WO2000032787A1

WO2000032787A1 - Therapeutic agents comprising an e3 ubiquitin ligase for use in degenerative disorders

Info

Publication number: WO2000032787A1
Application number: PCT/GB1999/003987
Authority: WO
Inventors: Alexander Fred Markham; Philip Alan Robinson
Original assignee: The University Of Leeds
Priority date: 1998-12-01
Filing date: 1999-11-30
Publication date: 2000-06-08
Also published as: AU1397500A; EP1135505A1; GB9826276D0

Abstract

An isolated nucleic acid molecule encoding an E2 associated protein, which acts in the role of an E3 ubiquitin ligase, wherein said isolated nucleic acid has a nucleotide sequence which hybridises to the nucleic acid shown in Figure 1. The invention also provides the polypeptide encoded by the nucleic acid and antibodies raised against the polypeptide.

Description

THERAPEUTIC AGENTS COMPRISING AN E3 UBIQUITIN LIGASE FOR USE IN DEGENARATIVE DISORDERS

The invention relates to the isolation of a nucleic acid molecule and the protein encoded thereby; and the use of these products as therapeutic agents particularly, but 5 not exclusively, in gene therapy.

The invention relates to a novel ubiquitination enzyme hereinafter referred to as Ring C enzyme. The ubiquitination pathway functions to target cellular proteins for degradation. The pathway is thought to operate in all cell types and is necessary for 0 cell viability. Ubiquitination is particularly important in the control of proliferation and differentiation; DNA repair; heat shock response; and organelle biogenesis. A functional de-ubiquitination system is also necessary for cell viability. Proteins with short half-life such as those which control progress through the cell cycle are targeted for degradation by ubiquitination. Abnormal and mutant proteins are processed in a 5 similar way. It is of note that many proteins are resistant to proteolytic digestion in the absence of ubiquitination.

The ubiquitination pathway involves a cyclical four-step process. The process includes a number of enzymes such as: ubiquitin activating enzymes (El), ubiquitin 0 conjugating enzymes (E2 or UBC), ubiquitin-protein ligases (E3), ubiquitin hydrolases and the proteasome.

The ubiquitination pathway first involves activation of ubiquitin by the enzyme El in an ATP-dependent manner. Activation involves the formation of a thioester between the active cysteine residue of El and the C-terminal glycine of ubiquitin. Once activated, the ubiquitin is transferred to a cysteine residue of a ubiquitin-conjugating enzyme (an E2 such as UBC4). The ubiquitin conjugating enzyme then catalyses the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino group of a lysine residue in a target protein. This is brought about by E3 ubiquitin ligases specifically binding to target proteins which are not otherwise recognised by E2's. In addition, ubiquitin also becomes conjugated to itself via a lysine residue at position 48 of ubiquitin, resulting in the formation of poly-ubiquitin chains. Poly-ubiquitinated proteins serve as targets that are recognised and degraded by an ATP-dependent proteinase complex, the proteasome.

Ubiquitin-conjugating enzymes or E2's comprise a family of proteins characterised by a highly conserved catalytic site. E2 proteins (UBC's) 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 and Arabidopsis, lends support to the contention that they provide an essential function in eukaryotic cell physiology.

The S.cerevisae E2 proteins encoded by RAD6 (ScUBC2) and CDC34 (ScUBC3) are essential genes required for DNA repair and cell cycle progression at the Gl/S boundary, respectively. Three other yeast E2 genes, ScUBC 1, 4 and 5 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 latter 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 of some of these yeast genes have been isolated. The human homologue of yeast CDC34 was cloned by functional complementation of temperature sensitive yeast strains. 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.

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.

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 with an E2 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. This is one example of dysfunction of the ubiquitination process leading to a disease state. There are likely to be many more genes involved in ubiquitination, and mutations in one of them, are characterised.

In 1965, the condition known as Angelman Syndrome (AS) was first described and since then a detailed thorough clinical phenotype has been established allowing the identification of subjects suffering from AS. The most common age of diagnosis of AS is between the ages of 3-7 years. Between these ages many of the behavioural and physical features of AS become apparent. Intially this disorder was considered rare. However it has become apparent that the disease is more common than was first thought.

