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Title: Novel screening methods for inhibitors of the human ubiquitin-conjugating enzymes
Author: Koszela, Joanna
ISNI:       0000 0004 5991 1159
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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The ubiquitin-proteasome system (UPS) controls the stability, activity and localisation of most of the proteome and regulates virtually all cellular processes through modification of proteins with ubiquitin. Ubiquitin conjugation is mediated by a conserved enzymatic cascade composed of E1, E2 and E3 enzymes, which cooperate to activate and transfer ubiquitin to substrate proteins. Dysfunction of the UPS is implicated in many disease states, including cancer, neurodegeneration, immune and cardiovascular disorders. Despite the central role of the UPS in cellular regulation, our understanding of the function, interactions and specificity of proteins that comprise the UPS is still limited. One approach to dissect and to study the UPS is to identify molecular probes, which can be used to specifically interrogate catalytic mechanisms and can be potentially considered as entry points for drug discovery. This work focuses on developing novel high-throughput screening methods for inhibitors of the ubiquitin-conjugating enzymes (E2s), using a unicellular organism Saccharomyces cerevisiae and in vitro technologies. S. cerevisiae is a model organism, commonly used in research as a valuable tool for genetic investigations and other high-throughput studies. In this work, we evaluated the toxicity of exogenously expressed human E2s on yeast cells and discovered that one of the E2s, Ube2U, significantly inhibited yeast growth. This inhibition was dependent on the Ube2U ubiquitin-conjugation activity, as demonstrated with a catalytically inactive Ube2U C89A control, which did not affect yeast growth. The growth defect induced by Ube2U allowed us to develop a screening setup for inhibitors of Ube2U, where the enzyme activity was coupled to cell growth readout. Potential Ube2U inhibitors would be identified as rescuers of the slow growing Ube2U-expressing yeast phenotype. Although screening methods in yeast are relatively straightforward to set up and run, the advantages of this system, namely simplicity of the detection signal and high-throughput, are limited by the fact that yeast is not a recognised large scale screening system in pharmaceutical industry, and that it is difficult to identify the target in a complex pathway such as the UPS. In vitro technologies are needed to provide the necessary structure-activity relationship for chemical optimisation. Therefore, we developed a novel, fluorescence-based, miniaturised assay technology, suitable for biochemical investigations and screening for inhibitors of a wide range of specific ubiquitination reactions within the UPS.
Supervisor: Auer, Manfred ; Walkinshaw, Malcolm Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: ubiquitination ; E2 enzymes ; high-throughput screening