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WO1998028436A1 - Detection de proteines - Google Patents

Detection de proteines Download PDF

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Publication number
WO1998028436A1
WO1998028436A1 PCT/GB1997/003468 GB9703468W WO9828436A1 WO 1998028436 A1 WO1998028436 A1 WO 1998028436A1 GB 9703468 W GB9703468 W GB 9703468W WO 9828436 A1 WO9828436 A1 WO 9828436A1
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WIPO (PCT)
Prior art keywords
protein
assay
ubiquitin
muscle
carrier
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PCT/GB1997/003468
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English (en)
Inventor
Brian Roy Holloway
Ian David Waddell
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Zeneca Limited
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Priority to JP52851898A priority Critical patent/JP2001506862A/ja
Priority to EP97950259A priority patent/EP0946745A1/fr
Publication of WO1998028436A1 publication Critical patent/WO1998028436A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates to assays suitable for screening inhibitors of muscle protein degradation, in particular to assays for screening inhibitors of proteins involved in ubiquitin-dependent proteolysis in eukaryotic cells. Such inhibitors may have utility in the treatment of disease states associated with proteolysis.
  • Eukaryotic cells contain three major proteolytic pathways namely the lysosomal, the calcium dependent and the ATP dependent pathways.
  • the lysosomal pathway mainly degrades extracellular proteins which have entered the cells via receptor mediated endocytosis or pinocytosis.
  • the calcium dependent proteases (the calpains) at present have no clear physiological role.
  • the ATP dependent pathway has long been known to play a role in the degradation of misfolded or mutant proteins, however it has only recently become clear that it selectively degrades intact cellular proteins, and thereby fulfills important roles in cellular regulation.
  • the ATP pathway is shown schematically in Fig. 1 which is explained as follows.
  • the protease activity for the ATP dependent pathway lies in a large multi-catalytic complex known as 26S proteosome. This consists of three multi-subunit complexes, namely a 700kDa 20S proteosome core particle and two 19S cap structures (reviewed by Peters J.M., Trends Biochem. Sci. (1994), 19, 377-382). Proteins destined for 26S proteosome degradation are usually modified by addition of ubiquitin (a 76 amino acid peptide) to specific lysine residues.
  • ubiquitin a 76 amino acid peptide
  • This ubiquitinylation reaction requires three classes of proteins called El, E2 and E3 (reviewed by Ciechanover A., Biol.Chem.Hoppe Seyler (1994), 375, 565-581).
  • El proteins catalyze the activation of ubiquitin to produce a thiol intermediate.
  • the E2 ubiquitin-carrier (or ubiquitin-conjugating) proteins transfer ubiquitin from El proteins to the E3 ubiquitin- protein ligase.
  • the E3 ligase catalyzes the formation of an isopeptide bond between the ubiquitin polypeptide and the protein substrate (although some E2 proteins can directly ubiquitinylate protein substrates).
  • Protein-bound ubiquitin may itself be ubiquitinylated, resulting in the formation of large protein-ubiquitin conjugates which are targets for destruction by the 26S proteosome (although such poly-ubiquitinylation is not essential for protein destruction to occur).
  • proteolysis is now emerging as an important mechanism of cellular regulation. For instance, it is needed by cells to adapt to a changing environment, as well as for the control of time-dependent cellular programs. Proteolysis offers advantages over other possible control mechanisms because it is fast, and thereby enables the cell to rapidly reduce the level of a defined protein. It is also irreversible, so ensuring the complete loss of function of the degraded protein. However, the danger for the cell of unspecific degradation of proteins means that proteolysis has to be very selective.
  • Ubiquitin-dependent proteolysis may also be implicated in the down regulation of signal transducing receptors.
  • the involvement of the ubiquitin conjugation system in the ligand-induced endocytosis and degradation of the growth hormone receptor may be of relevance in cachetic situations.
  • the yeast gene RAD6 encodes for E2 which appears essential for the repair of DNA caused by ultra-violet induced damage.
  • the RAD6 yeast enzymes are homologous to the rabbit E2i4k and the human ubiquitin-carrier E2i7k proteins (Schneider R., Eckerskorn C, Lottspeich F. & Schweiger, EMBO J. (1990), 9, 1431-1435; Wing S., Dumas F. & Banville D., J. Biol. Chem. (1992), 267, 6495-6501).
  • the mRNA encoding the rat homologue of human E2i7k protein has been shown to be raised 3-fold in atrophying muscles obtained from starved (Wing S. & Banville D., Am. J. Physiol. (1994), 267, E39-E48) and hindlimb suspension (Taillandier D et al, Biochem. J. (1996), 316, 65-72) rat models of cachexia.
