Identifying Novel Regulators of Intrinsic Apoptosis
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2023-05-26.
© 2021 Shuai Huang
Abstract: Apoptosis is a conserved cellular process of programmed cell death. The intrinsic pathway of apoptosis is principally regulated by three functional and structural subgroups of the BCL-2 protein family: pro-survival proteins, pro-apoptotic effector proteins and apoptotic initiator BH3-only proteins. The interactions between these proteins on the mitochondrial outer membrane (MOM) determine whether a cell survives or dies. In healthy cells, pro-survival proteins inhibit the pro-apoptotic effector proteins BAK and BAX. In response to death stimuli, BH3-only proteins are activated to promote apoptosis either by activating BAK and BAX or neutralising pro-survival proteins. As well as BCL-2 family proteins, non-BCL-2 proteins such as VDAC2 are emerging as important regulators of apoptosis. However, the different impacts are observed in different contexts – with VDAC2 promoting apoptosis in some cases and restricting apoptosis in others. For BAK-dependent apoptosis, VDAC2 inhibits BAK function by forming complexes with BAK on the MOM. Therefore, loss of VDAC2 sensitises the cells to apoptotic stimuli in some contexts. This suggests that there may be proteins that accelerate BAK-dependent apoptosis in VDAC2-deficient cells. A previous genome-wide CRISPR-Cas9 library screen was performed in Bax-/-Vdac2-/-Mcl-1-/- MEFs to identify genes that when deleted inhibited the response to BH3-mimetics. In this screen, the absence of MCL1 enabled BH3-mimetic drug ABT-737 that inhibits BCL-2, BCL-XL and BCL-W to drive apoptosis in these cells, while the absence of BAX and VDAC2 ensured that apoptosis was mediated by BAK and controlled independently of VDAC2. From this screen, three proteins involved in ubiquitin signalling were identified as potential candidates: MARCHF5, UBQLN1 and USP24. In Chapter 3, I focused on validating each of these candidates in BAK-dependent apoptosis and identified that deletion of Marchf5 in Bax-/-Vdac2-/-Mcl-1-/- MEFs had the greatest impact on BAK-driven apoptosis. The effect of Marchf5/MARCHF5 on BAK function was further explored across different contexts using both Mcl1+/+ and Mcl1-/- MEFs and two human cell lines (HeLa and KMS-12-PE). Consistent with the initial validation results, deleting Marchf5/MARCHF5 in both murine and human cells provided long-term protection from BAK-driven apoptosis induced by BH3 mimetic drugs. Having successfully identified MARCHF5 as an important regulator of BAK-dependent apoptosis in Chapter 3, I further investigated how MARCHF5 modulated BAK apoptotic function in Chapter 4. My research indicated that BAK adopted an activated conformation and formed stable Mode 2 inhibitory complexes with MCL-1 and BCL-XL in MARCHF5-deficient cells, thereby providing protection from BH3 mimetic drugs. Given that MARCHF5 is an E3 ubiquitin ligase, I further identified that the drug resistance observed upon MARCHF5 deletion could be phenocopied by abrogating MARCHF5 enzymatic activity. Finally, in Chapter 5 I performed proteomics and functional CRISPR/Cas9 genetic screens to identify substrates of MARCHF5 that could account for the mechanism driving BAK into Mode 2 complexes in MARCHF5-deficient cells. Through these complementary approaches, several potential candidates were revealed, that may represent novel mediators of BAK apoptotic activity.
KeywordsCell death; Apoptosis; BAK; BH3 mimetic drug; E3 ubiquitin ligase; MARCHF5
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