Medical Biology - Theses
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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.
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ItemControl of the Intrinsic Pathway of Apoptosis( 2019)Apoptosis is a cellular process of programmed cell death. The intrinsic pathway of apoptosis is triggered by mitochondrial outer membrane permeabilization, a point of no return that coincides with the release of cytochrome c into the cytosol where it activates the main effectors of cellular destruction: the caspases. The mitochondrial pathway that is centered on MOMP is tightly regulated by BCL2 family proteins, which includes some members that promote apoptosis and others that inhibit it. The interplay between these proteins with opposing roles determines whether a cell will die or survive. In a healthy cell, pro-survival BCL2 proteins inhibit the effector proteins BAX and BAK. BH3-only proteins are activated in response to cellular stress and promote apoptosis by neutralizing pro-survival proteins. Targeting BCL2 proteins to provoke apoptotic cell death has proven to be a successful strategy for cancer therapy with the BCL2-selective drug venetoclax exhibiting remarkable efficacy in treating cancers that rely on BCL2 for their survival. MCL1, a protein related to BCL2, is likewise critical for the survival of many cancer cells, making it another attractive anti-cancer drug target. Selective MCL1 inhibitors have been developed and are currently being evaluated in clinical trials to establish their safety and efficacy. Safety is a particular concern for MCL1 inhibitors because MCL1 is also essential for the survival of many cells in critical organs and tissues throughout the body. It remains to be seen if a sufficient therapeutic window will exist when MCL1 is targeted systemically. An alternative and potentially safer strategy to modulate MCL1 survival function would be to target pathways that regulate its activity in particular contexts. In Chapter 3 and 4, I focus on one such mechanism of MCL1 regulation: its turnover by the ubiquitin proteasome system. My work in Chapter 3 elucidated details of how MCL1 protein turnover is regulated by BH3-only protein NOXA. Using CRISPR-Cas9 screen, I discovered that the mitochondrial E3 ligase MARCH5, the E2 conjugating enzyme UBE2K and the mitochondrial outer membrane protein MTCH2 co-operate to mark MCL1 for degradation by the proteasome. I also demonstrated that this pathway is constitutively active in cells where NOXA is abundantly expressed and showed that manipulating NOXA expression in those cells impacts on MCL1 survival function. Having successfully demonstrated the power of CRISPR-Cas9 screen in Chapter 3, I undertook further screens in Chapter 4 to identify proteins, such as deubiquininating enzymes (DUBs), that might serve to enhance MCL1 protein stability. I did not identify any strong hits from these screens, possibly because multiple DUBs act redundantly on MCL1. Consistent with this hypothesis, only mild impacts on MCL1 protein stability were observed upon deleting DUBs previously reported to act on MCL1. Finally, in Chapter 5, I investigated how BH3 mimetics mimic the activity of BH3-only proteins to induce apoptosis. I studied how selective BH3 mimetic compounds perturb interactions throughout the BCL2 protein network beyond their direct protein targets. I showed that these second order impacts are crucial for effective killing. Apoptosis induced by the BCL2 selective inhibitor venetoclax, for example, typically also involves inhibition of MCL1. The impact on MCL1 in this context occurs as a consequence of displacing BH3-only proteins normally bound to BCL2.
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ItemDefining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection( 2019)Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.
