Medical Biology - Theses
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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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ItemInhibitor of APoptosis proteins (IAPs) and SHARPIN regulate the immune response in the skin to limit inflammation and maintain homeostasis( 2018)The skin is a remarkable organ, a barricade between our vulnerable insides and a constantly changing environment full of physical, chemical, and biological aggressors. Maintenance of barrier integrity, immune surveillance, and rapid response are fundamental, and this multifaceted protection is orchestrated by the epithelial barrier and immune cells. Acute and chronic inflammatory skin diseases can arise due to abnormal over-reactions to the changing environment. A number of these diseases have been associated with genetic aberrations of the TNF super family and innate receptors signalling. My PhD studies have focused on the role of particular E3 ligases in regulating inflammatory signalling in skin homeostasis and inflammation. Inhibitor of APoptosis proteins (IAPs) and the Linear Ubiquitin-chain Assembly Complex (LUBAC) are E3 ubiquitin ligases that play crucial roles in innate immunity by regulating cell death and survival pathways from the TNF and pattern recognition receptor families. Genetic or pharmacological disruption of the IAPs or LUBAC member SHARPIN induce dermatological phenotypes with interesting parallels to a variety of human skin diseases. To investigate the contribution of immune cells to the Sharpincpdm cutaneous phenotype I utilised the transgenic Diphtheria Toxin Receptor (DTR) system to specifically ablate particular immune cell subsets in-vivo. I have found that Langerhans cells play a pivotal role in the cell death mediated skin disease that arises in Sharpin mutant mice, placing them as a potential cellular source of pathogenic TNF in the Sharpincpdm skin, and highlighting a T-cell independent role for Langerhans cells in driving skin inflammation. Epidermal specific genetic deletion of the cellular IAPs (cIAPs) resulted in early post-natal lethality due to widespread dermatoses. Pharmacological loss of cIAP1, cIAP2 and XIAP by subcutaneous injection of an IAP antagonist drug (smac-mimetic; SM) into mice induced a Toxic Epidermal Necrolysis (TEN) like local inflammatory lesion characterised by keratinocyte cell death, immune cell infiltration, and increased production of pro-inflammatory cytokines. Both the genetic and pharmacological phenotypes can be ameliorated by the loss of a single allele of RIPK1. I have conducted a screen injecting SM into a panel of knock-out and mutant mouse strains in order dissect the complex set of interactions initiated by injection of SM and leading to the TEN like lesional response. I found that disruption of IAPs leads to a breakdown in immune tolerance to commensal microorganisms, which can then initiate inflammatory responses in the skin. A full response to SM depends on interactions between innate immune signalling pathways, immune cells, and the microbiota, nicely highlighting the multifaceted processes involved in skin inflammation and cell death.
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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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ItemA CRISPR/Cas9-based investigation of inflammasomes in infectious disease and autoinflammation( 2017)Inflammasomes are a family of innate immune signalling platforms that are activated in response to tissue damage or infection. Inflammasome stimulation results in activation of the inflammatory protease caspase-1, which induces a lytic cell death program known as pyroptosis, and maturation and release of the pro-inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. The potent inflammatory cascade triggered through activation of the inflammasomes is protective against many bacterial pathogens that either invade host cells or produce toxins that deregulate key homeostatic mechanisms within innate immune cells such as monocytes and macrophages. De-regulation of inflammasome signalling, such as gain-of-function mutations in inflammasome components, can result in autoinflammatory pathology. In order to investigate the function and regulation of inflammasomes, Clustered, Regularly Interspersed, Short, Palindromic Repeats (CRISPR)/Cas9 gene editing technology has been utilised to delete various inflammasome components from human myeloid cell lines or from mice. The alternative inflammatory caspases, caspase-11 in mice and caspases-4 and -5 in humans are activated directly by cytoplasmic lipopolysaccharide (LPS), a key component of the cell wall of gram-negative bacteria. These caspases are able to induce pyroptosis independently of caspase-1, but are only able to trigger IL-1β and IL-18 release in a caspase-1-dependent manner. In this thesis, the roles of caspase-4 and caspase-5 in the response to cytoplasmic lipopolysaccharide (LPS) and invasive gram-negative bacteria have been investigated in a human monocytic cell line. While both caspases responded to infection with live