EP1070131A1

EP1070131A1 - Ubiquitin conjugating enzyme

Info

Publication number: EP1070131A1
Application number: EP99911938A
Authority: EP
Inventors: Alexander Fred The University of Leeds MARKHAM; Philip Alan The University of Leeds ROBINSON
Original assignee: University of Leeds
Current assignee: University of Leeds
Priority date: 1998-03-27
Filing date: 1999-03-23
Publication date: 2001-01-24
Also published as: AU3045099A; WO1999050421A1

Abstract

The invention concerns the isolation of the nucleic acid or protein or polypeptide molecule of the human ubiquitin conjugating enzyme termed hUBC7 and the subsequent exploitation of same with a view to treating clinical conditions which result from hUBC7 mediated protein degradation.

Description

UBIQUITIN CONJUGATING ENZYME

The present invention relates to the regulation of biochemical pathways, in particular to the ubiquitination of proteins, and modulation and/or amelioration of clinical conditions related to the dysfunction of proteins involved in ubiquitination.

Many aspects of a cell^'s physiology are controlled by the selective degradation of proteins. The process does not only involve the removal of misfolded or mutant proteins but also the regulation of steady state levels of many important proteins involved in modulating various biochemical processes in the cell. Examples of this include Gl and mitotic cyclins. the yeast MATcc2 repressor and the p53 tumour suppressor protein. An important pathway for the selective removal of protein involves a highly conserved 75 amino acid cellular polypeptide called ubiquitin.

The role of ubiquitin in targeting protein for degradation involves the specific ligation of ubiquitin to the group of lysine residues in proteins that are to be removed from the cell. The ubiquitin tag determines the fate of the protein and results in its selective proteolysis. Recently a number of factors have been isolated and shown to be involved in the ubiquitination process.

The initial step in the addition of ubiquitin to protein is the activation of ubiquitin by the ubiquitin activating enzyme, El. This is an ATP dependent step resulting in the formation of a thioester bond between the carboxyl terminal glycine of ubiquitin and the active site cysteine residue of El . Activated ubiquitin then interacts with a second factor, the E2 protein. A thioester bond forms between the activated glycine residue in ubiquitin and a cysteine residue in a specific E2 protein. The E2 proteins represent a family of closely related proteins encoded by different genes that confer specificity in the proteolytic process. The ligation of ubiquitin to target proteins is effected by the involvement of a further factor, the ubiquitin protein ligase, E3. The exact role of E3 in this process is not clear. However, E3 completes the final step in the ubiquitination of target proteins by adding ubiquitin via the amino group on lysine residues in proteins to be targeted for degradation. Moreover. E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi- subunit proteasome. In summary. El activated ubiquitin is targeted to specific cellular proteins by E2. E3 is a co-factor that interacts both with the E2/ubiquitin complex and proteins that have been targeted for proteolysis.

E2 proteins (UBCs) represent a family of closely related proteins common in many if not all eukaryotic cells. The high degree of sequence conservation of this family of proteins in species as diverse as Saccharomyces cerevisae, Drosophila melanogaster and Arabidopsis thaliana, lends support to the contention they provide an essential function in eukaryotic cell physiology.

The S.cerevisae E2 proteins encoded by RAD6 (ScUBC2) (1) and CDC34 (ScUBC3) (2) are essential genes required for DNA repair and cell cycle progression at the Gl/S boundary, respectively. Three yeast E2 genes, ScUBC 1, 4 and 5 (3) have also been described that are specifically involved in the selective proteolysis of yeast proteins. The production of null mutations in any two of these genes results in a severe compromise of protein degradation. The deletion of all three of these genes is lethal. Interestingly, ScUBC 4 and 5 are heat shock inducible and creation of null mutations in either of these genes results in constitutive expression of other heat shock genes.

Human homologues to some of these yeast genes have been isolated. The human homologue of yeast CDC34 was cloned by functional complementation of temperature sensitive yeast strains (4). Curiously, the isolation of human homologues to yeast RAD6 identified 2 cDNAs designated HHR6A and HHR6B. They show 95% homology with each other and a surprisingly high level of sequence identity with S.cerevisae RAD6 protein (70% at the protein sequence level).

The yeast genetic studies have shown E2s to be essential for normal physiological function and cell viability. However few specific targets have been identified in vivo. lysine residues in proteins to be targeted for degradation. Moreover. E3 is able to add ubiquitin to ubiquitin molecules already attached to target proteins thereby resulting in polyubiquitinated proteins that are ultimately degraded by the multi- subunit proteasome. In summary. El activated ubiquitin is targeted to specific cellular proteins by E2. E3 is a co-factor that interacts both with the E2/ubiquitin complex and proteins that have been targeted for proteolysis.

The yeast genetic studies have shown E2s to be essential for normal physiological function and cell viability. However few specific targets have been identified in vivo. Examples include actin. the yeast MATα2 transcriptional repressor, histones H2A and H2B. several cell surface receptors and some cyclins. Perhaps the most prominent example of a cellular target for ubiquitin conjugation is the p53 tumour suppressor protein (5).

