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Title: Investigating apoptosis regulation in mitosis through proximity labelling techniques
Author: Pedley, Robert
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2019
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Cytotoxic chemotherapies remain an important component of modern cancer therapy. These drugs typically kill cells through the intrinsic apoptotic pathway. The point of no return in this pathway is the permeabilisation of the outer mitochondrial membrane, an event that is regulated by members of the Bcl-2 family of proteins. How close a cell's mitochondria are to undergoing outer membrane permeabilisation is referred to as 'apoptotic priming'. How primed a cell is influences its response to apoptotic stimuli such as chemotherapy. Individual cells dynamically adjust their levels of apoptotic priming in response to changing cellular conditions such as DNA damage or cell cycle progression. However, precisely how cells coordinate these changes remains unclear. It has recently been identified that the pro-apoptotic Bcl-2 family protein, Bid, becomes phosphorylated on entry to mitosis and increases apoptotic priming until phosphorylation is lost on transition to anaphase. Thus, mitosis provides an ideal model to study how cells are able to dynamically alter apoptotic priming with precise temporal control. To this end we have coupled proximity biotin labelling (BioID) techniques with mass spectrometry to perform unbiased proteomics screens with Bid as the bait. By performing screens in mitotic and non-mitotic conditions we have identified that VDAC2 is essential for Bid-phosphorylation dependent changes in apoptotic priming during mitosis. This result reveals VDAC2 as a key scaffold protein in coordinating apoptotic priming during mitosis, and highlights how non-Bcl-2 family proteins play a central role in the intrinsic apoptotic pathway, but are often overlooked. Furthermore, the results in this thesis demonstrate that the use of enzyme-based proximity labelling techniques are an effective, unbiased method for capturing the wider Bcl-2 family protein interactions in situ, and represents a powerful new approach for studying these proteins in an impartial context.
Supervisor: Gilmore, Andrew ; Brennan, Keith ; Clarke, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available