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ItemAnalysing the impact of the absence of CARD containing caspases on different forms of cell death( 2018)Cell death is an important process during embryogenesis as well as tissue homeostasis in the adult. Apoptosis, pyroptosis and necroptosis are three of the major programmed cell death pathways. Dysregulation of either of these cell death pathways can promote the development of a variety of diseases, such as cancer or autoimmune pathologies. Cysteine-dependent aspartate-specific proteases, known as caspases, exert key functions in all of these cell death pathways. Of note, certain caspases have been shown to play a role in more than one cell death pathway. This thesis presents the functional analysis of different caspases, in particular caspase activation and recruitment domain (CARD) containing caspases and their contributions to the pyroptotic, apoptotic and other cell death pathways. We have generated a novel triple knockout mouse strain deficient for the CARD containing caspases-1, -11 and -12. We initially used this strain to improve our understanding on the contributions of caspases-1, -11 and-12 to sepsis and different forms of cell death. Previous studies have suggested a role for caspase-12 in endoplasmic reticulum (ER) stress-induced cell death. However, we were not able to attribute a role of caspase-12 to sepsis or ER stress-induced apoptosis in vitro and in vivo. In Chapter 4 we present a study on the roles of different caspases as well as RipK3 during Salmonella infection in vitro and in vivo. There is evidence for a substantial functional overlap between different cell death pathways in the cellular response to pathogens, such as Salmonella. We examined this functional overlap of different cell death processes in the organismal and cellular response to infection by generating mice deficient for multiple caspases and also RipK3, an essential mediator of necroptotic cell death. Upon infection with S. Typhimurium SL1344 strain, primary myeloid cells from caspase-1/11/12/8 RipK3-/- mice showed marked resistance to cell death and survived even at high bacterial loads for up to 24 hours. When infecting the caspase-1/11/12/8 RipK3-/- mice with the vaccine Salmonella Typhimurium strain, they were not able to clear the bacteria from primary organs. Collectively, these findings provide evidence that there is substantial functional overlap between the different cell death pathways and hence the caspases involved in these processes in the cellular as well as organismal response to infection with S. Typhimurium and possibly other pathogens. Lastly, I generated mice lacking all murine CARD containing caspases, i.e. caspase-1, -11, -12, -2 and -9. These preliminary analyses revealed no major defects when comparing the embryonic development of mice lacking caspases-1, -11, -12, -2 and -9 to wildtype. Furthermore, we isolated haematopoietic stem and progenitor cells (HSPCs) from foetal livers derived from caspase-1/11/12/2/9 deficient mice and reconstituted lethally irradiated wildtype mice. Surprisingly, we did not find notable defects in the lymphoid and myeloid compartments in the caspase-1/11/12/2/9 deficient mice at steady state. In thymocyte cell death assays, cells from the quintuple caspase knockout mice still could undergo cell death, induced by the cytotoxic agent ionomycin, albeit at a delayed rate.
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ItemDissecting the role of TNF signalling in Mycobacterium tuberculosis disease pathogenesis to identify novel therapeutic targets( 2018)Mycobacterium tuberculosis (Mtb) is a formidable public health challenge, with a global epidemic, fuelled partly by rampant antibiotic resistance, that has the medical community grappling with more infected individuals than at any other time in history. Mtb is remarkable in its ability to efficiently disarm its primary host cell, the macrophage. One of our most crucial immunological defences against this highly skilled pathogen is the cytokine tumour necrosis factor (TNF), which can promote either cell survival or programmed cell death via apoptosis or necroptosis, depending on the cellular context. Given this essential role, TNF and its downstream pathways represent attractive therapeutic targets for tuberculosis (TB). Despite decades of research, however, fundamental insights into the means by which TNF mediates host protection remain elusive and have been hampered by reports of a pathological role of this cytokine in TB. The aim of this thesis is to systematically dissect the various components of TNF signalling and their impact on Mtb disease outcomes in order to identify aspects of the pathway that may be amenable to therapeutic intervention. This is achieved using a cutting-edge genetic approach and physiologically-relevant animal models of TB. Recent work suggested that TNF induces programmed forms of necrosis in Mtb-infected macrophages, thus promoting Mtb pathogenesis by facilitating mycobacterial escape and dissemination. In Chapters 3 and 4, I show that neither necroptosis, dependent on mixed lineage kinase domain-like (MLKL), nor a previously-undescribed death modality dependent on receptor-interacting protein kinase 3 (RIPK3) and B cell lymphoma-extra large (BCL-XL), are responsible for macrophage death during Mtb infection, and do not contribute to disease progression. This is in spite of the observation that the former pathway is strongly primed upon infection, suggesting that necroptosis is favoured by Mtb but ultimately restricted by the host. In contrast to lytic death, apoptosis of infected cells is considered beneficial to the host as the process is intrinsically microbicidal. In Chapter 5, I show that TNF is the primary death ligand driving the extrinsic apoptotic death pathway in infected macrophages during Mtb infection. Furthermore, I demonstrate that this pathway is beneficial in terms of eliminating intracellular bacilli and promoting the activation of adaptive immunity. Having established that apoptosis is protective, I postulate in Chapter 6 that the ability to pharmacologically modulate this process presents a potential therapeutic opportunity. Inhibitor of apoptosis (IAP) protein antagonists promote programmed cell death upon death ligand stimulation. I show that clinical-stage IAP antagonists selectively promote the apoptotic death of Mtb-infected macrophages in mice, and that this promotes the clearance of Mtb. I also extend these findings to infections caused by Burkholderia pseudomallei, in which a single dose of IAP antagonists completely eliminated the pathogen from the lungs. In summary, this thesis demonstrates that host TNF overwhelmingly promotes signalling pathways that are protective against Mtb. This refutes prior work suggesting that regulated necrosis is induced by TNF, and that advocated for the use of inhibitors of these pathways for the treatment of TB. The insights gained from this work have, however, led to the identification of a viable therapeutic strategy for Mtb and other intracellular pathogens, based on the finding that TNF-driven apoptosis of infected cells is beneficial to the host and can be harnessed with clinical-stage pharmaceuticals.
