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UBC Theses and Dissertations

A mitochondrial terminomics technique to study human cell death during bacterial infection Marshall, Natalie Claire

Abstract

Mitochondria are essential for human health. While mitochondria are primarily known for their essential role in generating cellular energy, these organelles play many vital roles in eukaryotic cells, including the cellular stress response, innate immunity, and the regulation of intrinsic apoptosis. Mitochondrial proteases are essential for mitochondrial function, including the import of 99% of the mitochondrial proteome, which often requires the proteolytic removal of a mitochondrial targeting sequence (MTS), creating a new protein amino terminus. However, little is known about how proteases regulate mitochondrial functions during health and disease because of the lack of tools able to examine global mitochondrial proteolysis and how it changes during mitochondrial processes. A novel terminomics workflow called ‘MS-TAILS’ was developed to address this gap by identifying changes in the mitochondrial terminal proteome, reflective of mitochondrial proteome-wide proteolysis. MS-TAILS identified the highest coverage of the human mitochondrial proteome and the most sites of import-associated MTS removal of any terminomics study to date, as well as 97 novel sites of mitochondrial proteolysis, demonstrating its utility to study mitochondrial proteolysis, proteases, and proteome import. MS-TAILS was applied to characterize mitochondrial changes during the induction of intrinsic apoptosis: a critical but poorly characterized mitochondrial process. MS-TAILS identified apoptosis-dependent changes in seven mitochondrial proteins not previously implicated in apoptosis, which may indicate conserved early apoptotic events. We examined the role of mitochondrial proteases in innate immunity by conducting the first terminomics study of microbial infection, identifying infection-specific mitochondrial changes during enteropathogenic E.coli (EPEC) infection: a pathogen that uses a type III secretion system (T3SS) to inject effectors into human cells and mitochondria to modulate apoptosis and immunity. The majority of infection- and T3SS effector-dependent mitochondrial changes were unique from canonical apoptosis events, suggesting that EPEC T3SS effectors mediate an infection-associated mitochondrial apoptosis pathway. These findings were examined in a broader context to demonstrate the impact of this thesis work on the field. Overall, this work provides a novel approach to study global dynamics in mitochondrial proteolysis between conditions and therefore addresses a technical gap to characterize mitochondrial proteases, processes, and pathologies, including and beyond apoptosis. Supplementary materials available at: http://hdl.handle.net/2429/66359

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Attribution-NonCommercial-NoDerivatives 4.0 International