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.
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ItemA genetics-based investigation into the regulation of RIPK1 and caspase-8 during cell death and disease( 2022)Cell death is a fundamental process needed for healthy development, immunity and life. The tight control and regulation of cell death signalling is important for cellular homeostasis, and the de-regulation of cell death is a hallmark of many diseases ranging from infection to cancer. Several regulated cell death (RCD) pathways have been described, with genetically encoded cell death signalling molecules and effectors dictating cellular fate. Some of these, such as necroptosis and pyroptosis, are highly inflammatory and immunomodulatory, while others, such as apoptosis, are generally considered non-inflammatory and tolerogenic. Caspase-8 is a critical cell death protein that also has a pleiotropic role in inflammation. Receptor interacting protein kinase (RIPK)1 liaises with external signals to control the death and inflammatory functions of caspase-8, but major gaps remain in our understanding of how RIPK1 regulates the death and non-death functions of caspase-8. Identifying and characterising the mechanisms that control caspase-8 activity is crucial to understanding how we might best therapeutically target cell death signalling to overcome relevant diseases. This PhD thesis explores the regulation of caspase-8 activity and identifies key upstream checkpoints to therapeutically intersect and modulate caspase-8 activity. Firstly, this thesis genetically delineates a unique caspase-8-dependent cell death triggered by combined signalling of host-derived interferon (IFN)-gamma and pathogen ligands that engage Toll-like receptors (TLRs). Experiments show that caspase-8 cell death signalling is licensed by nitric oxide (NO), which is produced by the inducible nitric oxide synthase (iNOS) protein. Physiologically, both caspase-8 and iNOS contributed to disease severity in a model of severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection, suggesting iNOS might licence damaging cell death and inflammation during coronavirus disease of 2019 (COVID-19). Secondly, the physiological role of Mind Bomb-2 (MIB2), a recently described pro-survival protein that prevents caspase-8 activation by RIPK1 in cancer cells, is examined using novel MIB2 gene targeted mice. This thesis reveals the physiological function of MIB2 in vivo and examines the function of MIB2 in both inflammation and cancer disease models to determine whether therapeutics designed to inhibit MIB2 could be used to safely activate caspase-8. These studies find that deficiency or inactivation of MIB2 is well-tolerated in mice and does not impact important biological processes including development, haematopoiesis, viability or fertility. Interestingly, challenging MIB2 knockout mice to drive excessive caspase-8 activity leads to enhanced cell death-induced dermatitis, while inactivation of MIB2 limits tumourigenesis in a model of inflammation-driven colorectal cancer. This thesis provides critical insight into the regulation of caspase-8 and uncovers distinct modes of regulation detailing how elevated NO or, inhibition of MIB2 contribute to excessive cell death and disease. This work aids the design of next generation treatments to overcome cell death resistance and transforms our understanding of how caspase-8 is regulated in inflammation and disease.
