Abstract
Silencing Defective 2 (SDE2) is an essential multifunctional protein involved in the DNA damage response, spliceosome assembly and ribosome biogenesis. It is expressed as a fusion protein containing an N-terminal ubiquitin-like domain (UBL), which is cleaved through deubiquitinase (DUB) driven cleavage. USP5 was recently identified as the DUB responsible for this cleavage and activation of the C-terminal domain. Here, I report the development of an activity-based probe (ABP) derived from the SDE2UBL, which selectively labels USP5. Through comparative, recombinant and cell lysate based, studies I reveal the distinct interaction preference of USP5 for the canonical substrate based ABP, Ub-PA, SDE2UBL-PA and a cross reactive UBL ABP, ISG15-PA.Additionally, I explore semi-synthetic strategies for developing further probes, particularly a cleavage reporter probe through incorporation of rhodamine 110. Exploration of alternative leaving groups including an azide and thiophenol, outside of the conventional MESNa thioester, did not provide adequate reaction yields. I also report the cross-reactivity of the viral protease LbPro with SDE2UBL and its ability to conjugate diglycine based warheads, however showed limited promise for the desired SDE2UBL-Rho. These findings, however unsuccessful highlight the range of strategies and provide the groundwork for means of future probe development.
| Date of Award | 2026 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Chiara Maniaci (Supervisor) & Satpal Virdee (Supervisor) |