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Quantitative Cell-based Protein Degradation Assays to Identify and Classify Drugs That Target the Ubiquitin-Proteasome System*

  • Tsui-Fen Chou
    Correspondence
    Supported by a 2008 Fellows Grant Program Award from the Multiple Myeloma Research Foundation, the Howard Hughes Medical Institute, and the Weston Havens Foundation. To whom correspondence may be addressed.
    Affiliations
    From the Division of Biology, Pasadena, California 91125
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  • Raymond J. Deshaies
    Correspondence
    Howard Hughes Medical Institute Investigator. To whom correspondence may be addressed.
    Affiliations
    From the Division of Biology, Pasadena, California 91125

    Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R03 MH085687. This work was also supported by the Howard Hughes Medical Institute.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental “Methods,” Figs. 1–6, Tables 1–3, and additional references.
Open AccessPublished:February 22, 2011DOI:https://doi.org/10.1074/jbc.M110.215319
      We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2VHL substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2VHL or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC50 = 1.7 μm) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system.

      Introduction

      The ubiquitin-proteasome system (UPS)
      The abbreviations used are: UPS, ubiquitin-proteasome system; ODC, ornithine decarboxylase; ERAD, endoplasmic reticulum-associated degradation; CHX, cycloheximide; Luc, luciferase; DMSO, dimethyl sulfoxide; UFD, ubiquitin fusion degradation; EerI, Eeyarestatin I; HTS, high-throughput screening; TCR, T-cell receptor.
      comprises one of the most important mechanisms for post-translational regulation of protein function in eukaryotic cells. The UPS comprises hundreds of enzymes that promote covalent attachment of ubiquitin and UBL (ubiquitin-like) proteins to target proteins, as well as enzymes that reverse the modification. Conjugation of ubiquitin to target proteins is a multistep process (
      • Weissman A.M.
      ,
      • Finley D.
      ,
      • Schrader E.K.
      • Harstad K.G.
      • Matouschek A.
      ,
      • Deshaies R.J.
      • Joazeiro C.A.
      ). First, ubiquitin is primed for transfer by the ubiquitin-activating enzyme (E1), to which it becomes attached via a thioester bond between the C terminus of ubiquitin and the active site cysteine of E1. Next, the activated ubiquitin is transferred from E1 to the active site cysteine of a ubiquitin-conjugating enzyme (E2). Finally, the E2∼ubiquitin thioester binds a ubiquitin ligase (E3), and the ubiquitin is transferred to the side chain amino group of a lysine residue on a substrate protein that is simultaneously bound to the same E3. Either this reaction can either terminate, or additional ubiquitin transfers can occur to ubiquitin itself, resulting in formation of a chain of ubiquitins attached to the substrate. Attachment of ubiquitin or UBL proteins to a target can yield diverse outcomes, including a change in the activity, localization, binding partners, or stability of the target. The most intensively studied consequence of ubiquitination is protein degradation. Polymerization of a chain linked together via Lys-48 of ubiquitin typically results in rapid degradation of the modified target by the 26 S proteasome. The proteasome binds proteins bearing Lys-48-linked ubiquitin chains and degrades the modified protein while recycling the ubiquitin for future use. The proteasome also degrades a handful of proteins, including ornithine decarboxylase (ODC), in a ubiquitin-independent manner (
      • Zhang M.
      • Pickart C.M.
      • Coffino P.
      ). Given the importance of the UPS to regulatory biology, there has been considerable interest in developing small molecule inhibitors as potential therapies for a range of human diseases. The UPS has been validated as an important target in cancer by clinical use of the proteasome inhibitor bortezomib (Velcade) for the treatment of multiple myeloma and mantle cell lymphoma (
      • Kane R.C.
      • Bross P.F.
      • Farrell A.T.
      • Pazdur R.
      ,
      • Colson K.
      • Doss D.S.
      • Swift R.
      • Tariman J.
      • Thomas T.E.
      ). The success of bortezomib has inspired interest in developing other UPS-directed drugs (
      • Eldridge A.G.
      • O'Brien T.
      ) that have greater efficacy but fewer side effects.
      The AAA (ATPase associated with diverse cellular activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (
      • Giaever G.
      • Chu A.M.
      • Ni L.
      • Connelly C.
      • Riles L.
      • Véronneau S.
      • Dow S.
      • Lucau-Danila A.
      • Anderson K.
      • André B.
      • Arkin A.P.
      • Astromoff A.
      • El-Bakkoury M.
      • Bangham R.
      • Benito R.
      • Brachat S.
      • Campanaro S.
      • Curtiss M.
      • Davis K.
      • Deutschbauer A.
      • Entian K.D.
      • Flaherty P.
      • Foury F.
      • Garfinkel D.J.
      • Gerstein M.
      • Gotte D.
      • Güldener U.
      • Hegemann J.H.
      • Hempel S.
      • Herman Z.
      • Jaramillo D.F.
      • Kelly D.E.
      • Kelly S.L.
      • Kötter P.
      • LaBonte D.