The human papilloma virus (HPV) types 16 and 18 have been shown to actively target the degradation of p53. In vitro experiments have shown that the viral encoded oncoprotein E6 interacts with an endogenous 1 OOkDa cellular protein termed E6- AP, for E6-associated protein. The combination of the viral E6 protein and the E6-AP protein is then able to recruit p53 leading to a rapid degradation of p53 via the ubiquitin system. In vitro the only requirements for the addition of ubiquitin to p53 are the presence of El, a specific E2, the 1 OOkDa E6-AP protein and the viral E6 protein. It is therefore thought that the E6-AP complex may functionally replace the endogenous E3 activity. Interestingly, the E2 activity responsible for the p53 specific addition of ubiquitin could be replaced in the in vitro assay by the Arabidopsis UBC8 protein. Accordingly the isolation of the cDNA for the human UBC8 homologue (UBC H5) confirmed that it had a high degree of sequence identity to Arabidopsis UBC8.

The involvement of E2 proteins in clinical conditions other than cancer is exemplified by the skin disorder endemic pemphigus foliaceus (EPF)(6). The pathological presentation of the disease is represented by skin blistering caused by the loss of cohesion between keratinocytes in the sub-corneal layer. In many EPF sufferers auto-antibodies are present in the circulation and are directed against epidermal antigens. The auto-antibodies are known to be pathogenic. The exact nature of the epitopes recognised by the EPF auto-antibodies is unclear. However EPF antisera have been used to screen epidermal expression libraries to clone cross- reacting cDNAs. One cDNA clone, EPF, was found to be recognised by 32% of EPF patients' antisera. The sequence of the cloned cDNA is homologous to the yeast UBC2 protein. Curiously, the E2 open reading frame and the EPF reactive auto- antibody epitopes are in different reading frames suggesting a bi-functional EPF mRNA molecule in which two open reading frames are expressed. The relevance of these polypeptides in either normal epidermal cells or EPF pathogenesis has not yet been determined.

The majority of ubiquitin conjugating enzymes are soluble proteins of the cytosol or nucleoplasm. An exception to this general rule is the recently isolated yeast gene ScUBCόp (7) which encodes a protein which localises to the endoplasmic reticulum (ER) and nuclear envelope. ScUBCόp functions in conjunction with ScUBC7p, the physical interaction of these proteins being suggested by the yeast 2-hybrid system. Contrastingly ScUBC7p is a soluble protein and has also been shown to be involved in cadmium resistance in yeast cells (8). Genetic evidence in yeast has implicated ScUBCόp in the degradation of mutated ER membrane proteins. These results have suggested the possible involvement of the ubiquitin degradation pathway in selective removal of misfolded or mutated proteins that are destined for insertion in the plasma membrane or secretion via Golgi vesicles. Two examples of the possible involvement of ubiquitination in the removal of mutated membrane or secretory proteins are exemplified by the cystic fibrosis transmembrane conductance regulator (CFTR) (9) and α-1-antitrypsin in PiZZ variant (10), respectively.

Defective CFTR has been identified as a molecule involved in the clinical manifestation of cystic fibrosis. It is thought to act either directly or indirectly to regulate chloride flux through epithelial cells that line, for example, the lungs and intestine. Multiple mutations in the CFTR gene have been characterised but the most extensively studied is that resulting in the removal of phenylalanine 508. Glycosylation of wild type CFTR occurs in the ER and Golgi apparatus prior to insertion into the cell membrane. Studies of Phe508 deleted CFTR reveal that the mutated protein fails to exit the ER and is degraded, via the ubiquitination process as a consequence of the quality control mechanism that functions in the ER. This is by no means the only CFTR mutation to be identified. However, the fate of other mutations in CFTR is often the same as in Phe508. Current opinion indicates that treatment involving the delivery of wild type CFTR to defective epithelial cells may lead to reductions in the clinical effects of the disease. Alternatively we propose that a means of bypassing the selective removal of mutated Phe508 CFTR (incorporation of mutated Phe508 CFTR into plasma membranes shows the mutated protein is able to function in the conductance of chloride ions and partial function of this protein prevents the disease state since heterozygotes do not exhibit the disease), may similarly lead to reductions in the clinical effects of the disease.

An example of a secretory protein that fails to exit the ER is the PiZ variant of -1 - antitrypsin (α-1 -AT) in familial α-1- antitrypsin deficiency, α-1 -AT is a protease inhibitor (Pi) found circulating in blood plasma. It blocks the proteolytic activity of trypsin and elastase in the lungs and liver. In 1 in 1800 live births a defect in this protein leads to its ubiquitination and degradation. Thus circulating levels fall and notably the defect is the most common genetic cause of liver disease in children. It is also implicated in chronic liver disease in adults and the development of emphysema. A specific defect, α 1-ATZ, PiZ is characterised by a single base substitution that results in the substitution of lysine for glutamate 342. This results in the retention of α-1 -AT PiZ in the ER and consequently a reduction in circulating α 1-AT to 10-15% of normal serum levels. The retention in ER ultimately leads to the removal of the mutated protein. The parallel with the CFTR protein is evident and the involvement of the ubiquitin process is suggested in α 1-AT deficiency.

