Contribution to Science from the Wilkinson Lab

1. Discovery of Ubiquitin's Role in Proteolysis.

In the late 1970’s Rose, Hershko, and Ciechanover (Nobel Prize, 2004) were deciphering the mechanisms regulating ATP-dependent proteolysis in cells. They had isolated and partially characterized a small protein that was covalently attached to target proteins for degradation. In the two JBC papers below I was able to show this small protein was identical to ubiquitin, first isolated by Gideon Goldstein in his search for thymopoeitin and shown by Goldknopf and Busch to be covalently attached to histone H2A. We went on to show the C-terminal GG residues were required for ubiquitin’s action, to determine the first x-ray structure of this highly conserved protein and used trypsin-catalyzed transpeptidation to synthesize C-terminal derivatives as substrates for a putative “deubiquitinating enzyme.”

    1. Wilkinson, K.D., Urban, M.K. and Haas, A.L. (1980) Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes.  J. Biol. Chem. 255, 7529-7532
    2. Wilkinson, K.D. and Audhya, T. (1981) Stimulation of ATP-dependent proteolysis requires ubiquitin with the COOH-terminal sequence Arg-Gly-Gly.  J. Biol. Chem. 256, 9235-9241.
    3. Vijay-Kumar, S., Bugg, C.E., Wilkinson, K.D., and Cook, W.J. (1985) Three-dimensional structure of ubiquitin at 2.8 A resolution. Proc. Natl. Acad. Sci. USA, 82, 3582-3585. PMC397829
    4. Wilkinson, K.D., Cox, M.J., Mayer, A.N., and Frey, T. (1986) Synthesis and characterization of ubiquitin ethyl ester, a new substrate for ubiquitin carboxyl-terminal hydrolase. Biochemistry 25, 6644-6649.

2. Cloning and Characterization of Deubiquitinating Enzymes.

Because ubiquitin was linked to both histone H2a and proteolytic targets via an isopeptide bond and it was known that ubiquitination of H2a was reversible, we reasoned that there must be proteases that hydrolyzed this isopeptide bond; Using the Ub C-terminal ester as a substrate we found several deubiquitinating enzyme (DUB) activities and cloned and characterized the first members of the Ub C-terminal Hydrolase gene family. Amazingly, this led to the identification of an extremely abundant family member, neuronal PGP9.5 that we now know as UCH-L1. We collaborated with Chris Hill to determine the first X-ray structure of a DUB and with Josh Wand to define the binding interaction between UCH-L3 and Ubiquitin. This structure revealed the importance of the “hydrophobic patch” on ubiquitin that is used in the vast majority of binding interactions.

    1. Wilkinson, K.D., Lee, K., Deshpande, S., Duerksen-Hughes, P.J., Boss, J.M., and Pohl, J. (1989) The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase. Science 246, 670-673.
    2. Mayer, A.N. and Wilkinson, K.D. (1989) Detection, resolution, and nomenclature of multiple ubiquitin carboxyl-terminal esterases from bovine calf thymus. Biochemistry  28, 166-172.
    3. Johnston, S.C., Larsen, C.N., Cook, W.J., Wilkinson, K.D., Hill, C.P. (1997) Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 A resolution. EMBO J.  16, 3787-3796. PMC1170002
    4. Wilkinson, K.D.,  Laleli-Sahin, E., Urbauer, J., Larsen, C.N., Shih, G.H., Haas, A.L., and Wand, A.J. (1999) The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3. J. Mol. Biol., 291, 1067-1077.

3. Mechanism of polyubiquitin disassembly

By the late 1980’s, Varshafsky’s group had shown that ubiquitin was synthesized by a poly-protein that must be processed to monomers and that the signal for degradation was K48-linked polyubiquitin. We next turned our attention to the DUB(s) with specificity for polyubiquitin and found USP5/UBP14 (Isopeptidase T), the enzyme responsible for disassembling the polyubiquitin signal after it had been released from substrates undergoing degradation by the proteasome. Uniquely, it trims chains from the proximal end utilizing a zinc finger domain that specifically binds the free C-terminus of a polyubiquitin chain. We identified this binding domain, solved its crystal structure bound to ubiquitin, and then mapped the binding domains for three other ubiquitins in a polyubiquitin chain,