AS is associated with a chromosomal disorder that results from a deletion in chromosome 15 (1-4). The deletion is identifiable in about 70% of individuals with AS. A putative AS gene has been identified as UBE3A, which encodes the human E6 associated protein, E6-AP. Mutations and truncations in this gene have been shown to be linked to AS. In addition mutations in the so called Imprinting Centre (a region of chromosome 15 involved in regulating the expression of UBE3A) have also been implicated in AS. However mutations in UBE3A are by no means the only cause of AS. In many cases chromosome rearrangement has resulted in deletion of the 15ql 1- 13 region of chromosome 15 which contain the UBE3A gene.

Individuals with AS exhibit a number of neurological disorders including feeding problems during infancy, delays in motor development, ataxic movement, seizures, mental retardation and a lack of expressive speech. AS sufferers are also characterised by having a wide mouth, tongue protrusion, irregular dentition, and a pointed chin. Because of the characteristic awkward gait AS has been referred to as the "Happy Puppet" syndrome. Current estimates of the prevalence of AS are lin 15000 to 1 in 30000 live births.

A number of drugs are available to control certain symptoms of AS. For example, anti-epileptic drugs such as Valproate and Topiramate have been shown to be effective for seizures resulting from AS.

It is clear that to ultimately control this condition it will be necessary to replace those functions lost as a consequence of the chromosome 15 gene deletions and/or mutations. It will be appreciated that one possible approach to restoring cell function would be in providing proteins which act downstream of enzymes such as UBE3A which, when itself mutated is known to induce serious neuronal developmental and degenerative diseases.

It is thus known that ubiquitin-conjugating enzymes or E2's when associated with E6 Associated Protein function in a manner to control neuronal development and as a result of this, mutations in E6 Associated Protein lead to severe brain disorders typically characterised by Angelman Syndrome.

We report herein the isolation of a novel protein termed Ring CI which associates with ubiquitin-conjugating enzymes so as to control neurone axonal extension/retraction. Ring CI is unrelated in primary sequence to UBE3A but may have related functions . It therefore follows that Ring CI enzyme has a major part to play in the establishment of neuronal connections and has a significant part to play in nerve cell development and the avoidance of degenerative diseases. It may also play an important role in the repair of neuronal damage, for example after a stroke. In view of these facts we consider that Ring CI enzyme is of therapeutic value in instances where the endogenous protein is not produced or is not functional. Alternatively, the Ring CI protein may be of therapeutic utility as an adjunct to neuronal or tissue regeneration when delivered at sites of damage. We particularly, but not exclusively, consider that the Ring C 1 gene has a role to play in gene therapy with a view to delivering the Ring CI nucleic acid molecule to a selected target site.

It is therefore an object of the invention to isolate a ubiquitination enzyme to use as a therapeutic agent.

According to a first aspect of the present invention there is provided an isolated nucleic acid encoding an E2 associated protein which acts in the role of an E3 ubiquitin-protein ligase, the nucleic acid may be selected from the group consisting of:

(a) DNA having the nucleotide sequence given herein as SEQ ID NO:l (which encodes the protein having the amino acid sequence given herein as SEQ ID NO:2), and which encode an E2 associated ubiquitin-protein ligase

(b) nucleic acids which hybridize to DNA of (a) above (e.g., under stringent conditions) and which encode an E2 associated ubiquitin ligase, 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 an E2 associated ubiquitin-protein ligase 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: l and encoding the protein given herein SEQ ID NO:2. 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 hybridize to DNA disclosed herein as SEQ ID NO: l (or fragments or derivatives thereof which serve as hybridization probes as discussed below) and which code on expression for a protein of the present invention (e.g., a protein according to SEQ ID NO:2) We report herein the isolation of a novel protein termed Ring CI which associates with ubiquitin- conjugating enzymes so as to control neurone axonal extension/retraction. Ring CI is unrelated in primary sequence to UBE3A but may have related functions.

