AbstractUbiquitylation is involved in almost all aspects of eukaryotic cell biology. Through the intrinsic nature of ubiquitin, a single molecule can be translated into a dizzying array of signals. As such, the ubiquitin code must be highly regulated. Deubiquitylases (DUBs) are the erasers of the ubiquitin system being able to trim ubiquitin signals, and even remove them entirely. DUBs are therefore essential in fine-tuning, or abolishing ubiquitylation. Being such important enzymes, it is important to fully understand them. The relatively recent discovery of two new classes of DUBs, the MINDY family and ZUP1, shows that we still have much to learn about this class of enzymes.
Being novel, unstudied DUBs, gaining mechanistic and biochemical insights into MINDY family members will improve our understanding of the DUB field more broadly. Using biochemical, biophysical, and structural approaches, the work highlighted in this thesis shows MINDY1/2 to be K48-specific DUBs that are adapted to cleave long polyUb chains. As such, the DUBs undergo a novel shift in cleavage mode depending on the length of the polyUb chain. In concert with this, MINDY1/2 are shown definitively to have 5 ubiquitin binding sites on their catalytic domains.
Meanwhile, work on MINDY3 highlights that it too is a K48-specific DUB that also prefers cleaving longer polyUb chains. Further, like MINDY1/2, the polyUb length- dependent switch in cleavage mode is also present in MINDY3. Using modelling approaches, it is tentatively concluded that MINDY3 has 6 ubiquitin binding sites. Of these sites, 3 are contributed by an EF-hand insertion in the catalytic domain, which appears to have lost the ability to bind calcium and now functions as a ubiquitin binding
domain (UBD). Further, the EF-hand mediates interactions with RAD23A/B, two functionally redundant proteins, which are found to bind to MINDY3 via their respective ubiquitin-like domains (UBL). This interaction is further probed to tease out the biological function of MINDY3 for which hints are provided of a role in DNA damage repair processes.
In addition to their importance to human biology, DUBs are also used by pathogens to exploit host ubiquitylation systems and stifle immune responses. SARS-CoV-2, the causative agent of COVID-19, has wreaked havoc on society. Interestingly, it possesses a papain-like protease (PLPro) domain that sits as a domain within non- structural protein 3 (NSP3) of SARS-CoV-2. PLPro plays a role in cleaving the viral polyprotein to liberate mature NSPs, but also has DUB activity. Much work has been done to characterise the PLPro domain of SARS-CoV-2 in isolation, but the question of whether other NSP3 domains influence PLPro activity remains unanswered. Work outlined in this thesis shows expression of an extended PLPro construct, NSP3core, that includes many other NSP3 domains. NSP3core is shown to be a more active DUB compared to PLPro, and importantly, NSP3core can cleave substrates mimicking the viral polypeptide, whilst PLPro alone cannot. This highlights the importance of studying protein domains in the context which they exist. Multiple strategies to inhibit the activity are also explored in this thesis including the development of a repurposing pipeline for FDA-approved small molecules, and the custom design of a nanobody as a tool to specifically study PLPro during viral infection. Overall, the work presented here shows the importance of DUBs to biology and why it is therefore important to understand how they function.
|Date of Award||2023|
|Supervisor||Yogesh Kulathu (Supervisor)|