It is evident from the foregoing description that the ubiquitin process is involved in many biochemical pathways that regulate the normal functioning of eukaryotic cells. The identification and analysis of some of the components involved in the ubiquitination of proteins destined for degradation will reveal essential functions that may be modulated to ameliorate clinical conditions related to the dysfunction of proteins involved in ubiquitination.

We herein describe the nucleic acid and amino acid sequence of a newly identified ubiquitin conjugating enzyme termed human UBC7, hUBC7. Using this information we have been able to identify a cDNA clone comprising human UBC7 and so isolate the corresponding gene and protein sequence.

It is therefore an object of this invention to provide isolated human UBC7 and to identify agents that may affect the functioning of human UBC7.

It is yet a further object of the invention to develop an in vitro assay for the screening of candidate ligands that may have an inhibitory effect on human UBC7.

According to one aspect of the present invention there is provided an isolated nucleic acid encoding a ubiquitin conjugating enzyme termed human UBC7, (hUBC7). The nucleic acid may be selected from the group consisting of:

(a) DNA having the nucleotide sequence given herein as Figure 1 and 2 (SEQ ID NO: 1 and 2), which encodes the protein having the amino acid sequence given herein as Figure 3 (SEQ ID NO:4); (b) nucleic acids which hybridise to DNA of (a) above (e.g., under stringent conditions) and which encode the ubiquitin conjugating enzyme UBC7; and (c) nucleic acids which differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and which encode the ubiquitin conjugating enzyme UBC7 encoded by a DNA of (a) or (b) above.

DNAs of the present invention include those coding for proteins homologous to, and having essentially the same biological properties as, the proteins disclosed herein, and particularly the DNA disclosed herein as SEQ ID NO: 1 and 2 and encoding the protein given herein SEQ ID NO: 3. This definition is intended to encompass natural allelic variations therein. Thus, isolated DNA or cloned genes of the present invention can be of any species of origin, including mouse, rat, rabbit, cat, porcine, and human, but are preferably of mammalian origin. Thus, DNAs which hybridise to DNA disclosed herein as SEQ ID NO: 1 and 2 (or fragments or derivatives thereof which serve as hybridisation probes as discussed below) and which code on expression for the ubiquitin conjugating enzyme UBC7 protein of the present invention (e.g.. a protein according to SEQ ID NO: 3)

Conditions which will permit other DNAs which code on expression for a protein of the present invention to hybridise to the DNA of SEQ ID NO: 1 and 2 disclosed herein can be determined in accordance with known techniques. For example, hybridisation of such sequences may be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% Formamide with 5x Denhardt's solution. 0.5% SDS and lx SSPE at 37°C; conditions represented by a wash stringency of 40-45%) Formamide with 5x Denhardt's solution, 0.5% SDS, and lx SSPE at 42°C; and conditions represented by a wash stringency of 50% Formamide with 5x Denhardt's solution, 0.5% SDS and lx SSPE at 42°C, respectively) to DNA of SEQ ID NO: land 2 disclosed herein in a standard hybridisation assay. See, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory). In general, sequences which code for proteins of the present invention and which hybridise to the DNA of SEQ ID NO: land 2disclosed herein will be at least 75% homologous. 85%) homologous, and even 95% homologous or more with SEQ ID NO: 1 and 2. Further, DNAs which code for proteins of the present invention, or DNAs which hybridise to that as SEQ ID NO: 1 and 2. but which differ in codon sequence from SEQ ID NO: 1 and 2 due to the degeneracy of the genetic code, are also an aspect of this invention. The degeneracy of the genetic code, which allows different nucleic acid sequences to code for the same protein or peptide. is well known in the literature. See, e.g., U.S. Patent No. 4.757,006 to Toole et al. at Col. 2, Table 1.

According to an alternative aspect of the invention there is provided a nucleic acid molecule that encodes at least part of UBC7: or a nucleic acid molecule that is complementary thereto and which hybridises under stringent conditions to the sequence presented in Figures 1 and 2, (SEQ ID NO: 1 and 2) or fragment of such nucleic acid molecule.

7 It will be apparent to one skilled in the art that stringent hybridisation conditions are represented by washing with 0.5 x SSPE and 0.1% sodium dodecyl sulphate, 65°C.

In a preferred embodiment of the invention the nucleic acid molecule is ideaily of human origin and is either cDNA or genomic DNA.

According to a further aspect of the invention there is provided a protein or peptide comprising the amino acid sequence shown in Figure 3 (SEQ ID NO: 4), or pan thereof having ubiquitin binding or modulating activity and/or being antigenic to anti-UCB7 antibodies, or a sequence homologous or analogous to the amino acid sequence shown in Figure 3 (SEQ ID NO: 4) or part thereof having ubiquitin binding or modulating activity and/or being antigenic to anti-UCB7 antibodies. Additionally the protein can comprise at least one epitope or antigenic determinant region so that antibodies can be raised against it.