  • starved Wing S. & Banville D., Am. J. Physiol. (1994), 267, E39-E48
  • hindlimb suspension Talandier D et al, Biochem. J. (1996), 316, 65-72
  • no work to date has shown whether such raised levels occur specifically in certain tissue types, or are more generally raised throughout many tissue types during muscle wasting.
  • a method or assay which will rapidly and conveniently determine inhibitors of ATP ubiquitin-dependent proteolysis will have utility in research and development, particularly in the development of pharmaceutical substances and compositions for the treatment of diseases such as cachexia. More particularly, a valuable assay would be able to identify those inhibitors which are specifically able to prevent or reduce only muscle protein degradation.
  • the plurality of E2 proteins in particular, and their expression in a range of tissue types makes difficult the development of an assay which is largely specific for inhibitors of muscle protein degradation. It will be appreciated that although an assay may highlight inhibitors which prevent muscle protein degradation, if the inhibitors are unspecific they may also interfere with protein regulation in other tissues.
  • the ATP ubiquitin-dependent proteolysis pathway is complex, and a suitable method or assay based on this pathway must overcome a number of problems before rapid and convenient screening for specific inhibitors of muscle protein degradation is possible. It is the aim of the present invention to deliver a practical method and assay which can identify inhibitors which are largely specific for muscle protein degradation.
  • inhibitors which are identified using our method or assay will be inhibitors of the human ubiquitin- carrier El protein, or of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle which is used in the method or assay.
  • the presence of a target protein in the method and assay of the invention provides for convenient measuring of the extent of ubiquitin transfer.
  • the present invention provides a method for specifically identifying inhibitors of muscle protein degradation, comprising contacting the inhibitor, in the absence of an E3 protein, with (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and then determining the extent of inhibition of ubiquitin transfer to a target protein.
  • ATP is necessary to initiate the ubiquitinylation reaction shown in Fig. 2.
  • a human ubiquitin-carrier E2 protein we mean, hereinabove in this disclosure and hereinafter, one with levels raised above those present in non-muscle cells and tissue.
  • Reference Example 2 illustrates examples of raised levels compared to a number of different non-muscle tissues. It can be seen, for example, in Reference Example 2 that levels in skeletal and/or cardiac muscle are raised by approximately two to three-fold compared to pancreas tissue, and by three to six-fold compared to placental tissue.
  • the present invention also provides a method for specifically identifying inhibitors of muscle protein degradation, as described hereinafter for the assay of the invention.
  • the present invention also provides an assay for specifically identifying inhibitors of muscle protein degradation, comprising (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein.
  • the present invention also provides an assay for specifically identifying inhibitors of muscle protein degradation, comprising, in the absence of an E3 protein, (i) ubiquitin, (ii) a human ubiquitin-carrier El protein and (iii) a human ubiquitin-carrier E2 protein, which E2 protein is able to directly ubiquitinylate protein substrates and is expressed in skeletal and/or cardiac muscle; and means for determining the extent of inhibition of ubiquitin transfer to a target protein.
  • rat models of cachexia show raised levels of mRNA's coding for the rat homologue of human E2i7k. We have confirmed this result in the denervated model of muscle atrophy - see Reference Example 1.
  • the human E2i7k ubiquitin-carrier protein is able to directly ubiquitinylate protein substrates. We have utilised this fact in the method and assay specifically described in our invention. It will be appreciated that the method and assay described specifically in the present invention can also be used with E2 proteins other than E2i7k which can directly ubiquitinylate protein substrates.
  • the present invention preferably provides a method for specifically identifying inhibitors of muscle protein degradation, as described above and hereinafter, wherein the human ubiquitin-carrier E2 protein is E2i7k .
  • the present invention also preferably provides an assay for specifically identifying inhibitors of muscle protein degradation, as described above and hereinafter, wherein the human ubiquitin-carrier E2 protein is E2i7k .