    1. Wilkinson, K.D., Tashayev, V.L, O'Connor, L.B., Larsen, C.N., Kasperek, E. and Pickart, C.M. (1995) Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry, 34, 14535-14546.
    2. Amerik, A.Y., Swaminathan, S., Krantz, B.A., Wilkinson, K.D., and Hochstrasser, M. (1997) In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome. EMBO J. 16, 4826-4838. PMC1170118
    3. Reyes, F. E., Horton, J. R., Mullally, J.E, Heroux, A., Cheng, X., and Wilkinson, K. D. (2006) The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell, 124, 1197-208.
    4. Reyes-Turcu, F.E., Shanks, J.R., Komander, D., Wilkinson, K.D. (2008) Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T. J Biol Chem. 283, 19581-92. PMC2443676

4. Recognition and specific binding of Polyubiquitin

Our definition of the binding domains of USP5 recognizing different subunits of a polyubiquitin chain led us to examine the specificity of a number of ubiquitin receptors.  Our long-time collaborator, David Komander, has extended this work to give a very rigorous and sophisticated understanding of how differently linked polyubiquitin chains are recognized.

    1. Reyes-Turcu, F.E. and Wilkinson, K.D. (2009) Polyubiquitin binding and disassembly by deubiquitinating enzymes. Chemical Reviews, 109, 1495-508. PMC2734106
    2. Komander, D., Reyes-Turcu, F.E., Odenwaelder, P., Wilkinson, K.D. and Barford, D. (2009) Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Reports, 10, 466-73. PMC2680876
    3. Fushman, D., Wilkinson, K.D. (2011) Structure and recognition of polyubiquitin chains of different lengths and linkage. F1000 Biol Rep.;3:26. PMID: 22162729; PMC3229271.
    4. Ye, Y., Akutsu, M., Reyes-Turcu, F., Enchev, R.I., Wilkinson, K.D., Komander, D. (2011) Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21. EMBO Rep. 12, 350-7. PMID: 21399617; PMC3077245.

5. C-terminal Derivatives of Ubiquitin as Substrates and Active Site Probes

In collaboration with Hidde Ploegh’s group we first used trypsin catalyzed transpeptidation (developed above) to synthesize an active site directed irreversible inhibitor of DUBs, ubiquitin vinyl sulfone. In a refinement of this technology we then applied the intein coupling method we had developed to look at C-terminal derivatives of other ubiquitin-like proteins. The intein method is a powerful approach that has been used many times to synthesize C-terminal derivatives of ubiquitin and ubiquitin-like proteins. It is now the preferred method used in commercial production of these reagents.

    1. Borodovsky, A., Kessler, B.M., Casgrande, R., Overkleeft, H.S., Wilkinson, K.D. and Ploegh, H.L. (2001) A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187-5196. PMC125629
    2. Borodovsky, A., Ovaa, H., Kolli, N., Gan-Erdene, T., Wilkinson, K.D., Ploegh, H.L., and Kessler, B.M. (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem Biol. 9, 1149-59.
    3. Hemelaar,  J., Borodovsky,  A., Kessler, B.M., Reverter , D., Cook, J., Kolli, N., Gan-Erdene, T., Wilkinson, K.D., Gill, G., Lima, C.D., Ploegh, H.L., Ovaa, H. (2004) Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol Cell Biol. 24, 84-95. PMC303361
    4. Wilkinson, K. D., Gan-Erdene, T. and Kolli, N. (2005) Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses. Methods Enzymol., 399, 37-51.

6. Role of BAP1 as a Tumor Suppressor

In the process of describing the human genes for UCH family DUBs we cloned and characterized the fourth member of this papain-like superfamiy, BRCA1-associated Protein 1 (BAP1), so named because it bound to the N-terminal RING domain of BRCA1. In the 1998 paper we, showed it was an active UCH, that cancer associated mutations had lost activity and that there was some genetic interactions that suggested it was a tumor suppressor. Ten years later no one had characterized its role in tumor suppression so we re-expressed Bap1 in NCI-H226, a mesothelioma that was null for BAP1, and found it killed the cells. We wenton to show it was an authentic tumor suppressor, required enzymatic DUB activity and nuclear localization. Subsequent work by many other groups has shown BAP1 regulates the G1-S checkpoint and its mutation is associated with uveal melanoma, mesothelioma, renal clear cell carcinoma, and atypical melanomas.

