Medical Biology - Theses
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ItemFunctional and structural characterisation of VDAC2 in BAK-mediated apoptosis( 2023-06)BAK and BAX are the executors of intrinsic apoptosis. Their activity is tightly regulated by their interactions with the BCL-2 family proteins, but also non-BCL 2 proteins including the mitochondrial Voltage-Dependent Anion Channel 2 (VDAC2). Whilst targeting their interactions with BCL-2 family proteins to manipulate apoptosis clinically to treat diseases including cancer and potentially degenerative diseases is receiving attention, their interaction with VDAC2 remains unexplored. VDAC2 is important for the targeting of both BAK and BAX to mitochondria where they execute their apoptotic function, and its interaction with BAK has recently emerged as a therapeutic target to manipulate BAK-mediated apoptosis. The Chapter 3 presents the intracellular evidence of how VDAC2 interacts with BAK to modulate BAK-driven apoptosis. I have identified key residues involved in the interaction between BAK and a cytosol-exposed region on VDAC2 using mutagenesis and obstructive cysteine labelling. Stabilisation of this interaction through mutagenesis of VDAC2 not only restrains BAK activity but is also sufficient to inhibit a cells response to BH3 mimetic compounds. Cysteine crosslinking experiments reveal that VDAC2 binds to BAK hydrophobic groove, which is to date the first example of a non alpha-helix binding to the BAK groove. The Chapter 4 details attempt to investigate the interaction between BAK and VDAC2 using recombinant proteins. Furthermore, given that BAK and VDAC2 are known to engage other proteins in the MOM, including VDACs 1 and 3, the Chapter 5 describes sample preparation and cryo-EM analysis of the complex isolated from mitochondria from mammalian cells to attempt to resolve the structure of this multi-protein complex. Here I report the preliminary cryo-EM data of the purified BAK–VDAC2 multi-complex. This thesis provides new insights into how BAK is regulated through its interaction with VDAC2 through both biochemical and structural perspectives, and can guide new avenues for potential therapeutic intervention.