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ItemStructural investigations of pro‑apoptotic Bcl‑2 family proteins( 2017)The Bcl‑2 protein family regulates the intrinsic apoptotic pathway through an intricate network of protein:protein and protein:membrane interactions. The pathway culminates in the permeabilisation of the mitochondrial outer membrane by the pro‑apoptotic effector proteins Bak and Bax, an event that irreversibly commits a cell to death. To facilitate membrane permeabilisation, Bak and Bax undergo a series of conformational changes to convert from inert monomers to membrane‑embedded homodimers that nucleate and propagate apoptotic oligomers. While great strides have been made in structurally characterising these conformational changes, questions remain surrounding homodimer interactions with the membrane, oligomerisation, and membrane pore formation. This thesis addresses these questions by providing structures of lipids bound to Bak BH3:groove core homodimers (Chapter 2). These are the first structures of any Bcl‑2 family protein in complex with lipid. They reveal symmetric binding sites for phospholipid headgroups and acyl chains. In one structure, adjacent Bak homodimers are cross‑linked by the acyl chains of single phospholipids, suggesting homodimer oligomerisation could be mediated by lipid. Bak oligomers could be dissociated with phospholipase A2, supporting a role for lipid in oligomer stability. Collectively, the structures presented here indicate that lipids may play a direct role in Bak oligomerisation. Like Bak, Bax homodimerises and oligomerises on the mitochondrial outer membrane. The original Bax BH3:groove core homodimer structure was solved as a GFP fusion at low resolution. Here, a tetrameric structure consisting of two Bax BH3:groove core homodimers alone was solved at high resolution (Chapter 3), providing details for canonical interactions in atomic detail. A crystal structure of Bax BH3:groove core homodimers containing lipid was also solved, although the structure could not be refined due to severe twinning. This result demonstrates that Bax core domains also associate with lipid, and provides a starting point for crystal optimisation. Pro‑survival Bcl‑2 family proteins antagonise the apoptotic function of Bak and Bax by preventing their activation and sequestering their activated forms. Sequestration of activated Bak and Bax in heterodimeric Mode 2 complexes involves binding of the Bak/Bax BH3 domain to a conserved hydrophobic groove. Beyond this, little is known regarding the topology of these complexes. The pro‑survival protein Bcl‑XL can undergo similar conformational changes to Bak and Bax, but whether it forms BH3:groove heterodimers with Bak/Bax was unknown. Using cysteine cross‑linking on mitochondria, I show that Bcl‑XL can form reciprocal BH3:groove heterodimers with Bax, and possibly Bak (Chapter 4). These results challenge a simplistic view of Mode 2 complexes, implicating more extensive interactions beyond the canonical BH3 in groove interface. Bok is a third potential pro‑apoptotic effector protein that shares sequence similarity with Bak and Bax, but its role in apoptosis remains unresolved. To investigate the structure and function of Bok, I developed a recombinant expression system to produce human, rat, and chicken Bok. The first crystal structure of Bok, from the chicken, reveals the canonical Bcl‑2 family fold, with deviations that may explain its proposed constitutive activity (Chapter 5). The structure paves the way for mutagenesis studies that will further our understanding of this enigmatic protein.
