Date of Award
Chemistry and Biochemistry
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This work is licensed under a Creative Commons Attribution 4.0 International License.
Ubiquitination is an essential post-translational event for cellular homeostasis. The attachment of ubiquitin (Ub) to a protein is, in some capacity, involved in every molecular process in the cell. Dysregulation of such has compelling links to cancers, ageing-related diseases, intellectual disorders, and many more pathologies. Currently being tested are a slew of therapies targeting players of Ub signaling, and while some have been clinically approved for E3 ligases that mediate Ub conjugation, therapies available for deubiquitinases (DUBs), which mediate deconjugation, lag behind. This is partly due to the complexity of these enzymes. This work focuses on characterizing the activity of one such DUB, USP8, and aims to also test the application of its Ub variant (UbV) inhibitor, known as UbV8.2, in biosensing and interactomics. USP8 is of particular interest for its demonstrated roles in glioblastoma, Parkinson's disease, and Cushing's disease. In addition, it is unique for being the only USP intramolecularly autoinhibited by its auxiliary domain, but how this occurs is not well understood. Here, we characterized the mechanism of autoinhibition, revealing a WW-like inhibitory domain that, due to its low affinity for the catalytic domain, functions only in cis. Modelling indicates it competes with Ub by binding the S1 pocket in a novel way. We speculate that USP8 evolved this mechanism for low-level basal activity. UbVs are engineered proteins that bind targets with exceptional potency. These tool molecules can probe DUB biology and drive the discovery of novel inhibitors. UbV8.2 is the only USP8 inhibitor of its kind. We recognized that these binders could be leveraged for broad applications, thus we tested UbV8.2 in a proof-of-concept study and developed a novel electronic biosensor capable of detecting analyte (USP8) with a sub-nanomolar limit of detection. Despite the usefulness of UbV8.2 in vitro, we wondered about its fidelity in more complex settings in vivo. The pipeline of UbV development is high throughput, leading to inconsistencies when validating binders during selection. As such, we sought a method to rigorously validate UbVs in a cellular context. Based on in vivo proximity biotinylation, we named the method uBioID—the biotinylation of proteins in proximity to the UbV when expressed inside the cell. Should the UbV co-localize and bind its endogenous target, only proteins in the vicinity of the target, such as physical interactors, will be biotinylated and detected using mass spectrometry. A comparative study of UbV8.2 and another well-characterized UbV, UbV7.2, was performed. The findings showed, under our conditions, UbV8.2 is inadequate in the cellular milieu, as it is unable to engage its endogenous target, USP8. On the other hand, UbV7.2 showed robust target engagement, thus facilitating the identification of potential interactors of its cognate target, USP7. uBioID presents as a platform for both UbV validation and target interactomics. Achieved here is a deeper understanding of DUB regulatory mechanisms, coupled with new applications and validation methods for UbVs, thus lending critical information to aid expansion of the druggable space for this recalcitrant enzyme class.
CABA, CODY, "Allosteric regulation of the deubiquitinase USP8 and applications of ubiquitin variant tool molecules." (2024). Electronic Theses and Dissertations. 9181.
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