Conditions which will permit other DNAs which code on expression for a protein of the present invention to hybridize to the DNA of SEQ ID NO:l disclosed herein can be determined in accordance with known techniques. For example, hybridization 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: l disclosed herein in a standard hybridization 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 hybridize to the DNA of SEQ ID NO: l disclosed herein will be at least 75% homologous, 85% homologous, and even 95% homologous or more with SEQ ID NO:l . Further. DNAs which code for proteins of the present invention, or DNAs which hybridize to that as SEQ ID NOT, but which differ in codon sequence from SEQ ID NO:l 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 a further aspect of the invention there is therefore provided an isolated nucleic acid molecule encoding an E2 associated protein, wherein said isolated nucleic acid molecule has a nucleotide sequence which hybridises to the nucleic acid shown in Figure 1 under high stringency conditions.

Ideally hybridisation occurs under stringent conditions such as lxSSC, 0.1%SDS, at 65° C.

Preferably, the nucleic acid is of human origin.

Preferably, the isolated nucleic acid is either cDNA or genomic DNA.

According to a yet further aspect of the invention there is provided an isolated polypeptide or fragment or analogue or derivative thereof encoded by the nucleic acid molecule according to the invention.

Preferably, the polypeptide or fragment or analogue or derivative thereof has E2 associated ubiquitin-protein ligase activity and/or is antigenic to anti-E2 associated ubiquitin-protein ligase antibodies.

According to a yet further aspect of the invention there is provided a delivery vehicle comprising the isolated nucleic acid molecule of the invention.

Reference herein to the term delivery vehicle is intended to include any vector whether a viral vector or otherwise for example, without limitation, an adenovirus, a retrovirus, a herpesvirus, a plasmid, a phage, a phagemid or a liposome. Ideally said delivery vehicle is adapted for administration, for example, but without limitation, by suitable formulation into a suspension.

More preferably, said delivery vehicle is adapted to deliver said nucleic acid molecule to selected tissue. Thus the delivery vehicle is provided with means to enable the nucleic acid molecule to be targeted to a specific site. The nature of the means comprises conventional technologies well known to those skilled in the art for example, without limitation, in the instance where the delivery vehicle is a viral vector said viral vector is provided with surface protein adapted to ensure the viral vector binds to and/or penetrates specific target tissues. Thus, in this way, the nucleic acid molecule of the invention can be used in gene therapy treatments.

According to a yet further aspect of the invention there is provided antibodies raised against the polypeptide, fragment, analogue, derivative or epitope of an E2 associated protein. Ideally, the antibodies are monoclonal and more ideally are human or are genetically engineered to be humanised.

It will be apparent to those skilled in the art that the antibodies of the invention can be used to determine the expression of the polypeptide of the invention in selected target tissue.

According to a further aspect of the invention there is provided a method for the treatment of neurological or degenerative disorders including stroke comprising administering to a patient suffering from a neurological or degenerative disorder including stroke, the nucleic acid molecule and/or polypeptide of the invention.

In this preferred embodiment of the invention said nucleic acid molecule is administered by the incorporation of said nucleic acid molecule into a delivery vehicle as herein described and ideally the method of treatment involves the use of gene therapy. According to a yet further aspect of the invention there is provided an isolated polypeptide or fragment or analogue or derivative thereof coded by the nucleic acid molecule of Figure 1 for use as a pharmaceutical.

According to a yet further aspect of the invention there is provided use of an isolated polypeptide or fragment or analogue or derivative thereof coded by the nucleic acid molecule of Figure 1 in the manufacture of a medicament for treating neurological or degenerative disorders including stroke.

Embodiments of the invention will now be described by way of example only with reference to the following figures wherein:

Figure 1 shows the cDNA of Ring CI (SEQ ID NO 1); and

Figure 2 shows the cDNA and predicted protein encoded by Ring CI (SEQ ID NO 2), and

Figure 3 shows the predicted protein encoded by Ring CI (SEQ ID NO 3).

Materials and Methods

Isolation of RingCl using the Yeast 2-Hybrid Selection Assay.

