitin 3 particulate fraction (from Example 5) + ubiquitin + El Example 7 Preparation of dominant negative mutants of HSUBC14 and HSUBC15 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 δ Reversal of ΔF508 phenotype HEK cells are transfected with an expression vector expressing ΔF508 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, ΔF508 protein is not expected to form the 7 kDa ladder characteristic of polyubiquitination. Example 9 Identification and cloning of HSUBClδ 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 Hδ5522 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-HsUBClδ. 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 (lδ.565 kDa) and shares 62% amino acid sequence identity and 75% homology with yeast Ubc7. This high level of homology predicts that HSUBClδ plays a role in human ER proteasome protein degradation analogous to the role played by Ubc7 in yeast. An alignment of HSUBClδ with the yeast protein Ubc7 is shown in Figure 6. Example 10 Expression and localization of HSUBClδ 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. To determine the intracellular localization of HSUBClδ, 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 OG4δδ (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. Example 11 Preparation of HSUBC18 The HSUBClδ 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 HSUBClδ. The pGEX-HSUBClδ plasmid was transformed into the E. coli strain BL21(DE3) (Novagen). Expression of the GST-HSUBClδ fusion was induced by the addition of 1 mM IPTG. An S100 fraction of bacterial cells expressing the GST-HSUBClδ 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-HSUBClδ fusion to generate GST and HSUBClδ. 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. HSUBClδ migrates at a slower rate in the presence of ubiquitin and ATP, and that effect is reversible by DTT. Reaction No. Proteins 1 El + ubiquitin 2 HSUBC18 + ubiquitin 3 HSUBC18 + ubiquitin + El Example 13 Preparation of dominant negative mutants of HSUBC18 The active site cysteine of HSUBC18, cloned as described in Example 9, was replaced by a serine using standard site-specific mutagenesis. 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. 4δ

Claims: What is claimed is: 1. Purified HSUBC14 protein. 2. The purified HSUBC14 protein according to claim 1 having the amino acid sequence set forth in SEQ ID NO: 2. 3. An HSUBC14 expression element. 4. The HSUBC14 expression element according to claim 3 selected from isolated or recombinant nucleic acid sequences encoding HSUBC14 or dominant negative mutants thereof, isolated or recombinant nucleic acid sequences specifically homologous or specifically complementary thereto, isolated or recombinant nucleic acids capable of expressing antisense transcripts targeted against HSUBC14, and vectors comprising any such nucleic acid sequences. 5. A method for identifying HSUBC14BMs comprising contacting purified HSUBC14 and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to HSUBC14. 6. An HSUBC14BM identified by the method according to claim 5. 7. A method for determining the presence or absence and/or quantity of HSUBC14 or HSUBC 1 /ubiquitin complex in a biological sample, the method comprising providing a detectable HSUBC14BM to a biological sample, allowing the detectable HSUBC14BM to bind to HSUBC14, or HSUBC 14 /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 HSUBC14BM and HSUBC14 or HSUBC 14 /ubiquitin complex. δ. A method for determining the presence or absence and/or quantity of HSUBC14 nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence or oligonucleotide which is specifically complementary to HSUBC14 nucleic acid. 9. A method for identifying modulating ligands of HSUBC14 comprising providing HSUBC14BMs to an assay system for participation of HSUBC14 in the ubiquitin-conjugation pathway, and determining whether such HSUBC 14BMs interfere with or enhance the ability of HSUBC14 to participate in the ubiquitin- conjugation pathway. 10. A modulating ligand of HSUBC14. 11. A modulating ligand of HSUBC14 identified by the method according to claim 9. 12. The modulating ligand of HSUBC 14 according to claim 10, which interacts with HSUBC14 to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBC14, and/or transfer of ubiquitin to an ER protein. 13. The modulating ligand of HSUBC14 according to claim 12, wherein the ER protein is CFTR or ΔF508. 14. An agent that interferes with the expression of the HSUBC 14 gene. 15. An oligonucleotide that is specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 1. 16. Purified HSUBC15 protein. 17. The purified HSUBC15 protein according to claim 16 having the amino acid sequence set forth in SEQ ID NO: 4. 18. An HSUBC15 expression element. 