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ItemThe role of RIPK3 ubiquitylation and MLKL signalling during cell death and autophagy( 2021)Receptor Interacting Serine/Threonine Kinase-3 (RIPK3) is essential for necroptosis, an inflammatory form of programmed cell death pathway implicated in innate immunity, kidney ischemia reperfusion injury, and systemic inflammatory response syndrome. In the classical model, cells committed to necroptosis phosphorylate RIPK1, which in turn drives RIPK3 phosphorylation and oligomerisation. Active RIPK3 oligomers subsequently phosphorylate mixed lineage kinase domain-like protein (MLKL) pseudokinase which induces its translocation to the plasma membrane. The necroptosis pathway culminates in MLKL perforating the plasma membrane as a prelude to cellular rupture and release of inflammatory cytokines and damage-associated molecular patterns to the extracellular milieu. In addition to being a pro-necroptotic kinase, RIPK3 is also capable of triggering apoptosis when its kinase activity is restrained. Moreover, numerous death-independent roles of RIPK3 have been described in the context of inflammation such as arthritis, viral infection, or colitis whereby RIPK3 either promotes or dampens the secretion of pro-inflammatory cytokines. Understanding the molecular regulation of RIPK3 will thereby facilitate the ongoing pre-clinical development of RIPK3 inhibitors. Like most proteins, post-translational modification (PTM) is a critical fine tuner of RIPK3 activities. Ubiquitylation, in particular, has recently garnered attention in the cell death field as loss of this PTM may result in hyperactive RIPK3 which consequently accelerates death and inflammation. However, the post-translational control of RIPK3 signalling is not fully understood. Using mass-spectrometry, I identified a novel ubiquitylation site on murine RIPK3 on lysine 469 (K469). Complementation of RIPK3-deficient cells with a RIPK3-K469R mutant demonstrated that the decoration of RIPK3 K469 by ubiquitin limits both RIPK3-mediated caspase-8 activation and apoptotic killing, in addition to RIPK3 autophosphorylation and MLKL-mediated necroptosis. Unexpectedly, the overall ubiquitylation of mutant RIPK3-K469R was enhanced, which largely resulted from additional RIPK3 ubiquitylation upstream on lysine 359 (K359). Loss of RIPK3-K359 ubiquitylation reduced RIPK3-K469R hyper-ubiquitylation and also RIPK3-K469R killing. Collectively, I therefore propose that ubiquitylation of RIPK3 on K469 functions to prevent RIPK3 hyper-ubiquitylation on alternate lysine residues, which otherwise promote RIPK3 oligomerisation and consequent cell death signalling. I further investigated the consequence of abolishing RIPK3 K469 ubiquitylation by generating Ripk3K469R/K469R mice. In agreement with in vitro findings, primary fibroblasts with mutant RIPK3-K469R enhanced apoptosis, and in vivo studies demonstrate that RIPK3-K469 ubiquitylation contributes to pathogen clearance. Specifically, when Ripk3K469R/K469R mice were challenged with Salmonella enterica serovar Typhimurium, bacterial loads in the spleen and liver were significantly increased relative to wildtype control animals. The increased bacterial burden in the mutant mice was consistent with reduced IFNg produced in the serum, while the elevated MCP-1 cytokine upon infection might be indicative of heightened immune infiltrates. Although necroptosis signalling clearly triggers cell death, how it might impact other cellular responses remains unclear. Therefore, to further delineate the functional outcomes of necroptotic activity I examined how its signalling impacts autophagy. The autophagy pathway is triggered when cells are deprived of nutrients. Although regarded as a pro-survival pathway which acts to recycle and remove damaged organelles, studies have recognised that autophagic pathways can impact cell death processes. In apoptosis, for instance, autophagy acts to limit pro-inflammatory IFN-b secretion, thus decreasing apoptotic immunogenicity. Nonetheless, little is known about the status of autophagy during necroptosis. I demonstrate through various genetic, imaging, and pharmacological approaches that active MLKL translocates to autophagic membranes during necroptosis. However, contrary to previous findings which reported the activation of autophagy upon necroptotic activity based on increased lipidated LC3B, a commonly used marker of autophagy induction, I challenged this conclusion by demonstrating that the accumulation of active LC3B during necroptosis is a consequence of reduced autophagic flux. Therefore, unlike apoptosis which proceeds in tandem with autophagy, the induction of necroptosis negates autophagy in an MLKL-dependent manner. While the function of MLKL-mediated autophagy inhibition warrants further investigation, I propose that attenuating autophagy during necroptosis contributes to the immunogenicity of this cell death modality by limiting the ability of the cell to clear damaged organelles and immunogenic molecules. Overall, my research has helped in outlining how a key necroptotic molecule RIPK3 is regulated post-translationally and how this is relevant in the context of microbial defence. I have also defined novel functional roles for necroptosis signalling in the regulation of autophagic responses. Understanding the molecular regulation of necroptosis signalling and how this cell death pathway is linked to other cellular responses, such as autophagy, is important for the accurate design of new therapeutics to target these pathways in pathological settings.