Human UbcH7 cDNA was cloned into the pGBT9 shuttle vector and used as bait in a yeast 2 hybrid system (Clontech Matchmaker) to select interacting clones from a human testis cDNA library cloned in the activation domain shuttle vector, pGAD424. A number of interacting clones were identified and sequenced. This allowed a full length cDNA clone to be isolated and sequenced in its entirety. No homology to the HECT protein homology domain, characteristic of E6-associated protein (E6-AP)/E3 homologues, was observed. A deletion series analysis of the RingCl clone identified a minimum interaction domain of 127 amino acid residues (residues 167 - 293), mediating the binding of this protein to human UbcH7.

Northern Blots

Northern Blot I [Clontech Ltd.] was probed with a ³²P-labelled RingCl cDNA probe. Northern blot analysis revealed the presence of 2 RingCl transcripts of approximately 2.7 and 6.5 kb in all tissues and a smaller species of 2.2 kb in testis. A particularly high level of expression of the 2.2kb. transcript was observed in testis.

Chromosomal localisation of RingCl

The NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2, version 2 (NIGMS Human Genetic Mutant Cell Repository, Coriell Institute for Medical Research, Camden, NJ 08103, USA) was screened by PCR using PCR primer pairs [ PAR012 - PAR 013: dACTAAGATATCACAACCATGAGCA (SEQ ID NO 4) and DATTTCACTGGCCTTGAATGTGGAC (SEQ ID NO 5), respectively] to amplify RingCl sequence 904bp to 1033bp. This sequence spans an intron. The RingCl gene mapped to chromosome 15. Fluorescence in situ hybridisation on metaphase spreads, prepared from cultured peripheral blood lymphocytes, using a probe prepared from an isolated human PI artificial chromosome containing the 3' end of the RingCl gene, indicated a chromosomal localisation of proximal 15q. Using the same primer pairs, screening of a radiation hybrid mapping panel by PCR indicated a location 2.84 cR from WI-3873 on proximal chromosome 15q.

Ubiquitin thioester adduct formation assays.

Baculovirus encoded wheat germ El, human E6-AP (95 kDa form) and RingCl were partially purified from infected High Five insect cells (Invitrogen). [ P]- radiolabelled ubiquitin from GST-ubiquitin was prepared essentially as described by Kaelin et al., (CetV 70:351-364.1992). The detection of E2-ubiquitin thioester adduct formation, and its capacity to catalyse the formation of an E6-AP-ubiquitin thioester, was performed essentially as described by Scheffner et al. (Proc.Natl Acad. Sci. USA 91, 8797-8801.1994). In brief, approximately 2 μg of radiolabelled ubiquitin, 100 ng of wheat germ El and 250 ng of E2, in the presence or absence of 1 μg of E6-AP and RingCl were incubated at 20°C for 10 min in a 100 μl reaction volume containing 25 mM Tris.HCl buffer, pH 7.6, containing 50 mM NaCl, 4 mM ATP, 10 mM MgCl₂, and 0.1 mM DTT. The reactions were equally divided into non-reducing (50 mM Tris.HCl buffer, pH6.8, containing 2% (w/v) sodium dodecyl sulphate [SDS], 4 M Urea, 10% (v/v) glycerol and 0.1% (w/v) bromophenol blue) or reducing (in which urea was replaced with 0.1M dithiothreitol [DTT]) SDS-polyacrylamide gel electrophoresis [SDS-PAGE] loading buffer and incubated at 30°C for 15 min or 100°C for 5 min respectively prior to SDS-PAGE. The reaction mixtures were resolved on 12% SDS-polyacrylamide gels at 4°C, which were then subject to autoradiography for band visualisation.

This in vitro biochemical data indicates that UbcH7 catalyses the formation of a mono-ubiquitin amide with RingCl . Furthermore, our in vitro competition studies indicate that UbcH7 interacts more stably with the Ring C protein than with, for example, E6-AP.