In a preferred embodiment of the invention there is provided a protein or peptide derived from the human UBC7 amino acid sequence of Figure 3 modified by substitution, deletion or addition of at least one amino acid. Ideally said modification produces a dominant negative effect on human UBC7 activity.

By way of example a mutated form of human UBC7 expressed in a cell containing wild type human UBC7 may produce a dominant/negative effect by binding tightly to accessory proteins, for example human UBC7, so as to prevent the normal interaction and function between wild-type human UBC7 and UBC6 thereby inhibiting human UBC7 activity.

According to a yet further aspect of the invention there is provided a means of manufacturing recombinant human UBC7 in prokaryotic or eukaryotic cells, wherein said means comprises a host cell including therein at least a part of the nucleic acid molecule shown in Figure 1 or 2. or a molecule complementary thereto and which

8 hybridises under stringent conditions to the nucleic acid of Figure 1 or 2. and suitable means to provide for expression of said human UBC7.

In a preferred embodiment of the invention said means for expression are well known in the art and. for example, include a secretion signal, whereby once said protein is expressed it is then secreted.

In a preferred embodiment of the invention said prokaryotic cells are E.coli.

In a further preferred embodiment of the invention said eukaryotic cells are fungal, insect, amphibian or mammalian cells.

According to a yet further aspect of the invention there is provided a recombinant vector containing a DNA sequence, represented in Figure 1 or 2 or part thereof.

In a preferred embodiment said recombinant vector is adapted, in a conventional manner, for expression of recombinant human UBC7 protein in either prokaryotic or eukaryotic cells.

In yet a further preferred embodiment said recombinant vector is engineered for repressible. inducible or constitutive expression, typically by use of expression sequences such as promoters that are characterised by repressible, inducible or constitutive expression.

According to a yet further aspect of the invention there is provided the use of recombinant human UBC7 protein in the production of antibodies to UBC7, ideally human UBC7.

According to a yet further aspect of the invention there is provided an antibody, or effective part thereof, raised against UBC7. and ideally human UBC7. ln a preferred embodiment said antibody is ideally monoclonal or an effective fragment thereof.

In a further preferred embodiment said monoclonal antibody recognises at least one UBC homologue. Ideally said monoclonal antibody is specific for UBC7 homologues, and more ideally still said monoclonal antibodies are specific for human UBC7.

According to a yet further aspect of the invention there is provided a method for the purification of a molecule selected from the group consisting of: recombinantly manufactured human UBC7 and homologues thereof; endogenously manufactured human UBC7 and homologues thereof and; effective parts of recombinantly or endogenously manufactured human UBC7 or homologues of either, the effective parts having ubiquitin binding or modulating activity, comprising the use of antibody affinity. Thus the method of purification can be directed towards recombinant or endogenous UBC7 or homologues of either, or fragments of recombinant or endogenous UBC7 or homologues of either, providing that the end product has effective ubiquitin binding or modulating activity. Reference herein to effective ubiquitin binding or modulating activity is intended to include homologues and/or fragments of UBC7.

A preferred method comprises affinity purification of UBC7, or a homologue and/or part thereof having ubiquitin binding or modulating activity, and which comprises the use of an inert matrix for example. Affigel™^* or a similar product, to which is coupled an antibody of the invention.

In a preferred embodiment the antibody can be polyclonal or monoclonal, but ideally monoclonal, or an effective part thereof.

In a yet further aspect of the invention there is provided a method for the identification and/or purification of proteins capable of interacting with human

10 L BC7 tor example human L BC6 homologues or analogues thereof which method Lompπset. a binding assav tor monitoring binding ot said protein to human L BC7 or fragment thereof

More preferably the method compπses the coupling ot recombinantly manufactured human UBC7 to an inert matπx. for example Affigel^**1* or a similar product The coupled hUBC7. or fragment thereof, under appropπate binding conditions functions as a ligand for binding cellular proteins The bound proteins are eluted from the bound human UBC7 by alteπng the binding conditions to disrupt any interaction between human UBC7 and cellular targets for human UBC7

In yet a further preferred embodiment of the invention said method is used to screen for molecules that bind the antibody of the invention.

According to a yet further aspect of the invention there is provided a method for screening molecules that interact with UBC7, ideally human UBC7, so as to affect the functional activity of same.

In this preferred method of the invention existing or novel molecules or compounds may be screened with a view to identifying, ideally, inhibitory agents that would be most suitable for use lntracellularly with a view to inhibiting or preventing the activity of UBC7. ideally hUBC7. and so inhibiting or preventing degradation of corresponding target proteins

According to a yet further aspect of the invention is provided an assay kit for the determination of UBC7. ideally hUBC7, activity in vitro

In a preferred embodiment the assay kit is composed of isolated rmcrosomes. recombinantly manufactured human UBC7 and includes other soluble factors required for UBC7 activity in vitro

11 The assa kit functions to determine the stability of the proteins identified as targets tor hUBC7 activity, for example CFTR protein or mutated variants of CFTR The removal of CFTR protein is monitored by ELISA assay using antibodies produced to human UBC7 The assay screens samples from cystic fibrosis patients to initially identify the defect in CFTR function and assist in the design of the correct prophylactic treatment for the patient.

