Research Interest-Prof. Benedikt Kessler

Ubiquitylation has emerged as a central process in the regulation of protein function. This modification has been implicated not just in the elimination of damaged proteins, but also in physiological proteolytic control of processes such as transcription, signal transduction, and cell cycle transitions and even a range of non-proteolytic targeting events. So far, analysis has principally focused on ubiquitin (ub) attachment, with several hundred ub conjugating enzymes characterized to date. Much less is known about enzymes that remove ub from substrate proteins, yet around a hundred genes have been identified, sharing consensus motifs for deubiquitylating enzymes (DUBs). Such diversity is inconsistent with a simple recycling function and strongly suggests a range of specific (but currently largely undiscovered) biological functions. Members of the DUB family are already known to contribute to neoplastic transformation and are implicated in neurodegenerative diseases, making them attractive targets for drug design.

UPS_SchemeThe role of the ubiquitin-proteasome system (UPS) in proteolysis and antigen presentation. Degradation of proteins is initiated by the attachment of poly-ubiquitin chains (top). Poly-ubiquitin serves as a specific signal for proteasome-dependent degradation. Proteolytic fragments are then further degraded into smaller peptides and individual amino acids, which can then serve for the synthesis of novel proteins. A small fraction of peptides are transported through the TAP complex into the ER, where they are loaded on to class I major histocompatibility molecules (MHC I) for the presentation to cytotoxic T lymphocytes at the cell surface. The protein degradation process is controlled at many different steps, including ubiquitin conjugating enzymes (E1,E2,E3), but also ubiquitin processing enzymes, which generate mono-ubiquitin, recycle ubiquitin and can regulate the fate of a protein substrate by removing ubiquitin prior to proteolysis. In particular, we are interested in studying enzymes with deubiquitylating activity (DUBs, indicated in red), and how they may control the lifespan of proteins and enzymes critical for cell signaling, growth and differentiation.

Ubiquitin mediated proteolysis

Proteolytic enzymes with Ub/Ubl processing activity appear to be more widespread than originally anticipated. It is therefore not surprising that bacterial and viral pathogens have exploited many ways to interfere with Ub/Ubl conjugation, but also de-conjugation. On one hand, pathogens were shown to manipulate host encoded enzymes. On the other hand, pathogen derived sequences of proteases specific for Ub/Ubls are emerging as a common feature shared by many viruses, bacteria and protozoa, and we are at an early stage of understanding how these proteases contribute to the pathogenesis of infection. Whereas some of these proteases share a common origin with mammalian cell encoded hydrolases with specific properties towards Ub/Ubls, most of them have ancient intrinsic functions, such as processing pathogen protein components, and may have acquired the specificity for Ub/Ubls by interacting with mammalian hosts and their immune system throughout evolution. Since many of these proteases are clearly distinct from their mammalian counterparts, they represent attractive targets for drug design against infectious diseases.

DUB_Mechanism_SchemeRecognition of ubiquitylated proteins by deubiquitylating enzymes (DUBs). Ubiquitin-specific proteases contain predominantly a cysteine in their catalytic site (dark grey), which undergoes a nucleophilic attack on the isopeptide bond of the ubiquitin C-terminus attached to a lysine residue of the protein substrate (light grey). This molecuar principle is also found for proteases specific for ubiquitin-like molecules (Ubls), such as SUMO, Nedd8, UCRP, FAT10 and ISG-15. New members of Ub/Ubl-specific proteases family are still being discovered and the biological function of most of them remains to be examined (Kessler et al., 2006).

 

Chemoproteomics for Ubiquitin and Protease Biology

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Figure 1: Molecular principle of activity-based probes (ABPs)
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Figure 2: Activity-based profiling of deubiquitinase (DUB) inhibitors in cells (Altun et al., 2012)

Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment (Figure 1). One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. In the Kessler laboratory, a family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors (Figure 2). 

Combining DUB active site probes with proteomics affords researchers, for the first time, the ability to generate DUB inhibition profiles and measure changes in the content of the substrates of DUBs in living cells (Figure 3) – a key translational step in the development of new drugs for the treatment of cancer, pathogen infection and neurodegenerative disorders.

In a mass spectrometry based workflow, two DUB inhibitors, PR-619 and P22077, were characterized in a cell culture model. Labeling of HEK293T cell lysates treated with inhibitors at varying concentrations was performed with the ubiquitin probes HAUbVME and HAUbBr2 to investigate DUB inhibition profiles. As a read-out of these assays, anti-HA immunoblotting can be performed. Alternatively, anti-HA-immunoprecipitation (IP) was combined with identification and label-free quantification by mass spectrometry based proteomics (Figure 3).

