Undergraduate Course

Department of Chemistry University of Oxford

CHEMISTRY PART II PROJECT 2017/2018

I would be able to take up to 2 students.

Title(s): How the ubiquitin system affects cancer cell metabolism: A chemical biology approach

Description of the projects:

Human neurodegenerative, infectious diseases and tumorigenesis are associated with alterations in ubiquitin pathways. Ubiquitin is a small 76 amino acid polypeptide that gets attached to proteins and controls their fate, turnover of biological activity. Over 10% of the genome encode for genes that either bind or manipulate ubiquitin to affect a large proportion of biological processes. In our group, we have developed activity-based chemical proteomics approaches to study the biological function of ubiquitin conjugating (E1/E2/E3 enzymes) and deconjugating enzymes (DUBs). Chemical modifications on ubiquitin, a 76 amino acid protein, allow a covalent attachment to DUBs and ubiquitin conjugating enzymes in a mechanism-based fashion. When combined with mass spectrometry, we can track active enzyme species on a systems-wide level in order to study their contributions to different physiological and pathological conditions such as tumorigenesis. Using this approach, we have discovered and characterised USP7 and USP47 as deubiquitinating enzymes involved in DNA repair mechanisms, and have delineated a chemoproteomics approach to screen small molecule inhibitors of these enzymes as potential new leads for the development of novel anti-cancer drugs.
Cancer cells also have an altered metabolism adapted to tumour cell growth. There is preliminary evidence that the Ubiquitin system is involved in its regulation.

Part II project 1 will involve chemistry & mass spectrometry development of ubiquitin based active site probes based on di-ubiquitin scaffolds to probe the specificity of DUBs in cancer cell under different physiological conditions, in particular where we have knocked out specific DUBs. It will be particularly interesting to study potential compensation mechanisms for loss of DUB function. In addition, metabolic profiles will be established in wildtype and cancer cells lacking specific DUBs by using mass spectrometry and compared to the DUB activity profiles.

Part II project 2 will also involve the synthesis of ubiquitin based active site probes in conjunction with project 1. In addition, it is planned to examine the effect of inhibiting deubiquitylating enzymes (DUBs) using small molecular compounds that we have available through the DUB Alliance (Cancer Research Technologies / Forma Therapeutics) on i) the cancer cell physiology/toxicity, ii) cell cycle progression and iii) general metabolic profile as measurable by GCxGC-MS.

Information obtained from both projects will further advance our understanding of the biology of DUBs in normal and disease-based physiology and possible links to cancer metabolism.

Techniques used in the projects:

This project will provide the opportunity for the application of chemistry on intact proteins, such as incorporation of unnatural amino acids into proteins, native chemical ligation, intein chemistry, “click”-chemistry and aspects of peptide synthesis. In addition, a thorough introduction into biochemistry, mass spectrometry (MS) and proteomics will be provided. This includes methods of protein biochemistry (protein expression, isolation, separation and analysis), chemical modification of proteins as well as protocols for analysis by tandem mass spectrometry (MS). In addition, small molecule mass spectrometry will also be used to measure a distinct set of cellular metabolites. The candidate will be carefully introduced and trained in all these methodologies as well as the data analysis of complex proteomics data sets by an experienced postdoctoral fellow in the laboratory. Tissue culture, expression and siRNA knockdown of proteins as well as immunoprecipitation methods will also be used in the context of this study.

Key references:

Pinto-Fernandez A, Kessler BM. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets. Front Genet. 2016 Jul 28;7:133

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.

McGouran JF, Gaertner SR, Altun M, Kramer HB, Kessler BM. Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem Biol. 2013 Dec 19;20(12):1447-55.

Valli A, Rodriguez M, Moutsianas L, Fischer R, Fedele V, Huang HL, Van Stiphout R, Jones D, Mccarthy M, Vinaxia M, Igarashi K, Sato M, Soga T, Buffa F, Mccullagh J, Yanes O, Harris A, Kessler B. Hypoxia induces a lipogenic cancer cell phenotype via HIF1α-dependent and -independent pathways. Oncotarget. 2015 Feb 10;6(4):1920-41

Name: Prof. Benedikt Kessler
Department: Target Discovery Institute (TDI), Nuffield Department of Medicine
Tel No: 01865 612 921
Email address: benedikt.kessler@ndm.ox.ac.uk
Lab Website: http://www.tdi.ox.ac.uk/mass-spectrometry