Thus, for example, in the instance where it was determined that cystic fibrosis was due to degradation of CFTR protein then agents suitable for use in preventing this degradation could be used in the treatment, such agents would include agents that interfere with the functioning of human UBC7. Alternatively, in the instance where it was determined that cystic fibrosis was caused by something other than degradation of CFTR protein then an alternative course of therapy would be required.

According to a yet further aspect of the invention there is provided the use of the said assay kit to screen for ligands that inhibit the activity of UBC7, ideally human UBC7.

According to a yet further aspect of the invention there is provided a pharmaceutical composition compnsing a UBC7 nucleic acid as herem descπbed or a UBC7 protein as herein descπbed. and optionally a pharmaceutically acceptable diluent, earner or excipient.

It will be apparent from the above that the invention concerns the isolation of the nucleic acid or amino acid molecule of hUBC7 and the subsequent exploitation of same with a view to treating clinical conditions which result from hUBC7 mediated protein degradation.

λn embodiment of the invention will now be described by way of example only with reference to the following figures.

12 Figure 1 represents the full-length cDNA sequence of Human UBC7. The translation initiation codon (ATG) and die translation termination codon (TGA) are indicated in bold;

Figure 2 represents genomic sequence of Human UBC7. The translation initiation codon (ATG), translation termination codon (TGA) and the single intron are indicated in bold;

Figure 3 represents the 165 amino acid protein sequence that is Human UBC7. Outlined in bold is the cysteine residue used for thioester formation with ubiquitin;

Figure 4 represents a northern blot of RNA samples extracted from various tissues and probed with ³²P- labelled hUBC7;

Figure 5 represents Fluorescence In situ Hybridisation (FISH) of metaphase spreads of human lymphoblastoid cells using digoxigenin labelled H22-1 λGET clone containing hUBC7; and

Figure 6 represents a comparison of hUBC7 with yeast UBC7 and UbcP3, Arabidopsis ArUBC7 and human skeletal muscle UBE2G. A consensus sequence is also deduced.

Materials and Methods

hUBC7 cDNA cloning and Sequencing.

PCR primers; dCAATGACGAAAGTGGAGCTA (SEQ ID NO:5); and dGGAGAATGCTGAGCTGCTTG (SEQ ID NO:6) were designed from the sequence of the H22-15 λGET clone and used to screen a human fetal brain λgtl 1 library (Clontech Lab. UK. Ltd.) that had been amplified and stored in aiiquots. The same procedure that had been employed to isolate the H22-15 λGET clone as

13 described above was used to isolate two hUBC7 cDNA clones. The inserts were amplified using vector specific (λgtl 1 forward and reverse) primers and Klentaq DNA polymera≤e (Clontech). Products were then sequenced using the Dye terminator Rhodamine Fluorescent sequencing kit (Perkin Elmer). Full length coding sequence was sub-cloned into the pET3A vector (Novagen) by PCR using Klentaq DNA polymerase.

Isolation of a hUBC7 λGET genomic clone.

A hUBC7 λGET clone was isolated by PCR using oligonucleotide primers dCGTCCAGAAGTCTCTGGGAC and dCAGCACAGAGCATCACTGTC (SEQ ID NOs: 7 and 8 respectively) using the method described by Ardley et al (11). This method was employed as it also identifies sequences representing pseudogenes, a characteristic of other E2s (12-15). In brief, 2 μl aiiquots of thirty five amplified pools (average titre >10⁷ pfϋ. / μl) of a human genomic DNA library, average insert size 10 -18 kb (partial Sau3A restriction enzyme digestion) prepared in the exon trap vector λGET (16) were screened by PCR. Each pool represents the amplification of 104 pfu from the primary unamplified λGET library.

For the secondary screen, 1 μl of 1/10³ dilution of a primary pool was then used to infect 200μl K 251 cells (OD^ 1.0). LB top agar (3 mi) containing 0.2% w/v maltose (Sambrook et al., 1989) was then added to infected K 251 ceils. Aiiquots of 30 μl were then distributed to each well of a 96.well microtiter plate, the wells of which already contained 150 μl bottom agar containing 0.2% w/v maltose (17). The 96 well plates were then incubated for 16hr. at 37 °C at which time confluent lysis was observed in all wells. After cooling for 30 min. at 4 °C, 40 μl λ diluent was added to each well and phage eluted for 60 min. Aiiquots (2 μl) were then removed for analysis by PCR using the same conditions described above. Eluates that give positive signals by PCR were stored at 4°C.

