Investigation of Novel HECTD1 Functions in Cellular Metabolism and Cell Death

: (Alternative Format Thesis)

Abstract

Ubiquitin is small post-translational modifier that is crucial for the regulation of protein fate and function. It prominently regulates protein turnover via the ubiquitin-proteasome system but also a plethora of other cellular processes and signalling pathways including the cell cycle, inflammation, DNA repair, apoptosis, and metabolism. It is conjugated to, mostly, substrate lysine residues through an enzymatic cascade involving E1-activating enzymes, E2-conjugating enzymes, and E3 ubiquitin ligases. Here, I investigated novel functions of the Homologous to the E6-AP Carboxyl Terminus (HECT)-type E3 ubiquitin ligase HECTD1 in cellular metabolism and cell death. We previously identified that HECTD1 depletion results in reduced cell proliferation and a mitotic defect. In addition, preliminary as well as published proteomics data highlighted enrichment of HECTD1 interactors in metabolic pathways. Thus, I set out to characterise the effect of HECTD1 depletion on cellular metabolism using the gold-standard Seahorse analyser. Strikingly, HECTD1 depletion resulted in decreased ATP levels and robust attenuation of basal glycolysis. This metabolic phenotype could be recapitulated by transfecting a HECTD1 specific ubiquitin variant that inhibits HECTD1 catalytic activity. It will be particularly interesting to further characterise the mechanism by which HECTD1 E3 ligase activity mediates its metabolic effect and to evaluate its therapeutic potential in glycolytic cancers.

HECTD1 has been previously shown to assemble K63-linked ubiquitin chains and we previously showed that the HECTD1 HECT domain preferentially assembles K29/K48-branched ubiquitin chains, in vitro at least. However, beyond linkage specificity many regulatory aspects remain to be defined. Proteolytic processing is an irreversible regulatory mechanism that is best characterised for caspase activation during cell death, however it is unclear if it also constitutes a regulatory mechanism for other proteins including E3 ligases. I probed the PROTOMAP database, which mapped the global topography of proteolytic cleavage events, for HECT E3 ligases cleaved during cell death and identified HECTD1 as a potential proteolytic substrate. I validated HECTD1 cleavage during extrinsic and intrinsic apoptotic cell death and mapped the single cleavage site to Asp1664. I further identified caspase 3 as sole caspase responsible for HECTD1 cleavage and also showed that full-length, not cleaved, HECTD1 mediates a pro-apoptotic effect by increasing caspase 3 activity via its E3 ligase activity. Thus, I identified a second, novel and functional role for HECTD1 but it will be important to further characterise through which mechanism and ubiquitin signals HECTD1 mediates its positive effect on apoptotic cell death.

Date of Award	22 Feb 2023
Original language	English
Awarding Institution	University of Bath
Supervisor	Julien Licchesi (Supervisor), Keith Vance (Supervisor), Florian Siebzehnrubl (Supervisor) & Kathreena M Kurian (Supervisor)