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ItemPro-apoptotic therapies for the treatment of Mycobacterium tuberculosis infection( 2017)One third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) has killed more than 1 billion people over the past two hundred years, surpassing mortality caused by all other pandemics and epidemics combined. Despite a concerted global effort to reduce transmission, Mtb infects an estimated 10.4 million people and kills 1.4 million people each year. Managing this condition is becoming increasingly challenging because Mtb is fast becoming resistant to all first line antibiotic therapies. Novel interventions beyond iterations on antibiotics are required. Understanding host-Mtb interactions, with a view to targeting host signalling pathways that the organism is reliant upon, is a tenable approach to combatting this deadly disease. Host cells are intolerant of intracellular organisms and consequently Mtb must prevent a cell from dying so that it has time to propagate and disseminate. Exactly how it does so is controversial and poorly understood. In my work, I sought to understand the role of apoptosis in Mtb disease pathogenesis. I dissected the role of the extrinsic apoptotic pathway and associated key molecular components including inhibitor of apoptosis (IAP) proteins. I also examined the role of the intrinsic apoptotic pathway and associated Bcl-2 family of proteins. I found that Mtb infected mouse and human macrophages showed major aberrations in the protein expression levels of IAP and Bcl-2 family molecules such that the stoichiometry of these proteins strongly favoured cell survival. I infected mice that were deficient in the three major mammalian IAPs (cIAP1, cIAP2 andXIAP) and found that in the absence of cIAP1, and to a lesser extent cIAP2, Mtb infected macrophages died and disease pathogenesis was strikingly altered. I then sought to reprogram the extrinsic apoptotic pathway to promote death of Mtb infected cells by using a clinical stage drug inhibitor of cIAPs. I was able to optimise a dosing regimen of the cIAP antagonist, birinapant, that proved efficacious in killing Mtb infected macrophages and in reducing bacterial loads in various strains of mice and in mice engrafted with a human immune system. An examination of the intrinsic cell death pathway also proved very interesting. Again, I used a combination of gene-targeted mice and clinical stage drugs to antagonise the function of several Bcl-2 family pro-survival proteins. Interfering with Bcl-xL function had no effect on Mtb disease pathogenesis whilst antagonising the function of Bcl-2 made the disease worse. Notably, as little as a 50% reduction in Mcl-1 function, examined using Mcl-1+/- mice, produced an improvement in Mtb infection outcomes. Given the success in defining targetable host cell pathways involved in TB pathogenesis, I next investigated if these insights were applicable to latent Mtb infection. A large proportion of people infected with Mtb may not progress to overt disease but remain latently infected and can reactivate disease under certain circumstances. There are well defined indications for treating some people who are latently infected. Treatment of latent infection suffers from the same shortcomings as treatment of active disease. I found that birinapant could be optimised to also impact on latent infection. The significance of my work includes providing valuable insights into how apoptosis plays a critical role in determining Mtb infection and disease outcomes. I believe that my work, for the first time, has identified the key molecular components that regulate cell survival / apoptosis signalling during Mtb infection. The importance and implications of these findings are underscored by my preclinical studies showing that these host cell molecules and pathways can be targeted using clinical stage drugs to promote clearance of Mtb infection and disease.