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Figure 3: Mass spectrometry-based DUB inhibitor profiling in cells (Altun et al., 2011)

Selected Recent Publications

TURNBULL AP, IOANNIDIS S, KRAJEWSKI WW, PINTO-FERNANDEZ A, HERIDE C, MARTIN ACL, TONKIN LM, TOWNSEND EC, BUKER SM, LANCIA DR, CARAVELLA JA, TOMS AV, CHARLTON TM, LAHDENRANTA J, WILKER E, FOLLOWS BC, EVANS NJ, STEAD L, ALLI C, ZARAYSKIY VV, TALBOT AC, BUCKMELTER AJ, WANG M, MCKINNON CL, SAAB F, MCGOURAN JF, CENTURY H, GERSCH M, PITTMAN MS, MARSHALL CG, RAYNHAM TM, SIMCOX M, STEWART LMD, MCLOUGHLIN SB, ESCOBEDO JA, BAIR KW, DINSMORE CJ, HAMMONDS TR, KIM S, URBÉ S, CLAGUE MJ, KESSLER BM, KOMANDER D . 2017. Molecular basis of USP7 inhibition by selective small-molecule inhibitors. Nature, 550 (7677), pp. 481-486. Read more

ELLIOTT PR, LESKE D, HRDINKA M, BAGOLA K, FIIL BK, MCLAUGHLIN SH, WAGSTAFF J, VOLKMAR N, CHRISTIANSON JC, KESSLER BM, FREUND SMV, KOMANDER D, GYRD-HANSEN M . 2016. SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling. Mol Cell, 63 (6), pp. 990-1005. Read more

WIJNHOVEN P, KONIETZNY R, BLACKFORD AN, TRAVERS J, KESSLER BM, NISHI R, JACKSON SP. 2015. USP4 Auto-Deubiquitylation Promotes Homologous Recombination. Mol Cell, 60(3), pp. 362-373. Read more

KESSLER BM. 2014. Selective and reversible inhibitors of ubiquitin-specific protease 7: a patent evaluation (WO2013030218). Expert Opin Ther Pat, 24 (5), pp. 597-602.  Read more

Kessler BM. Ubiquitin - Omics reveals novel networks and associations with human disease. Curr Opin Chem Biol 2013. View Article

Chauhan D, Tian Z, Nicholson B, Kumar KG, Zhou B, Carrasco R, McDermott JL, Leach CA, Fulcinniti M, Kodrasov MP, Weinstock J, Kingsbury WD, Hideshima T, Shah PK, Minvielle S, Altun M, Kessler BM, Orlowski R, Richardson P, Munshi N, Anderson KC. A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell. 2012 Sep 11;22(3):345-58. View Article

McGouran JF, Kramer HB, Mackeen MM, di Gleria K, Altun M, Kessler BM. Fluorescence-based active site probes for profiling deubiquitinating enzymes. Org Biomol Chem. Org Biomol Chem. 2012 May 7;10(17):3379-83. . View Article

Khoronenkova SV, Dianova II, Ternette N, Kessler BM, Parsons JL, Dianov GL. 2012. ATM-Dependent Downregulation of USP7/HAUSP by PPM1G Activates p53 Response to DNA Damage. Mol Cell. 2012 Mar 30;45(6):801-13. View Article

Altun M, Kramer HB, Willems LI, McDermott JL, Leach CA, Goldenberg SJ, Suresh Kumar KG, Konietzny R, Fischer R, Kogan E, Mackeen MM, McGouran J, Khoronenkova SV, Parsons J, Dianov GL, Nicholson B, Kessler BM. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem Biol. 2011. 18(11):1401-1412. View Article. Press Release

Edelmann MJ, Nicholson B, Kessler BM. Pharmacological targets in the ubiquitin system offer new ways of treating cancer, neurodegenerative disorders and infectious diseases. Expert Rev Mol Med. 2011 Nov 17;13:e35. View Article.

Kessler BM, Edelmann MJ. PTMs in Conversation: Activity and Function of Deubiquitinating Enzymes Regulated via Post-Translational Modifications. Cell Biochem Biophys. 2011 Jun;60(1-2):21-38. View article

Parsons JL, Dianova II, Khoronenkova SV, Edelmann MJ, Kessler BM, Dianov GL. USP47 Is a Deubiquitylating Enzyme that Regulates Base Excision Repair by Controlling Steady-State Levels of DNA Polymerase β. Mol Cell. 2011 Mar 4;41(5):609-15. View Article