  • the present invention also provides an assay, as described above and hereinafter, for use in the method of the invention.
  • the present invention also provides an assay, as described above and hereinafter, for use with an inhibitor of human ubiquitin-carrier El protein, and/or for use with an inhibitor of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle that is used in the assay.
  • Inhibitors identified using the method and assay of the present invention are provided as a further feature. Such inhibitors may act on one, or a number, of the components in the ubiquitinylation reaction of Fig.2, and be used as such.
  • the present invention also provides an assay, as described above and hereinafter, together with an inhibitor of human ubiquitin-carrier El protein, and/or together with an inhibitor of the specific human E2 ubiquitin-carrier protein expressed in skeletal and/or cardiac muscle that is used in the assay.
  • the means for determining the extent of inhibition of ubiquitin transfer to a target protein is determined by a scintillation proximity assay (SPA), by an enzyme linked immuno- sorbent assay (ELISA) or by a radio-immuno assay (RIA).
  • SPA scintillation proximity assay
  • ELISA enzyme linked immuno- sorbent assay
  • RIA radio-immuno assay
  • the target protein used is histone 2A, biotinylated histone 2A, troponin T or ⁇ actin.
  • the target protein used is histone 2A.
  • the target protein used is biotinylated histone 2A.
  • the El and E2 proteins used are obtained by recombinant means.
  • a gel-based version of the assay described in the present invention it is possible to determine whether inhibitors specifically block the El and/or the E2 protein.
  • the gel-based assay thus allows identification of the stage in the ubiquitinylation reaction of Fig.2 at which the inhibitor is acting. Therefore, in a yet further aspect of the invention a gel-based version of the method and assay is used. Such an assay is run using standard reagents and conditions.
  • An attractive stage at which to inhibit the ubiquitinylation reaction of Fig.2 is at the stage of transfer of ubiquitin to the E2 protein, or at the stage of transfer of ubiquitin to the target protein.
  • the latter stage is particularly attractive for inhibition as an inhibitor acting at this stage is most likely to be substrate and/or enzyme specific.
  • the presence of a target protein in our assay allows such inhibitors to be identified.
  • Human skeletal muscle cDNA clones encoding both the human El and E2i7k 5 ubiquitin-carrier proteins were obtained by Polymerase Chain Reaction (PCR) of human skeletal muscle polyA" mRNA (Clontech. Palo Alto USA). These were expressed from the in- house vector pTB375NBSE (modified to maximise yield by incorporating tetracycline, rather than ampicillin resistance) in BL21 cells, in such a way as to produce recombinant protein containing a 6-histidine tag immediately adjacent to the N-terminal methionine.
  • PCR Polymerase Chain Reaction
  • pTB375NBSE vector 10 commercial alternative to the pTB375NBSE vector is pET21a, available from Novagen, R&D Systems Europe Ltd., UK (Cat. No. 69762-1).
  • BL21 cells are also commercially available from Novagen (Cat. No. 69443-1).
  • the 6-His tag was used to aid purification of the recombinant protein using a nickel chelating column.
  • the El protein was obtained by a slow- growing process at 20°C as originally described by La Vallie et al (Bio/Technology 1993, 11,
  • the E2 protein was obtained by conventional growing techniques at 37°C with 0.4mM IPTG. Further details are provided below in the section marked "Examples”.
  • the ELISA technique was modified such that it too could be used as an alternative high throughput screen or as a secondary screen in place of SPA.
  • Target proteins were "fixed" to the bottom of a 96-well ELISA plate in the presence of PEI (polyethylenimine).
  • PEI polyethylenimine
  • Ubiquitinylated target protein was detected using a horse radish peroxidase linked polyclonal antibody to poly-ubiquitin. Horseradish peroxidase was detected using either ATBS or ECL (enhanced chemiluminesence) detection system.
  • the SPA or ELISA assays are adapted to a radio-immuno assay system.
  • E2i7k antibodies or fragments thereof Purified recombinant 6His-tagged E2l7 protein was used in conjunction with Freund's adjuvant to produce antibodies in New Zealand white rabbits.
  • the anti-sera ie. E2i7k antibodies or fragments thereof was used to decrease the inappropriately enhanced degradation of normal proteins which occurs in certain pathological conditions including cachexia.
  • J Bluescript II vectors A recombinant cloning vector system similar to that employed by Yanisch-perron C. et al (Gene (1985), 33, 109-119) comprising a colEI-based replicon bearing a polylinker DNA fragment containing multiple unique restriction sites, flanked by bacteriophage T3 and T7 promoter sequences; a filamentous phage origin of replication and an ampicillin drug resistance marker gene. 2 Hvbond-N + (TM)