Results

In order to identify novel proteins, including ubiquitin-protein ligases (E3s), which interact with UbcH7, we used UbcH7 as a bait in a yeast two-hybrid screen of a human testis cDNA library. We identified a novel human protein. RingCl, which interacts specifically and at high affinity with the human ubiquitin conjugating enzyme, UbcH7. To our surprise, a Drosophila homologue, 'ariadne', displays an unexpected level of similarity to the RingCl clone, and functions in axonal pathfinding in the fruitfly. The deduced amino acid sequence of RingCl is 557 amino acid residues long. A deletion series analysis of the interaction of RingCl with UbcH7 indicated a minimum interacting domain of 127 amino acid residues. This binding domain encompasses the N-terminal RING domain (residues 186-236) and part of the IBR (in between RING) domain (residues 257-316) (1). The C-terminal RING domain plays no role in this interaction. This binding domain demonstrates no homology to the known HECT ubiquitin protein-ligase homology domain.

Recently, the first report of a human genetic disorder resulting from an abnormality of a member of the ubiquitin-dependent proteolytic pathway was described. Loss of function of the E6-AP gene, UBE3A, through deletion, truncating mutations (frameshift, nonsense and missense mutations), or as a result of imprinting anomalies at chromosome 15ql 1-13, results in Angelman syndrome (2-5). Angelman syndrome is characterised clinically by severe mental retardation, absence of speech, frequent smiling and laughter, and a high incidence of seizures. The gene is transcribed from both parental alleles in lymphocytes but only from the maternal allele in brain. Paternal non-dysjunction is therefore associated with disease. Characterization of the gene encoding RingCl has indicated that it is located telomeric of UBE3A on chromosome 15. Why the Angelman syndrome develops from the loss of E6-AP function is unclear as the endogenous cellular targets for this E3 have not been identified. E6-AP mutations may prevent interactions with UbcH7.

The RingCl protein contains two classical CX₂CX₁₃CXHX₂CX₂CX,₉CX₄C (residues 186-236) and CX₂CX₉CXHX₂CX₄CX₄CX₂C (residues 344-375) RING zinc-finger domain structures (consensus CX₂CX(_9-39)CX₍i_-3)HX_(2-3)CX₂CX_4-48CX₂ - C (6,7)) surrounding an IBR domain (residues 257-316). RING finger domains mediate both protein-DNA interactions and protein-protein interactions (6). This RTNG-IBR- RING domain structure is found in a number of other proteins located on databases (1). We propose that this domain structure will modulate interaction between these proteins and UbcH7 or other E2s and that their function is dependent upon these interactions.

There is a growing body of literature indicating that many functionally important proteins, including Radl8, BAP1, HAUSP. PML, PARKIN and Apcl l , contain RING finger domains and inter alia interact with members of the ubiquitin or ubiquitin-like pathway (8-15). Rad6 a yeast E2 forms a heterodimeric complex with Radl 8 that possesses DNA binding and ATP hydrolytic activities (8). BAP1 is a ubiquitin carboxy-terminal hydrolase that binds to the native breast/ovarian cancer susceptibility gene product BRCA1, but not to mutated germ-line mutants (9). HAUSP is a human ubiquitin hydrolase that interacts with a herpesvirus regulatory protein (10,1 1). PML protein displays tumour supressor activity and is found covalently bound to members of the sentrin family (sentrins are ubiquitin-like molecules that are also found conjugated to proteins) (12,13). Mutations to the Parkin gene have been reported to be responsible for the pathogenesis of autosomal recessive juvenile Parkinsonism (14). This gene also contains a ubiquitin-like domain at its N- terminus and a RING domain at its C-terminus. Ape 11 is a phylogenetically conserved member of the multi-protein E3 complex called the "anaphase promoting complex" (APC) (15). The E6 proteins of HPVs also contain a putative metal binding domain of structure CX₂CHX₃CXι₈CX₅CXHCX₃C (16).