For tertiary screening, serial dilutions of phage from positive wells were used to

14 infect 70μl K 251 cells as described above. The same procedure was then used to screen eluates of a 16hr incubation phage lysate as described above for the secondary screening. Those wells corresponding to the greatest dilution that gave a positive signal by PCR were chosen for further screening. This tertiary PCR screening procedure was then repeated. Phage from positive wells were then plated at low dilution on 8 cm plates so that individual plaques were well separated. Individual plaques were then picked and eluted into 100 μl λ diluent for tertiary analysis by PCR. Phagemid DNA was excised from the λGET clone H22-15 by infecting BNN132 cells, which express ere recombinase (16)

Northern Blotting.

Human multi-tissue Northern Blots I and II (Clontech Laboratories UK Ltd) were probed with 3 p_ι_ab_elled hUBC7 DNA probe prepared by Random priming (Megaprime - Amersham) of cloned hUBC7 cDNA.

Chromosomal Localisation ofhUBC7 by Somatic Cell Hybrid Panel Screening and Radiation Hybrid Mapping.

Oligonucleotide primer pair dGCTGAAACCAGCAGTTCATGGC (SEQ ID NO:9) & dCTGTGCTGCAGCTACAGG (SEQ ID NO:10)was used to screen the NIGMS human/rodent somatic cell hybrid panel No2, version 2( Cornell Inst for Medical Research USA). This primer pair amplified a 14lbp product found in the 3' UTR. PCR cycling conditions were as follows: 94°C for 30s, 58°C for 30s, 72°C for 90sec for 30 cycles followed by a final extension at 72°C for 5 min. The Genebridge 4 Radiation hybrid-mapping panel (Human Genome Mapping Project) was screened using the same PCR primers and the identical PCR amplification conditions.

15 Fluoresence In Situ Hybndisanon (FISH).

FISH was performed with biotinylated phagemid DNA probes on metaphase preparations. In brief, H22-15 λGET phagemid DNA was labelled with Digoxigenin using the random-primed DNA labelling kit and Digoxigenin-11-dUTP (Boehringer Mannheim Ltd. Lewes. U.K.). FISH was performed using metaphase spreads prepared from cultured peripheral blood lymphocytes (18,19). Any repetitive elements in the probe solution were competed out by incubation with human Cot-1 DNA (BRL-Life Technologies) at 37"C. Hybridisation was performed under a sealed glass coverslip at 37°C in a moist chamber for 16h.

Slides were washed three times (5 min. per wash) with 50% formamide in 2x SSC at 45°C and. three times (5 min. per wash) with 0. lx SSC at 60 °C. Signal detection and amplification were with sheep anti-digoxigenin Fab fragments (Boehringer Mannheim) and anti-sheep IgG FITC (Sigma). Slides were mounted with Vectashield (Vector Laboratories) containing DAPI and visualised with a Zeiss Axioscop fluorescence microscope with a CCD camera linked to an image analysis system (Vysis, UK)

Production of Antisera to Human UBC7.

A peptide corresponding to the sequence C-K-M-W-R-D-D-R-E-Q-F-Y-K-I-A-K-Q- I-V-Q-K (SEQ ID NO: 1 1) was synthesised using solid-phase peptide synthesis methodology and purified by reverse phase high performance liquid chromatography. It was then N-terminally conjugated to keyhole limpet haemocyanin using m- maieimidobenzoyl-N- hydroxysuccinimide ester (MBS). This conjugate was then used to inject rabbits (x3) over a period of 3 months to generate antisera.

16 RESULTS

Isolation of hUBC cDNA

Two hUBC" cDNA clones, both of approximate size 2.1 kbp. were isolated from screening the λgtl 1 human fetal brain cDNA library. The difference in size reflected differences in length of the 3' UTR. The sequence and deduced protein sequence is described in Figure 1 and Figure 3. The observed sequence is homologous to yeast UBC7 (Accession No. S28951) see Figure 6. Database scanning also revealed high degrees of homology to other database entries such as yeast UbcP3 (Accession No. D85544). Arabidopsis ArUBC7 (Accession No. P42747) and the skeletal muscle- specific E2, UBE2G (Accession No. D78514) (Figure 6).

We also isolated a λGET clone containing the 3' sequence of the gene. This sequence most likely represents the transcribed sequence as it matches the sequence of the isolated cDNA clones as well as intronic sequence located at bp383 (Figure 2 and Figure 3).

From the Clontech Northern blot I analysis a major transcript of 3.5kb was observed in muscle, heart, brain, liver, lung, kidney and pancreas (Figure 4). Particularly relatively high levels were observed in muscle and heart. Screening of Clontech Northern blot II indicated that this gene is also transcribed in spleen, testis, prostate, thymus and ovary (data not shown) but in relatively lower amounts compared to muscle and heart.

Chromosomal location ofhUBC7

Screening of the NIGMS somatic cell hybrid panel No2. version 2 indicated a single chromosomal locus for hUBC7 on chromosome 21. Sub-chromosomal localisation by FISH also indicated a single locus on chromosome 21 at 21q22 (Figure 5).

17 Screening of the Genebridge 4 Radiation Hybrid mapping panel further confirmed this locus at chromosome 21q22.3 to qter [21.07 cR from WI-3679 ( LOD >3.0)].