  • a supported nylon-66 membrane with a pore size of 0.45 microns used for the immobilisation of nucleic acids by either UN. cross linking or oven baking and supplied by
  • Poly A + mR ⁇ A was prepared directly from ⁇ lxl 0 8 C2C12 cells using a FastTrack mR ⁇ A isolation kit (Invitrogen).
  • Rat tissue total mR ⁇ A was prepared by polytron homogenisation in 4M guanidine isothiocyanate, 2.5mM citrate, 0.5% Sarkosyl (SDS), lOOmM ⁇ -mercaptoethanol, followed by centrifugation through 5.7M CsCl, 25mM sodium acetate at 135,000g (max).
  • Rat poly-A + was obtained using a FastTrack mR ⁇ A isolation kit
  • E coli transformations were generally carried out by electroporation. 400 ml cultures of strains DH5 ⁇ or BL21(DE3) were grown in L-broth to an OD 600 of 0.5 and harvested at 2,000g. The cells were washed twice in ice-cold deionised water, resuspended in 1ml 10% glycerol and stored in aliquots at -70°C. Ligation mixes were desalted using millipore V series membranes (0.0025mm) pore size).
  • RNA samples were analysed by electrophoresis on 1% denaturing formaldehyde agarose gels in MOPS buffer (Sambrook J., Fritsch E.F. & Maniatis T. in "Molecular Cloning, A laboratory manual", 2 nd Ed. (1989), Cold Spring Harbour Laboratory Press) and transferred onto hybond N * (Amersham). Probes were labelled with 2 P by random hexamer priming, and hybridisations were carried out in 0.28M sodium phosphate (pH 7.2), 5xDenhardt's solution, 10% dextran sulphate. 0.1% SDS at 65°C.
  • Membranes were washed to a final stringency of 0.2xSSC,0.1% SDS at 65°C. To control for loading variations, blots were stripped after autoradiography by boiling in 0.1%SDS, and then rehybridised using a probe containing 1.2kb of a rat glyceraldehyde-3 -phosphate dehydrogenase cDNA (GAPDH) (Fort P., Marty L., Pieechaczyk M., El Sabrouty S., Jeanteur P. & Blanchard J.M., Nucleic Acid Res. ( 1985), 13, 1431-1442).
  • GPDH rat glyceraldehyde-3 -phosphate dehydrogenase cDNA
  • RNA's from human tissues was carried out using a panel of commercially available pre-blotted RNAs (Clontech Laboratories, Palo Alto, Ca USA). 8 Ubiquitin Conjugation Assay Conjugation assays were performed at 37°C for 2 hours.
  • the reaction mixtures contained 50mM Tris-HCl (pH 7.5), 2mM ATP, 5mM MgCl 2 , 0.5mM DTT, recombinant El, recombinant E2, 7k , l ⁇ g 125 I-Ubiquitin (Amersham) and 2 ⁇ g Histone (Sigma).
  • reaction products were resolved by SDS-PAGE (12%) acrylamide gels, BIO-RAD) and, after drying the gel, visualised by autoradiography.
  • Histone2A was biotinylated using a kit purchased from Boehringer Mannheim (cat. no. 1418165) and was used according to the manufacturer's instructions. In summary, free amino groups of the target protein (histone2A in this case) were reacted with D-biotinyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (biotin-7-NHS) by forming a stable amide bond. Non-reacted biotin-7-NHS was separated on a Sephadex G-25 column. The molar concentrations used were 4mg Histone2A in 1ml of phosphate buffered saline to which was added 20mg/ml biotin-7-NHS. The incubation was carried out at ambient temperature for 2 hours with gentle shaking. K) Scintillation proximity assay (SPA)
  • Conjugation assays were performed at room temperature for 2 hours.
  • the reaction mixtures contained 50mM Tris-HCl (pH 7.5), 2mM ATP, 5mM MgCl 2 , 0.5mM DTT, recombinant El. recombinant E2, 7k , l ⁇ g l25 I-Ubiquitin (Amersham) and 2 ⁇ g of biotinylated Histone (Sigma) to give a final volume of lOO ⁇ l.
  • reactions were terminated by addition of lOmMEDTA and O.lmg/well avidin linked SPA beads (Amersham Int)
  • Lane 1 is inervated rat soleus total RNA (2 ⁇ g),
  • Lane 2 is denervated rat soleus total RNA (2 ⁇ g)
  • Lane 3 is denervated rat soleus total (2 ⁇ g) + lmg/kg Clenbuterol and
  • Lane 4 is C2C12 cell control mRNA (2 ⁇ g ).
  • Series 1 is the mean of 4 determinations and Series 2 demonstrates the standard deviation in the measurements.
  • Reference Example 2 Northern blotting of E2 abundance levels in different tissue types.
  • Northern blotting was carried out as described above using full length E2, 7k labelled by random priming.
  • the human multiple (healthy) tissue blot was obtained from Clontech and contained 2 ⁇ g of polyA + mRNA from each of the following tissues:- Lane 1 pancreas, Lane 2 kidney, Lane 3 skeletal muscle, Lane 4 liver, Lane 5 lung, Lane 6 placenta, Lane 7 brain and Lane 8 heart.
  • Fig.5 shows (prepared in a similar manner to that described for Reference Example 1 , and again showing the standard deviation in the measurements).
  • a dose-response curve was produced by measuring the (SPA) count at different concentrations of the Me-ubiquitin inhibitor to give an IC 50 (the concentration necessary to produce 50% inhibition of the uninhibited signal) of 3.3xlO "6 M (based on 100% chemical methylation of ubiquitin).