    1. Jensen, D.E., Proctor, M., Marquis, S.T., Perry, H., Gardner, 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., and Rauscher, III., F.J.  (1998) BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression. Oncogene 16, 1097-1112.
    2. Ventii, K.H., Devi, N.S., Friedrich, K.L., Chernova, T.A., Van Meir, E.G., and Wilkinson K.D. (2008) BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization. Cancer Research, 68, 6953-62. PMC2736608
    3. Eletr, Z.M., Wilkinson, K.D. (2011) An emerging model for BAP1's role in regulating cell cycle progression. Cell Biochem. Biophys. 60, 3-11. PMID: 21484256; PMC3128820.
    4. Eletr, Z.M., Wilkinson, K.D. (2013) BAP1 is phosphorylated at serine 592 in S-phase following DNA damage..FEBS Lett. 587(24):3906-11. PubMed PMID: 24211834. 

7. Ubiquitin pathway in neurological disease.

Intrigued by the fact that UCH-L1 (PGP9.5) was very abundant in brain, we began to examine histochemical sections and soon realized it was often concentrated in lesions associated with protein deposits of Parkinson’s, Alzheimer’s and Huntingtins diseases. A great number of investigators have since shown it is an important DUB whose loss leads to profound effects on neurological structure and function. Specifically, it is tightly associated with the cells attempt to deal with the consequences of amyloid and polyglutamine deposits, probably via the ubiquitin proteasome system (UPS) and/or autophagy.

    1. Lowe, J., McDermott, H., Landon, M., Mayer, R.J. and Wilkinson, K.D. (1990) Ubiquitin carboxyl-terminal hydrolase (PGP 9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. J. Pathology, 161, 153-160.
    2. Leroy, E., Boyer, B., Auburger, G., Leube, B., Ulm, G., Mezey, E., Harta, G., Brownstein, M.J., Jonnalagadda, S., Chernova, T., Dehejia, A., Lavedan, C., Gasser, T., Steinbach, P., Wilkinson, K.D. and Polymeropoulos, M.H. (1998) The ubiquitin pathway in Parkinson's disease. Nature, 395, 451-2.
    3. Olzmann, J.A., Brown, K., Wilkinson, K.D., Rees, H.D., Huai, Q., Ke, H., Levey, A.I., Li, L., and Chin, L.S. (2004) Familial Parkinson's disease-associated L166P mutation disrupts DJ-1 protein folding and function. J Biol Chem. 279, 8506-8515.
    4. Kaytor, M.D., Wilkinson, K. D., and Warren, S. T. (2004) Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity. J Neurochem. 89, 962-73.

8. Yeast Prions as models of polyglutamine diseases

In an attempt to find a simpler system with which to study the role of UPS in dealing with the consequences of amyloid or polyglutamine inclusions we recently turned to yeast prions, self propagating amyloids that usually involve a polyglutamine containing prion domain. We have found that disturbing protein homeostasis via the UPS we can modulate the formation and the propagation of these prions. We noted that overexpression of Lsb2, a ubiquitin and actin binding protein, enhanced prionogenesis.Lsb2 is overexpressed during heat shock, accumulates in cytoskeletal patches at localized high concentrations and appears to recruit other Q-rich proteins that can then form prions. This may be a generalized stress response that triggers rather non-selective prion formation. This would provide a source of non-mendellian variation that could result in an adaptive advantage during stress.

    1. Allen, K. D, Wegrzyn, R. D., Chernova, T. A., Muller, S., Newnam, G. P., Winslett, P. A., Wittich, K. B., Wilkinson, K. D., Chernoff, Y. O. (2005) Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+]. Genetics. 169, 1227-42. PMC1449557
    2. Allen, K.D., Chernova, T.A., Tennant, E.P., Wilkinson, K.D., Chernoff, Y.O. (2007) Effects of ubiquitin system alterations on the formation and loss of a yeast prion. J. Biol. Chem., 282, 3004-13.
    3. Chernova, T.A., Romanyuk, A.V., Karpova, T.S., Shanks, J.R., Ali, M., Moffatt, N., Howie, R.L., O'Dell, A., McNally, J.G., Liebman, S.W., Chernoff, Y.O., Wilkinson, K.D. (2011) Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton. Mol Cell. 43, 242-52. PMID: 21777813; PMC3151368
    4. Ali, M., Chernova, T.A., Newnam, G.P., Yin, L., Shanks, J., Karpova, T.S., Lee, A,. Laur, O., Subramanian, S., Kim, D., McNally, J.G., Seyfried, N.T., .Chernoff ,Y.O., Wilkinson, K.D. Stress-dependent proteolytic processing of the actin assembly protein Lsb1 modulates a yeast prion. J Biol Chem. 2014 Oct 3;289(40):27625-39.