A loss or mutation of RingCl would alter the degree of RingCl/ UbcH7 <→ubiquitin- protein ligase <->target protein interaction and ultimately disrupt biological functions such as axonal pathfinding and tissue regeneration. Hence human diseases in which the gene encoding the RingCl gene is mutated or malfunctional are likely to develop the same or a complex phenotype. Likewise, inappropriate changes in the levels of RingCl expression in other circumstances (eg tissue degeneration, infection, trauma) may result in CNS malfunction. The cumulative evidence suggests that RING proteins may mediate E1-E2-E3 -target protein interactions thereby representing a mechanism by which different proteins are specifically selected for degradation. Moreover, reactivation of the axonal path finding mechanisms may be essential in processes such as the recover}' from acute injuries to the nervous system, such as trauma or stroke. Although it may be difficult to induce post-mitotic, highly differentiated cells such as neurones to resume cell division to replace damaged tissue, it may be the case that function can be restored to a clinically useful extent if surviving cells can be induced to establish new axonal networks. Our observation that Ring CI is present at the tips of migrating axons suggests that therapeutic provision of this gene or its encoded protein may be a valuable treatment modality also in stroke and head injury.

References

1. Morett E. and Bork P. (1999) A novel transactivafion domain in Parkin. TIBS 24, 229-231. 2. Kishino T., Lalande M. and Wagstaff J. (1997) UBE3A/E6-AP mutations cause Angelman Syndrome. Nature Genet. 15, 70-73.

3. Matsuura T. Sutcliffe J.S., Fang P.,. Galjaard R-J., Jiang Y-h., Benton C.S., Rommens J.M. and Beaudet A.L. (1997) De Novo truncating mutations in E6- AP ubiquitin -protein ligase gene (UBE3A) in Angelman Syndrome. Nature Genet. 15, 74-77.

4. Rougeulle C, Glatt H. and Lalande M. (1997) The Angelman Syndrome candidate gene, UBE3A/E6-AP, is imprinted in Brain. Nature Genetics 17, 14- 15.

5. Burger J. Buiting K., Dittrich B., Groβ S., Lich C, Sperling K.. Horsthemke B. and Reis A. (1997) Different mechanisms and recurrence risks of imprinting defects in Angelman Syndrome. Am J. Human Genet. 61, 88-93

6. Bordin K.L.B. and Freemont P.S. (1996) The ring finger domain - a recent example of a sequence-structure family. Curr. Opin. Struct. Biol. 6, 395-401.

7. Saurin A.J., Borden K.L.B. , Boddy M.N. and Freemont P.S. (1996) Does this have a familiar ring. Trends Biochem Sci. 21, 208-214.

8. Bailly V., Lauder S., Prakash S. and Prakash L. Yeast DNA repair proteins Radό and Radl8 form a heterodimer that has ubiquitin conjugating, DNA binding and ATP hydrolytic activities. J. Biol. Chem 272, 23360-23365. (1997) 9. Jensen D.E., Procter M., Marquis S.T., Gardner H.P., Ha S.I., Chodosh L.A., Ishov A.M., Tommerup N., Vissing H., Sekido Y., Minna J., Borodovsky A., Schultz D.C., Wilkinson K.D., Maul G.G., Barlev N., Berger S.L., Prendergast G.C., Rauscher F.J. (1998) BAP1 : a novel ubiquitin hydrolase which binds the BRCA1 ring finger and enhances BRCA1 -mediated cell growth suppression. Oncogene 16, 1097-11 12. 10. Everett R.D., Meredith M., Orr A., Cross A., Kathoria M. and Parkinson J.

(1997) A novel ubiquitin-specific protease is dynamically associated with the PML nuclear domain and binds to herpesvirus regulatory protein. EMBO J. 16, 566-577. 11. Robinson P.A., Lomonte P., Leek J.P., Markham A.F. and Everett R.D (1998) Assignment of herpesvirus-associated ubiquitin-specific protease gene, HAUSP, to human chromosome band 16pl3.3 by in situ hybridization. Cytogenet. Cell Genet. In press (1999).

12. Hemenway C.S., Halligan B.W. and Lev)' L.S. (1998) The Bmi-1 oncoprotein interacts with dinG and Mph2: the role of RING finger domains. Oncogene

16, 2541-2547.