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Abstract

L'invention a pour objet un procédé et un dosage (y compris un dosage sur gel) pour identifier, de manière spécifique, les inhibiteurs de la dégradation des protéines musculaires. Ce procédé consiste (i) à utiliser de l'ubiquitine, (ii) une protéine E1 porteuse d'ubiquitine de l'homme et (iii) une protéine E2 porteuse d'ubiquitine de l'homme. Cette dernière protéine permet d'ubiquitinyler directement les substrats protéiques et est exprimée dans le musque squelettique et/ou cardiaque (par exemple E217k). L'invention a aussi pour objet des moyens pour déterminer l'ampleur de l'inhibition du transfert de l'ubiquitine (par exemple, détection de proximité par scintillation) vers une protéine cible (par exemple, histone biotinylée 2A).
PCT/GB1997/003468 1996-12-20 1997-12-16 Detection de proteines WO1998028436A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP52851898A JP2001506862A (ja) 1996-12-20 1997-12-16 タンパク質分解阻害物質のスクリーニングアッセイ
EP97950259A EP0946745A1 (fr) 1996-12-20 1997-12-16 Detection de proteines

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GBGB9626603.6A GB9626603D0 (en) 1996-12-20 1996-12-20 Protein assay
GB9626603.6 1996-12-20

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0654530A2 (fr) * 1993-06-21 1995-05-24 Rappaport Family Institute For Research In The Medical Science L'enzyme E2-F1, porteur d'ubiquitine, sa purification, sa production, et son utilisation
WO1995018974A2 (fr) * 1994-01-04 1995-07-13 Mitotix, Inc. Enzymes conjuguant l'ubiquitine
WO1996033286A1 (fr) * 1995-04-20 1996-10-24 President And Fellows Of Harvard College Methode et reactifs permettant de detecter la degradation par l'ubiquitine de proteines regulatrices du cycle cellulaire
WO1997037027A1 (fr) * 1996-04-01 1997-10-09 President And Fellows Of Harvard College Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0654530A2 (fr) * 1993-06-21 1995-05-24 Rappaport Family Institute For Research In The Medical Science L'enzyme E2-F1, porteur d'ubiquitine, sa purification, sa production, et son utilisation
WO1995018974A2 (fr) * 1994-01-04 1995-07-13 Mitotix, Inc. Enzymes conjuguant l'ubiquitine
WO1996033286A1 (fr) * 1995-04-20 1996-10-24 President And Fellows Of Harvard College Methode et reactifs permettant de detecter la degradation par l'ubiquitine de proteines regulatrices du cycle cellulaire
WO1997037027A1 (fr) * 1996-04-01 1997-10-09 President And Fellows Of Harvard College Nouveaux polypeptides porteurs d'ubiquitine et selectionnant les cyclines

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COSTELLI P. ET AL: "Muscle Protein Waste In Tumor Bearing Rats is Effectively Antagonised by a beta-2 Adrenergic Agonist", J. CLIN. INVEST., vol. 95, 1995, pages 2367 - 2372, XP002059174 *
PALLARES-TRUJILLO J. ET AL: "The Ubiquitin System: A Role in Disease?", MEDICINAL RESEARCH REVIEWS, vol. 17, no. 2, March 1997 (1997-03-01), pages 139 - 161, XP002059175 *
TAWA N.E. ET AL: "Inhibitors of the Proteosome Reduce the Accellerated Proteolysis in Atropying Rat Skeletal Muscles", J. CLIN. INVEST., vol. 100, no. 1, July 1997 (1997-07-01), pages 197 - 203, XP002059173 *

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EP0946745A1 (fr) 1999-10-06
GB2320570B (en) 1999-01-27
GB9626603D0 (en) 1997-02-05
GB9726589D0 (en) 1998-02-18
JP2001506862A (ja) 2001-05-29

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