DISCUSSION

We have described the isolation of the human homologue for yeast UBC7. Both FISH analysis and Radiation Hybrid mapping panel confirm a single chromosomal location to the telomeric end of the chromosome 21q.

A major transcript of 3.5kb was observed in muscle and heart. hUBC7 together with UBE2G (20) probably are the major E2 molecules that regulate protein turnover in muscle tissue. In the fasting state, sepsis, cachexia or uncontrolled diabetes meilitus the breakdown of muscle protein is increased (21-26). These stress induced muscle wasting diseases are the result not of decreased protein synthesis but of an increased level of protein degradation. Degradation of protein is by the ubiquitination- proteasome pathway. These E2 molecules may therefore have a major role in controlling the rate of protein degradation in these diseases. The inference is that blocking of their activity in these tissues may prevent muscle-wasting diseases.

Mutation of E2 molecules can have severe consequences for eukaryotic organisms. For example, a single amino acid mutation [P97S] in the Drosophila bendless E2 altered the connectivity between subsets of neurones and manifested as visual system defects (27.28). Similarly, it has been demonstrated that decreasing the levels of the mouse homologue of the human ubiquitin conjugating enzyme. UbcH7 to approximately 30% of its normal level is embryonically lethal (29). This indicates that minor changes in the level of expression of particular E2 moieties can have major implications to the survival or behaviour of whole organisms. It is of interest therefore that the gene encoding hUBC7 lies at the telomeric end of chromosome 21q. Overexpression of this protein may also have a major biological effect. Hence, a 50%) over-expression of hUBC7 that may occur in brains of Trisomy 21 Downs Syndrome patients due to the presence of three copies of the gene may be responsible for some of the phenotypic features of this condition.

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1 1. Ardley, H.C., Moynihan, T.P., Thompson. J., Leek, J.P., Markham, A. F. and Robinson. P.A (1997) Cytogen. Cell Genet. 79, 188-192. 12. Moynihan T.P.. Cole C.G., Dunham I., O'Neil L.. Markham A.F., Robinson P.A. (1998) Genomics 51, 124-128.

13. Moynihan, T.P., Markham. A.F. and Robinson, P.A. (1996) Nucl. Acids Res. 24, 4094-4095.

14. Moynihan T.P.. Ardley H.C., Leek J.P., Thompson J.. Brindle N.S.. Markham A.F. and Robinson P.A. (1996) Mammalian Genome 7, 520-525.

15. Robinson. P.A.. Leek. J.P., Thompson, J., Carr. I. M.. Bailey A.. Moynihan, T.P., Coletta P.L., Lench, N.J., and Markham. A.F. (1995) Mammalian Genome. 6, 725-731.

16. Nehls. M., Pfeifer, D. and Boehm, T. (1994). Oncogene 9, 2169-2175. 17. Sambrook, J.. Fritsch. .F and Maniatis. T. Molecular cloning: a laboratory manual. 2nd ed. (Cold Spring Harbor. N.Y.. Cold Spring Harbour Laboratory

Press) (1989). 18. Landegent. J.E.. Dewal. N.J.I., Dirks. R.W.. Baas F. and Van der Ploeg, M.

(1987). Hum. Genet. 11, 336-370. 19. Jalal. S.M., Law. M.E., Christensen. E.R.. Spurbeck. J.L. and Dewald. G.W.

(1993). Amer. J. Med. Gen. 46, 98-103.

20

Claims

20. Watanabe. T. .. Kawai. A.. Fujiwara. T., Maekavva. H.. Hirai Y.. Nakamura Y.. and Takahashi E. ( 1996) Cytogenet. Cell Genet. 74. 146-148. 21. Mitch. W.E. and Goldberg, A.L. ( 1996) New Engi. J Med. 335, 1897-1905. 22. Temparis. S.. Aseni, M., Taillander. D., et al. (1994) Cancer Res. 54, 5568- 5573. 23. Price, S.R.. Bailey J.L., Wang X., et al (1996) J. Clin. Invest. 98, 1703-1708. 24. Solomon. V. and Goldberg, A.L. (1996) J. Biol. Chem. 271, 26690-26697. 25. Argiles J.M. and Lopez-Soriano F.J. (1996) TiPS 17, 223-226. 26. Haas, A.L., Baboshina O., Williams B., and Schwartz, L.M. (1995) J. Biol. Chem. 270, 9407-9412. 27. Muralidhar, M.G. and Thomas, J.B. (1993) Neuron 11, 253-266. 28. Oh, C.E., McMahon, R, Benzer, S., et al. (1994) J. Neurosci. 14, 3166-3179. 29. Harbers K. Muller U., Grams A., et al. (1996) Proc. Natl. Acad. Sci. 93, 12412-12417. 21 Claims

1. A nucleic acid encoding a ubiquitin conjugating enzyme termed human UBC7, (hUBCT), the nucleic acid may be selected from the group consisting of: (a) DNA having the nucleotide sequence given herein as Figure 1 and 2

(SEQ ID NO: 1 and 2), which encodes the protein having the amino acid sequence given herein as Figure 3 (SEQ ID NO:4);

(b) nucleic acids which hybridise to DNA of (a) above (e.g., under stringent conditions) and which encode the ubiquitin conjugating enzyme UBC7; and

(c) nucleic acids which differ from the DNA of (a) or (b) above due to die degeneracy of the genetic code, and which encode the ubiquitin conjugating enzyme UBC7 encoded by a DNA of (a) or (b) above.