13. Kamitani T., Nguyen H.P., Kito K., Fukuda-Kamitani T. and Yeh E.T.H.

(1998) Covalent modification of PML by the sentrin family of ubiquitin-like proteins. J. Biol. Chem. 31 17-3120. 14. Kitada T., Asakawa S., Hattori N., Matsumine H., Yamamura Y., Minoshima S., Yokochi M., Mizuno Y. and Shimizu N. (1998) Mutations in the Parkin gene cause autosomal recessive juvenile Parkinsonism. Nature 392, 605-608.

15. Zachariae W., Shevchenko A., Andrews P.D., Ciosk R., Galova M., Stark M.J.R., Mann M. and Nasmyth K.(1998) Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins. Science 279, 1216-1219.

16. Huibregtse J.M., Maki C.G. and Howley P.M. (1998) Ubiquitination of the p53 Tumour suppressor. In Ubiquitin and the biology of the cell . (Eds J-M. Peters, J.R. Harris and D. Finley.) Plenum Press, New York. pp. 324-339.

Claims

1. An isolated nucleic acid encoding an E2 associated protein which acts in the role of an E3 ubiquitin ligase, the nucleic acid may be selected from the group consisting of:

(a) DNA having the nucleotide sequence given herein as SEQ ID NO:l (which encodes the protein having the amino acid sequence given herein as SEQ ID NO:2), and which encode an E2 associated ubiquitin-protein ligase. (d) nucleic acids which hybridize to DNA of (a) above (e.g., under stringent conditions) and which encode an E2 associated ubiquitin- protein ligase, and (e) nucleic acids which differ from the DNA of (a) or (b) above due to the degeneracy of the genetic code, and which encode an E2 associated ubiquitin-protein ligase encoded by a DNA of (a) or (b) above.

2. A nucleic acid that encodes at least part of an E2 associated ubiquitin-protein ligase or a nucleic acid that is complementary thereto and which hybridises under stringent conditions to the sequence presented in Figure 1 or fragments of such nucleic acid molecules.

3. A nucleic acid according to either preceding claim which hybridises under stringent conditions of washing with lxSSC, 0.1%SDS, at 65° C.

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

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

6. An isolated polypeptide or fragment or analogue or derivative thereof comprising the sequence as shown in Figure 2. or fragment or analogue or derivative thereof which has E2 associated ubiquitin ligase activity and/or is antigenic to anti- E2 associated ubiquitin ligase antibodies.

7. A deliver)' vehicle comprising the isolated nucleic acid molecule according to Claim 1.

8. A delivery vehicle according to Claim 7 wherein the vehicle is a viral vector, an adenovirus, a retrovirus, a heφesvirus, a plasmid, a phage, a phagemid or a liposome.

9. A delivery vehicle according to either Claim 7 or 8 further including a suitable formulation for the production of a suspension.

10. A delivery vehicle according to any of Claims 7-9 further wherein a surface protein of the viral vector is adapted to ensure the viral vector binds to and/or penetrates specific target tissues.

11. Use of a delivery vehicle according to any of Claims 7-19 in gene therapy treatments.

12. Antibodies raised against the polypeptide, fragment, analogue, derivative or epitope of an E2 associated protein.

13. Antibodies according to Claim 12 wherein the antibodies are monoclonal

14. Antibodies according to either Claim 12 or 13 wherein the antibodies are human or are genetically engineered to be humanised.

15. Antibodies according to any of Claims 12-14 for use in determining expression of the polypeptide fragment, analogue, derivative or epitope of an E2 associated protein in a selected target tissue.

16. A method of treatment of neurological or degenerative disorders including stroke comprising administering to a patient suffering from a neurological or degenerative disorder including stroke, the nucleic acid molecule according to Claim 1 and/or polypeptide according to Claim 6 and/or the deliver)' vehicle according to Claim 7.

17. A method of treatment according to Claim 16 and optionally further including any of the preferred features of Claims 2-5 or 8-11.

18. An isolated polypeptide or fragment or analogue or derivative thereof coded by the nucleic acid molecule of Claim 1 for use as a pharmaceutical.

19. An isolated polypeptide or fragment or analogue or derivative thereof coded by the nucleic acid molecule of Claim 1 , for use in the manufacture of a medicament, the medicament being for treating neurological or degenerative disorders including stroke.