19. The HSUBC15 expression element according to claim 18 selected from isolated or recombinant nucleic acid sequences encoding HSUBC15 or dominant negative mutants thereof, isolated or recombinant nucleic acid sequences specifically homologous or specifically complementary thereto, isolated or recombinant nucleic acids capable of expressing antisense transcripts targeted against HSUBC15, and vectors comprising any such nucleic acid sequences. 20. A method for identifying HSUBC15BMs comprising contacting purified HSUBC15 and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to HSUBC15. 21. An HSUBC 15BM identified by the method according to claim 20. 22. A method for determining the presence or absence and/or quantity of HSUBC15 or HSUBC15/ubiquitin complex in a biological sample, the method comprising providing a detectable HSUBC 15BM to a biological sample, allowing the detectable HSUBC15BM to bind to HSUBC15, or HSUBC15/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 HSUBC15BM and HSUBC15 or HSUBC15/ubiquitin complex. 23. A method for determining the presence or absence and/or quantity of HSUBC15 nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence or oligonucleotide which is specifically complementary to HSUBC15 nucleic acid. 24. A method for identifying modulating ligands of HSUBC15 comprising providing HSUBC15BMs to an assay system for participation of HSUBC15 in the ubiquitin-activation/ conjugation pathway, and determining whether such HSUBC15BMs interfere with or enhance the ability of HSUBC15 to participate in the ubiquitin-activation/conjugation pathway. 25. A modulating ligand of HSUBC15. 26. A modulating ligand of HSUBC15 identified by the method according to claim 24. 27. The modulating ligand of HSUBC15 according to claim 25, which interacts with HSUBC15 to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBC15 and/or transfer of ubiquitin to an ER protein. 2δ. The modulating ligand of HSUBC 15 according to claim 27, wherein the ER protein is CFTR or Δ F50δ. 29. An agent that interferes with the expression of the HSUBC 15 gene. 30. An oligonucleotide that is specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 3. 31. Purified HSUBC18 protein. 32. The purified HSUBClδ protein according to -claim 31 having the amino acid sequence set forth in SEQ ID NO: 6. 33. An HSUBClδ expression element. 34. The HSUBClδ expression element according to claim 33 selected from isolated or recombinant nucleic acid sequences encoding HSUBClδ or dominant negative mutants thereof, isolated or recombinant nucleic acid sequences specifically homologous or specifically complementary thereto, isolated or recombinant nucleic acids capable of expressing antisense transcripts targeted against HSUBC18, and vectors comprising any such nucleic acid sequences.. 35. A method for identifying HSUBC lδBMs comprising contacting purified HSUBClδ and populations of molecules or mixed populations of molecules and determining the presence of molecules which bind specifically to HSUBC18. 36. An HSUBC18BM identified by the method according to claim 35. 37. A method for determining the presence or absence and/or quantity of HSUBClδ or HSUBClδ/ubiquitin complex in a biological sample, the method comprising providing a detectable HSUBClδBM to a biological sample, allowing the detectable HSUBClδBM to bind to HSUBClδ, or HSUBClδ/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 HSUBClδBM and HSUBC18 or HSUBC 18 /ubiquitin complex. 38. A method for determining the presence or absence and/or quantity of HSUBClδ nucleic acid in a biological sample comprising providing to the biological sample a nucleic acid sequence or oligonucleotide which is specifically complementary to HSUBClδ nucleic acid. 39. A method for identifying modulating ligands of HSUBC 18 comprising providing HSUBC18BMs to an assay system for participation of HSUBClδ in the ubiquitin-conjugation pathway, and determining whether such HSUBClδBMs interfere with or enhance the ability of HSUBClδ to participate in the ubiquitin- conjugation pathway. 40. A modulating ligand of HSUBClδ. 41. A modulating ligand of HSUBClδ identified by the method according to claim 39. 42. The modulating ligand of HSUBC18 according to claim 40, which interacts with HSUBC18 to inhibit or enhance the formation of a thioester bond between ubiquitin and HSUBC18, and/or transfer of ubiquitin to an ER protein. 43. The modulating ligand of HSUBC 18 according to claim 40, wherein the ER protein is CFTR or ΔF508. 44. An agent that interferes with the expression of the HSUBClδ gene. 45. An oligonucleotide that is specifically complementary to a portion of the nucleotide sequence set forth in SEQ ID NO: 5.

Inventors: CHAU VINCENT, CHAU, VINCENT
Applicants: LEUKOSITE INC, LEUKOSITE, INC.
Application year: 1999
Main classification: C12N15
IPC codes: A61K31, A61K45, A61K48, A61P1, A61P11, A61P35, C07K14, C12N9, C12N15, C12Q1