2. A nucleic acid molecule that encodes at least part of UBC7 or a nucleic acid molecule that is complementary thereto and which hybridises under stringent conditions to the sequence presented in Figures 1 and 2 or fragment of such nucleic acid molecules.

3. A nucleic acid molecule according to either Claim 1 or Claim 2 which hybridises under the stringent hybridisation conditions of washing with 0.5 x SSPE and 0.1% sodium dodecyl sulphate, 65°C.

4. A nucleic acid moiecule according to any preceding claim which is of human origin.

5. A nucleic acid molecule according to any preceding claim wherein the isolated nucleic acid molecule is either cDNA or genomic DNA.

6. A protein or peptide comprising the amino acid sequence shown in Figure 3. or part thereof having ubiquitin binding or modulating activity and/or being antigenic to αntι-L^'BC7 antibodies, or a sequence homologous to the amino acid sequence shown in Figure 3 or part thereof having ubiquitin binding or modulating activity and/or being antigenic to antι-UBC7 antibodies.

7 A protein or peptide according to Claim 6 wherein said protein is the human L^'BC7 ammo acid sequence of Figure 3 modified by substitution, deletion or addition of at least one amino acid.

8. A protein or peptide according to Claim 7 wherein said modification produces a dominant/negative effect on human UBC7 activity.

9 A means of manufacturing recombinant human UBC7 in prokaryotic or eukaryotic cells, wherein said means comprises a host cell including therein at least a part of the nucleic acid molecule shown in Figure 1 or 2, or a molecule complementary thereto and which hybridises under stringent conditions to the nucleic acid of Figure 1 or 2. and suitable means to provide for expression of said human UBC7.

10. A means according to Claim 9 further including a secretion signal, whereby once said protein is expressed it is then secreted.

11. A means according to either of Claims 9 or 10 wherein said prokaryotic cells are E.coli.

12. A means according to either of Claims 9 or 10 wherein said said eukaryotic cells are fungal, insect, amphibian or mammalian cells.

13. A recombinant vector containing a DNA sequence according to any of Claims 1-

5.

23

14. A vector according to Claim 13 adapted for expression of recombinant human L^'BC7 protein in either prokaryotic or eukaryotic cells.

15. A vector according to either of Claims 13 or 14 wherein the vector is engineered for repressible. inducible or constitutive expression by use of expression sequences such as promoters that are characterised by repressible. inducible or constitutive expression.

16. Use of recombinant human UBC7 protein in the production of antibodies to UBC7.

17. An antibody raised against UBC7. or effective part thereof.

18. An antibody according to Claim 17 wherein said antibody is raised against human UBC7, or effective part thereof.

19. An antibody according to either Claim 17 or 18 that is monoclonal or an effective fragment thereof.

20. An antibody according to any of Claims 17-19 wherein said antibody recognises at least one UBC7 homologue.

21. An antibody according to any of Claims 17-20 wherein said antibody is specific for at least one UBC7 homologue.

22. An antibody according to any of Claims 17-21 wherein said antibody is specific for human UBC7.

23. A method for the purification of a molecule selected from the group consisting of: recombinantly manufactured human UBC7 and homologues thereof: endogenously manufactured human UBC7 and homologues thereof and; effective

24 parts of recombinantly or endogenously manufactured human UBC7 or homologues o( either, the effective parts having ubiquitin binding or modulating activity, comprising the use of antibody affinity.

24. A method according to Claim 23 which comprises affinity purification of UBC7 or a homologue and/or part thereof having ubiquitin binding or modulating activity and which comprises the use of an inert matrix for example, AffigeF^m* or a similar product, to which is coupled an antibody according to any of Claims 17-22.

25. A method according to either of Claims 23 or 24 wherein the UBC7 homologue and/or part thereof having ubiquitin binding or modulating activity, are human.

26. A method according to any of Claims 23-25 wherein the antibody is polyclonal or monoclonal, or an effective part thereof.

27. A method for the identification and/or purification of proteins or fragments thereof that are capable of interacting with human UBC7, for example human UBC6 homologues or analogues thereof, which method comprises a binding assay for monitoring binding of said protein to human UBC7.

28. A method according to Claim 27 in which the binding assay uses human UBC7 or a fragment thereof which is coupled to an inert matrix.

29. A method according to either of Claims 27 or 28 for identifying molecules that bind an antibody according to any of Claims 17-22.

30. A method for screening molecules that interact with human UBC7 so as to affect the functional activity of same, wherein existing or novel compounds are screened for their inhibition or modulation of UBC7 activity whereby degradation of target proteins is assessed.

25