Medical Biology - Theses
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ItemIdentifying and characterising novel regulators of TRAIL-induced cell death and cholangitis-like liver injury( 2022)Primary sclerosing cholangitis (PSC) is a progressive, idiopathic cholangiopathy characterised by chronic inflammation of the biliary epithelium and cholestasis. PSC promotes fibrotic scarring of the intrahepatic and extrahepatic bile ducts often leading to premature death due to irreversible liver damage. Chronic persistent inflammation in the biliary tree further predisposes to the development of malignant cholangiocarcinoma (CCA). Tumour necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL)/TRAIL-receptor-mediated signalling was shown to play a substantial role in the pathogenesis of human sclerosing cholangitis-like disease in mice with TRAIL- mediated apoptosis contributing to the disease. However, the etiology and exact pathogenic mechanisms of TRAIL-dependent PSC, or inflammation-associated cholangiocarcinogenesis are largely unclear. Various genetic and environmental factors have been reported to play role in the pathogenesis of PSC. Recently, mutations in ZFYVE19 gene (protein name: ANCHR) were described as a novel cause of neonatal sclerosing cholangitis and hepatic fibrosis termed ZFYVE19 disease. However, the mechanism by which ZFYVE19/ANCHR is involved in the pathogenesis of sclerosing cholangiopathy in these patients has not been yet explored. In Chapters 3 & 4 of my thesis, I identify and characterise two novel regulators of TRAIL-induced cell death, the Abscission/NoCut Checkpoint Regulator (ANCHR/ZFYVE19) and its interacting protein E3 ligase Mind Bomb 2 (MIB2) and show that the loss of ANCHR or MIB2 sensitises TRAIL-resistant cancer cells to caspase-8- dependent death. Moreover, loss of ANCHR alone sensitises CCA cells to death in vitro. Given that the loss of ZFYVE19/ANCHR in a cohort of patients was associated with PSC and cholestasis, and TRAIL/TRAIL-R-mediated apoptosis has been suggested to play an essential role in a PSC-like disease in mice, I further interrogate the physiological consequences of ANCHR loss in mice, particularly focusing on TRAIL-mediated cell death in the liver. In Chapters 3 & 4 I demonstrate a role for ANCHR in limiting TRAIL-induced cell death in vivo and show that loss of ANCHR in mice sensitises to TRAIL-mediated liver cell death. A significant increase in liver cell death in Zfyve19 knock-out mutant mice is observed compared to the wild-type mice after anti-TRAIL-R2 monoclonal antibody (MD5- 1) injection, with concurrent increase in cholangiocyte cell death, suggesting a role for ANCHR in limiting the TRAIL-mediated cholangitis in mice by limiting TRAIL-induced cell death in the liver. Lastly, in the Chapter 5 of this thesis, I demonstrate, as a proof-of-concept, that ANCHR and MIB2 can be efficiently targeted and degraded using the emerging degradation tag (dTAG) PROteolysis-TArgeting Chimera molecules (PROTACs). Our preliminary results serve as a basis for future research and suggest that anti-apoptotic ANCHR and MIB2 are feasible targets for target-specific protein degradation for development of future TRAIL therapeutics. Overall, this thesis expands our understanding on how TRAIL-signalling is regulated and provides a mechanism for the interplay between ZFYVE19/ANCHR loss and TRAIL- mediated PSC-like liver disease. Furthermore, our studies provide correlative evidence for the relationship between the PSC pathology seen in patients carrying bi-allelic nonsense mutations in the ZFYVE19 gene and an overactive TRAIL signalling or overactive liver sensitivity to endogenous TRAIL.
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ItemIdentifying Novel Regulators of Intrinsic Apoptosis( 2021)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.