gram-negative bacteria, free LPS that was transfected into the cytoplasm activated only caspase-4. This suggests that caspases-4 and -5 may be activated by distinct stimuli or through different mechanisms. This work also interrogates the role of the inflammasome-forming receptor pyrin, in both autoinflammatory disease and the anti-bacterial immune response. A serine to arginine mutation in pyrin at amino acid position 242 results in a newly described autoinflammatory condition known as Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). A monocytic cell line expressing the S242R mutant of pyrin has been created and it was demonstrated that this mutation results in spontaneous inflammasome activity. Under homeostatic conditions, serine 242 is phosphorylated and interacts with the 14-3-3 family of adapter proteins to keep pyrin inactive. Deletion of specific 14-3-3 isoforms also resulted in spontaneous production of mature IL-1β. Finally, the expression of pyrin in various myeloid compartments and its role in in vivo models of bacterial infection have been investigated using a pyrin-deficient mouse line. Two isoforms of pyrin were detected that were differentially expressed among myeloid populations. Additionally, no role for the pyrin inflammasome was observed in a Dextran Sodium Sulfate (DSS)-induced colitis model, or Citrobacter rodentium, Salmonella Typhimurium or Mycobacterium tuberculosis infection models.
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ItemNeutrophil extracellular trap-associated cell death - role in gout and relationship to alternate forms of cell death( 2017)Cell death has emerged as a critical process in many facets of human disease, ranging from cancer to inflammation and cardiovascular disease. One such modality, Neutrophil Extracellular Trap (NET)-related cell death, or NETosis, is a form of cell death with potential implications in a wide range of human conditions but, at present, understanding of the mechanisms of NETosis and its physiologic and pathological consequences is limited. Finding the key mechanisms underlying NETosis will illuminate roles for NETosis in human diseases and animal models of these conditions, and provide targets for intervention. This thesis examines NETosis in the context of the human inflammatory disease, gout, and explores the relationship between NETosis and the other main types of cell death - necroptosis and apoptosis. First, I developed a novel time-lapse imaging-based quantitative analysis of NETosis. The assay combines multi-well format live-cell microscopy technique with computerized analysis to obtain detailed kinetic information about the two key components of NETosis - cell death and chromatin decondensation. The technique allowed me to demonstrate that different NETosis stimuli, namely phorbol myristate acetate and monosodium urate crystals, exhibit different kinetics of cell death. Exploring the differences further, I found that crystals induce NETosis through a nicotinamide adenine dinucleotide phosphate oxidase-independent pathway that is distinct from PMA-induced NETosis. I also observed that both forms of NETosis depend on neutrophil elastase activity to cause chromatin decondensation, but not cell death. I observed NET-like structures in samples taken from gout patients both during the acute inflammation of a gout attack and from the uninflamed crystal-rich tophus tissue, a feature of chronic gout. The NETs released during MSU-induced NETosis were resistant to serum nucleases relative to PMA-induced NETs, suggesting that these DNA structures may persist within tissues, as seen in the patient samples. This nuclease resistance was at least partially attributable to the presence of increased actin in MSU-induced NETs relative to PMA-induced NETs, as identified using proteomic techniques. Given the presence of NETs in both inflammatory and uninflamed contexts, I demonstrated that the presence of NETs dampens the IL-1β responses of macrophage-like cells and reduces crystal-induced macrophage cell death. I further demonstrated that NETosis is distinct from both apoptosis and necroptosis. Unexpectedly, two MLKL inhibitors did inhibit NETosis, but this is an indirect effect, dependent on accelerating apoptosis. This finding highlighted that over time in culture, neutrophils lose the ability to release NETs when stimulated with PMA and this loss of “NET competence” is mediated by caspase activation. Using the power of a live cell imaging approach to simultaneously quantify cell death and NET release, my studies have advanced our fundamental understanding of the mechanistic differences underlying NETosis induced by different stimuli. Further, by applying this assay, I was able to establish that proteins within the apoptosis and necroptosis pathways that had been previously attributed roles in the NETosis pathway, were in fact dispensable. Consequently, I anticipate this assay and the mechanistic insights it provides will play an important part in advancing our understanding of the NETosis pathway.
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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.