      • Lamb D.C.
      • Lan N.
      • Liang H.
      • Liao H.
      • Liu L.
      • Luo C.
      • Lussier M.
      • Mao R.
      • Menard P.
      • Ooi S.L.
      • Revuelta J.L.
      • Roberts C.J.
      • Rose M.
      • Ross-Macdonald P.
      • Scherens B.
      • Schimmack G.
      • Shafer B.
      • Shoemaker D.D.
      • Sookhai-Mahadeo S.
      • Storms R.K.
      • Strathern J.N.
      • Valle G.
      • Voet M.
      • Volckaert G.
      • Wang C.Y.
      • Ward T.R.
      • Wilhelmy J.
      • Winzeler E.A.
      • Yang Y.
      • Yen G.
      • Youngman E.
      • Yu K.
      • Bussey H.
      • Boeke J.D.
      • Snyder M.
      • Philippsen P.
      • Davis R.W.
      • Johnston M.
      ) and mice (
      • Müller J.M.
      • Deinhardt K.
      • Rosewell I.
      • Warren G.
      • Shima D.T.
      ). p97 is overexpressed in several cancers, supporting the idea that it could be a target of general importance in oncology (
      • Yamamoto S.
      • Tomita Y.
      • Hoshida Y.
      • Iizuka N.
      • Kidogami S.
      • Miyata H.
      • Takiguchi S.
      • Fujiwara Y.
      • Yasuda T.
      • Yano M.
      • Nakamori S.
      • Sakon M.
      • Monden M.
      • Aozasa K.
      ,
      • Yamamoto S.
      • Tomita Y.
      • Nakamori S.
      • Hoshida Y.
      • Nagano H.
      • Dono K.
      • Umeshita K.
      • Sakon M.
      • Monden M.
      • Aozasa K.
      ). Loss-of-function studies in model organisms indicate that p97 plays a critical role in a broad array of cellular processes including Golgi membrane reassembly (
      • Rabouille C.
      • Levine T.P.
      • Peters J.M.
      • Warren G.
      ), membrane transport (
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ,
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ), degradation of misfolded membrane and secretory proteins by the UPS (
      • Golbik R.
      • Lupas A.N.
      • Koretke K.K.
      • Baumeister W.
      • Peters J.
      ,
      • Richly H.
      • Rape M.
      • Braun S.
      • Rumpf S.
      • Hoege C.
      • Jentsch S.
      ), regulation of myofibril assembly (
      • Janiesch P.C.
      • Kim J.
      • Mouysset J.
      • Barikbin R.
      • Lochmüller H.
      • Cassata G.
      • Krause S.
      • Hoppe T.
      ), cell division (
      • Cao K.
      • Nakajima R.
      • Meyer H.H.
      • Zheng Y.
      ), and formation of protein aggregates (
      • Boyault C.
      • Gilquin B.
      • Zhang Y.
      • Rybin V.
      • Garman E.
      • Meyer-Klaucke W.
      • Matthias P.
      • Müller C.W.
      • Khochbin S.
      ,
      • Boyault C.
      • Zhang Y.
      • Fritah S.
      • Caron C.
      • Gilquin B.
      • Kwon S.H.
      • Garrido C.
      • Yao T.P.
      • Vourc'h C.
      • Matthias P.
      • Khochbin S.
      ,
      • Ju J.S.
      • Miller S.E.
      • Hanson P.I.
      • Weihl C.C.
      ). This broad range of functions is thought to derive from the ability of p97 to unfold proteins or disassemble protein complexes. Several factors make p97 an intriguing target for the development of drugs to treat cancer. First, elevated expression levels of p97 have been associated with poor prognosis of cancer (
      • Yamamoto S.
      • Tomita Y.
      • Hoshida Y.
      • Iizuka N.
      • Monden M.
      • Yamamoto S.
      • Iuchi K.
      • Aozasa K.
      ,
      • Tsujimoto Y.
      • Tomita Y.
      • Hoshida Y.
      • Kono T.
      • Oka T.
      • Yamamoto S.
      • Nonomura N.
      • Okuyama A.
      • Aozasa K.
      ). Second, p97 is an ATP hydrolase, and thus in theory, it should be druggable. Third, p97 is essential, and thus, p97 inhibitors should have antiproliferative activity. In addition, p97 is essential for endoplasmic reticulum-associated degradation (ERAD) (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ,
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ,
      • Neuber O.
      • Jarosch E.
      • Volkwein C.
      • Walter J.
      • Sommer T.
      ). Blockade of ERAD is thought to be a key mechanism underlying the anticancer effects of bortezomib (
      • Nawrocki S.T.
      • Carew J.S.
      • Dunner Jr., K.
      • Boise L.H.
      • Chiao P.J.
      • Huang P.
      • Abbruzzese J.L.
      • McConkey D.J.
      ). Given that p97 is implicated in ERAD but otherwise has a more restricted role in the UPS compared with the proteasome, it is possible that drugs that target p97 might retain much of the efficacy of bortezomib but with less toxicity.

      Acknowledgments

      We thank M. Smythe and C. Crews for YU101, A. M. Weissman for PYR-41, Y. Ye for EerI, Millennium Pharmaceuticals for MLN4924, K. Vousden for JNJ26854165, J. Huang for SMER3, and C. C. Wu for 3,4-methylenedioxycinnamic acid (compound 18). We thank P. I. Hanson, A. T. Brunger, and A. L. King for providing plasmids and M. G. Masucci, N. P. Dantuma, R. R. Kopito, and D. Baltimore for providing cell lines. We thank F. Parlati for critical reading of the manuscript; H. Park, R. Oania, and D. Shimoda for technical assistance; and the members of the Molecular Libraries Probe Production Centers Network at The Scripps Research Institute and Kansas University for advice and guidance on the implementation of the ATPase assay and discussions.

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