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Definitions

• 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.
• El ubiquitin activating enzymes
• E2 or UBC ubiquitin 0 conjugating enzymes
• E3 ubiquitin-protein ligases
• 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 ⁇ -amino group of a lysine residue in a target protein.
• a ubiquitin-conjugating enzyme an E2 such as UBC4
• E3 ubiquitin ligases specifically binding to target proteins which are not otherwise recognised by E2's.
• 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 represent a family of closely related proteins common in many if not all eukaryotic cells.
• 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.
• 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).
• 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 MAT ⁇ 2 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.
• HPV human papilloma virus
• E6-AP endogenous 1 OOkDa cellular protein termed E6-AP, (for E6-associated protein).
• E6-AP endogenous 1 OOkDa cellular protein termed E6-AP
• 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.
• 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.
• AS Angelman Syndrome
• 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.
• 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.
• mutations in UBE3A are by no means the only cause of AS.
• chromosome rearrangement has resulted in deletion of the 15ql 1- 13 region of chromosome 15 which contain the UBE3A gene.
• 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.
• AS neurostyresib
• anti-epileptic drugs such as Valproate and Topiramate have been shown to be effective for seizures resulting from AS.
• Ring CI 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.
• Ring CI enzyme is of therapeutic value in instances where the endogenous protein is not produced or is not functional.
• the Ring CI protein may be of therapeutic utility as an adjunct to neuronal or tissue regeneration when delivered at sites of damage.
• 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.
• 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:
• nucleic acids which hybridize to DNA of (a) above (e.g., under stringent conditions) and which encode an E2 associated ubiquitin ligase
• 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.
• 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.
• Ring CI 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)
• a protein of the present invention e.g., a protein according to SEQ ID NO:2
• 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.
• 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.
• 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.
• hybridisation occurs under stringent conditions such as lxSSC, 0.1%SDS, at 65° C.
• the nucleic acid is of human origin.
• the isolated nucleic acid is either cDNA or genomic DNA.
• an isolated polypeptide or fragment or analogue or derivative thereof encoded by the nucleic acid molecule according to the invention is provided.
• 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.
• a delivery vehicle comprising the isolated nucleic acid molecule of the invention.
• 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.
• said delivery vehicle is adapted for administration, for example, but without limitation, by suitable formulation into a suspension.
• said delivery vehicle is adapted to deliver said nucleic acid molecule to selected tissue.
• 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.
• the nucleic acid molecule of the invention can be used in gene therapy treatments.
• antibodies raised against the polypeptide, fragment, analogue, derivative or epitope of an E2 associated protein are monoclonal and more ideally are human or are genetically engineered to be humanised.
• antibodies of the invention can be used to determine the expression of the polypeptide of the invention in selected target tissue.
• 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.
• 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.
• Figure 1 shows the cDNA of Ring CI (SEQ ID NO 1);
• Figure 2 shows the cDNA and predicted protein encoded by Ring CI (SEQ ID NO 2)
• Figure 3 shows the predicted protein encoded by Ring CI (SEQ ID NO 3).
• 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.
• E6-AP E6-associated protein
• Northern Blot I [Clontech Ltd.] was probed with a 32 P-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.
• NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 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.
• screening of a radiation hybrid mapping panel by PCR indicated a location 2.84 cR from WI-3873 on proximal chromosome 15q.
• 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 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.
• the RingCl protein contains two classical CX 2 CX 13 CXHX 2 CX 2 CX, 9 CX 4 C (residues 186-236) and CX 2 CX 9 CXHX 2 CX 4 CX 4 CX 2 C (residues 344-375)
• RING zinc-finger domain structures (consensus CX 2 CX( 9-39 )CX ( i -3) HX (2-3) CX 2 CX 4-48 CX 2 - 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.
• Radl8 BAP1
• HAUSP HAUSP
• 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).
• APC anaphase promoting complex
• the E6 proteins of HPVs also contain a putative metal binding domain of structure CX 2 CHX 3 CX ⁇ 8 CX 5 CXHCX 3 C (16).
• RingCl 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.
• human diseases in which the gene encoding the RingCl gene is mutated or malfunctional are likely to develop the same or a complex phenotype.
• inappropriate changes in the levels of RingCl expression in other circumstances eg tissue degeneration, infection, trauma
• 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.
• 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.
• acute injuries to the nervous system such as trauma or stroke.
• highly differentiated cells such as neurones to resume cell division to replace damaged tissue
• function can be restored to a clinically useful extent if surviving cells can be induced to establish new axonal networks.
• 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.

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