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ItemInvestigation of cell death pathways in response to TNF and IFNγ( 2017)During my PhD I investigated the regulation of the TNF and IFNγ signalling pathways and their ability to induce cell death. IFNγ is a critical cytokine in the immune response against viral and intracellular bacterial infections. It has also been associated with auto-inflammatory and auto-immune disorders (Pollard et al., 2013; Zhang, 2007), where it was found upregulated together with other pro-inflammatory cytokines like TNF (Ohmori et al., 1997). TNF signalling and the mechanism of cell death induction downstream of the TNF receptor complex has been investigated in detail over the past 4 decades. Although IFNγ was first described 50 years ago, and before TNF, significantly fewer IFNγ signalling components have been discovered compared to the TNF signalling complex. Nevertheless, both cytokines induce equally potent and potentially dangerous systemic responses at low concentrations and must be tightly regulated. I therefore hypothesised that additional IFNγ signalling regulators must exist. In order to discover such novel regulators of the IFNγ signalling pathway I enriched for the IFNγ receptor and identified binding partners using mass spectrometry. Using this approach I identified SPTLC1 and 2, which are two subunits forming the serine palmitoyltransferase, directly interacting with the IFNγ receptor chain 2 (IFNGR2) constitutively. Weak interaction between SPTLC1/2 and IFNGR1, however, was only detected upon IFNGR complex formation induced by IFNγ stimulation suggesting that IFNGR1 interacts with SPTLC1/2 indirectly via IFNGR2. SPTLC2 deficient single cell mouse dermal fibroblast showed either normal or increased phosphorylation of STAT1 upon IFNγ stimulation and lack of SPTLC2 had no impact on transcription of classical IFN target genes. Secondly, I investigated the mechanism of cell death induced by IFN in combination with Smac-mimetics, a group of small molecule inhibitors of the inhibitor of apoptosis proteins (IAPs), which have been heavily investigated in context of TNF signalling. Previous studies revealed that inhibition of IAPs renders cells sensitive to TNF induced cell death, which is primarily apoptosis mediated by caspase-8. However, inhibition of caspase-8 by caspase inhibitors triggers an alternative cell death pathway called necroptosis. Here I found that the combination of IFN/Smac-mimetic had a similar impact on survival and, more precisely, induced RIPK3 dependent caspase-8 mediated apoptosis in mouse dermal fibroblasts. Surprisingly, IFN/Smac-mimetic induced killing in HT29 cells was not blocked by deleting caspase-8 and effectors of the necroptotic pathway like RIPK3 and MLKL. In contrast, deficiency of RIPK1 largely protected cells from IFN induced cell death, indicating that a novel form of RIPK1 dependent cell death was being induced. In trying to discover the mechanism we observed that caspase-10 was significantly upregulated by IFN and activated by IFN/Smac-mimetic treatment. HT29 cells deficient for caspase-10, caspase-8 and either MLKL or RIPK3 were completely resistant to IFN/Smac-mimetic revealing an important role for caspase-10 in IFN/Smac-mimetic induced killing. Thirdly I focused on the activation and function of MLKL, the most downstream member of the necroptotic pathway known. Necroptosis has been best studied downstream of the TNF signalling complex, upon IAP and caspase inhibition. We and others propose a model where phosphorylation of the MLKL pseudokinase domain by RIPK3 triggers a molecular switch, leading to exposure of MLKL’s N-terminal four-helix bundle domain, its oligomerisation, membrane translocation, and ultimately cell death. We additionally identified novel phosphorylation sites S158, S228, S248. By mutating these sites and overexpressing phosphomimetic and -ablating MLKL mutants in Mlkl-/- or Ripk3-/-/Mlkl-/- deficient murine fibroblasts I demonstrated that these sites influence MLKL activity and discovered a potential inhibitory effect of RIPK3 on cell death induced by MLKL. Finally, I examined the evolutionarily conservation of the necroptosis inducing activity of MLKL by analysing the function of MLKL orthologs. While the intrinsic ability to lyse membranes, which was tested in liposome assays, is highly conserved, several MLKL orthologs including human MLKL failed to induce cell death when expressed in murine fibroblasts. This suggests the presence of additional poorly conserved, species-specific factors that inhibit or activate MLKL.
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ItemStructural transitions during cell death: bak activation and oligomerisation( 2015)Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This thesis investigates structural transitions occurring to Bak during apoptosis. I present crystal structures of a Bak core/latch dimer and demonstrate the dissociation of the core and latch domains upon Bak activation. I provide the first high-resolution details for the core domain dimer, a subunit upon which the larger Bak oligomer builds. Cellular assays, guided by the presented crystal structures, confirm the physiological relevance of these key events in the intrinsic apoptotic pathway (Chapter 2). I also describe the first crystal structures of Bak in complex with the BH3-domain of Bim (Chapter 3). These studies complement previous work performed on Bax and support an analogous mechanism of activation and oligomerisation. Certain detergents have been reported to activate Bak in vitro. Here I demonstrate that some detergents can oligomerise Bak and/or promote hetero-complexes between Bak and the pro-survival protein Mcl-1. I describe the production of homo-oligomeric and hetero-oligomeric complexes of Bak, which may be amenable to structural studies (Chapter 4). The literature on apoptosis assumes that mouse and human Bak are analogous in structure and therefore function. Here I report structural differences between Bak homologs from these two species (Chapter 5). These differences exist at the site of ligand binding, yet it remains unclear whether they result in functional variations. These data may aid in the development of novel agonists and antagonists of Bak, and could prove fundamental to designing murine-based pre-clinical trials. The BH3-only protein from the Schistosoma mansoni worm has been identified as a potential direct activator of Bak and Bax. This direct activator BH3-only protein is unique as it only binds to one of the pro-survival proteins (Mcl-1). Here I describe the crystal structure of the Schistosoma BH3-domain (sBH3) in complex with Bax. Liposome release assays demonstrate that sBH3 can directly activate Bak and Bax and induce the formation of membrane permeabilising oligomers (Chapter 6). These studies support current models for the activation and oligomerisation of Bax. Defining the structural characteristics of the intrinsic apoptotic pathway provides novel opportunities for drug design. Organic agonists of Bak may prove useful for the treatment of cancers, while antagonists could provide therapy for diseases characterised by excessive cell death.
