Cowan, Emma
(2023)
STRUCTURAL AND BIOPHYSICAL ANALYSIS OF UBE3A AND PARTNER PROTEIN COMPLEXES.
PhD thesis, University of Nottingham.
Abstract
UBE3A is a human E3 ubiquitin ligase, responsible for the transfer of ubiquitin onto substrates to target them for degradation and other cellular processes. UBE3A has been implicated in a range of neurodevelopmental disorders, most notably Angelman Syndrome, but also Dup15Q syndrome, autism spectrum disorders, and schizotypies. Alongside this, it has also been shown to be involved in the oncogenesis of many different types of cancer, including prostate cancer, small-cell lung cancer, B-cell lymphomas, and most notably HPV-associated oropharyngeal cancer and cervical carcinomas. More recent studies have even shown a role of UBE3A in cardiomyopathies, Alzheimers, and the immune response to HIV, as well as identifying it as part of a diverse range of cellular signalling processes.
Despite the huge clinical significance of UBE3A, a full-length structure for the enzyme is not currently available. The catalytic HECT domain was solved by x-ray crystallography in 1999, but a large distance between the catalytic site and the binding site of the cognate E2 enzyme have raised more questions about the mechanism of UBE3A activity than answers. Various studies in the intervening years have revealed more insights into UBE3A’s activity and interaction with cellular partners, but a full structure would enable a much better understanding of its mechanism. This is key for designing small molecules to counteract some of the effects of UBE3A mutations or oncogenic signalling disruptions, but also just for understanding how UBE3A works and how its loss may be compensated for in the majority of Angelman Syndrome cases.
In this work, I used a range of biophysical techniques, biochemical assays, and structural techniques in an attempt to characterise UBE3A and its interactions with partner proteins as completely as possible. Although many of the questions that I attempted to address remain unanswered, I was able to reconstitute a low-resolution cryo-EM map of isolated full-length UBE3A, even lower resolution models of UBE3A in complex with two of its binding partners, and a high resolution model of the RLD2 domain of the HERC2 protein. I was also able to demonstrate some of the properties of these interactions through SV-AUC, ITC, CD, and crosslinking and co-purification experiments. The different forms of UBE3A, both alone and in the presence of its binding partners, were subjected to in vitro assays in an attempt to determine the effects of complex formation of the ubiquitin ligase activity of UBE3A.
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