Decoding the Ubiquitin System Landscape: Regulation of UBE2N Through a Conserved Ubiquitination Site and Identifying Novel Oncogenic E3 Ligases
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2025
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Melbourne Authors
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Medical Biology
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PhD thesis
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Open Access
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© 2025 Tamer Aly ABDELNAAEM
Abstract
Ubiquitin is a small 8.6 kDa protein which acts as a modifier of other proteins to influence a myriad of outcomes such as protein stability, degradation, activity, and localisation. The ubiquitination cascade is comprised of E1 ubiquitin-activating, E2 ubiquitin-conjugating, and E3 ubiquitin-ligase enzymes, working in sequential harmony to orchestrate the ubiquitination of substrates. Deregulation of any of the members of the ubiquitination cascade can result in catastrophic outcomes for cells and ensuring a robust ubiquitin system is essential for the fitness of all cells.
In Chapter 2, I focus on the second member of the ubiquitination cascade, the E2 ubiquitin-conjugating enzymes. Often overlooked in research, E2s are key regulators of the ubiquitin system and are finely regulated to ensure their activity is kept in check. A method of regulation of E2s is ubiquitination. A conserved ubiquitination site adjacent to the E2s’ catalytic cysteine has been reported to be autoubiquitinated, which affects the activity of the E2. I utilise a mix of in vitro and in-cell assays to study this ubiquitination site in E2s, focusing primarily on UBE2N. I report the importance of this conserved ubiquitination site for the full activity of UBE2N to assemble free ubiquitin chains in vitro, and its mutation restricts UBE2N function in inflammatory signalling.
In Chapter 3, I focus on the final and largest member of the ubiquitination cascade, the vast family of E3 ligases. Recently, E3s have garnered much attention as drug targets for cancer therapy. Controlling a wealth of biological contexts, E3s are lucrative targets for drug development. In this chapter, I investigate E3 ligases in a holistic manner using a bioinformatics analysis to filter out and classify E3 ligases that hold high potential as anticancer targets. I classify the E3s into distinct groups that share similar therapeutic profiles in cancer, suggesting the creation of groups of interchangeable drug targets. I show preliminary validation of some of the targets and elucidate the strength of our dataset for cancer research.
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E2, Autoubiquitination, UBE2N, Inflammation, E3, Cancer