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ItemStudies of the role of Mcl-1 in haemopoiesis and leukaemia( 2015)Cell death by apoptosis plays a critical role during embryonic development and in maintaining tissue homeostasis. Consequently, defective apoptosis can lead to degenerative diseases, autoimmunity and tumour development. In mammals, there are two converging apoptosis pathways: the ‘extrinsic’ pathway, which is triggered by engagement of cell surface ‘death receptors’ such as Fas; and the ‘intrinsic’ pathway, which is triggered by diverse cellular stresses, and is regulated by pro- and anti-apoptotic members of the Bcl-2 family of proteins. The principal focus of my studies is Mcl-1, an inhibitor of the intrinsic apoptosis pathway. Mcl-1 is overexpressed in a variety of cancers, including acute myeloid leukaemia (AML) where high levels of Mcl-1 are associated with poor prognosis and drug resistance. Using mouse genetic models, I have investigated the consequences of overexpression of Mcl-1 for haemopoiesis and autoimmunity (Part I) and for the development and treatment of AML (Part II). I. To determine the impact of simultaneously inhibiting the intrinsic apoptosis pathway via overexpression of Mcl-1 and the extrinsic apoptosis pathway via a non-functional Fas receptor, mcl-1 transgenic mice were crossed with faslpr/lpr mice. The combined mutations had little impact on myelopoiesis apart from an increase in macrophages, mainly in the spleen. All major lymphoid subsets were elevated, however, including the “unusual” T cells characteristic of faslpr/lpr mice. Furthermore, the onset of autoimmune disease was markedly accelerated. Thus, consistent with other genetic studies, the intrinsic and extrinsic apoptosis pathways synergise to control autoimmunity. II. To determine the impact of Mcl-1 in AML, I used a mouse model induced by retroviral expression of MLL-AF9, the fusion oncoprotein created by the t(9;11) translocation often found in childhood and treatment-induced adult AML. Overexpression of Mcl-1 or its pro-survival relative, BCL-2, increased the leukaemic burden in the spleen and blood of sick mice although it did not accelerate morbidity. AMLs overexpressing Mcl-1 or BCL-2 tended to have a higher proportion of mature cells compared to ‘wild type’ MLL-AF9 leukaemias. Unlike ‘wild type’ MLL-AF9 leukaemias, which were readily transplantable in non-irradiated recipients, most MLL-AF9 leukaemias overexpressing Mcl-1 and many overexpressing BCL-2 would only transplant if injected into lightly-irradiated recipients. Possible reasons for this unexpected result are discussed. In vitro experiments using short-term lines derived from primary tumours demonstrated that overexpression of Mcl-1 or BCL-2 in MLL-AF9 tumours increased resistance to standard drugs used to treat AML in the clinic. However, even those overexpressing Mcl-1 or BCL-2 were sensitive to the proteasome inhibitor, bortezomib, and to various CDK inhibitors as single agents. The addition of the BH3-mimetic ABT-737 enhanced the response of MLL-AF9 AMLs of all genotypes to standard therapeutics. In contrast, when added to bortezomib or CDK inhibitors, ABT-737 only enhanced the sensitivity of the AMLs that overexpressed BCL-2. Future studies will compare the efficacy of these drug regimens in vivo in transplanted syngeneic immuno-competent mice.