Caspases are cysteine proteases best known for their controlling roles in apoptosis and inflammation. Caspase-6 has recently been shown to play a key role in the cleavage of neurodegenerative substrates that causes Huntington and Alzheimer's Disease, heightening interest in caspase-6 and making it a drug target. All thirteen human caspases have related specificities for binding and cleaving substrate, so achieving caspase-specific regulation at the active site has been extremely challenging if not impossible. We have determined the structures of four unliganded forms of caspase-6, which attain a novel helical structure not observed in any other caspases. In this conformation, rotation of the 90's helix results in formation of a cavity that can function as an allosteric site, locking caspase-6 into an inactive conformation. We are using this cavity to look for chemical ligands that target this cavity and maintain caspase-6 in the inactive, helical conformation. We found that known allosteric inhibitors of caspase-3 and -7 also inhibit caspase-6 through a cavity at the dimer interface. We have determined new structures of a phosphomimetic state and a zinc-bound conformation of caspase-6, which show the molecular details of two additional allosteric sites. The phosphomimetic form of caspase-6 inactivates caspase-6 by disrupting formation of the substrate binding-groove by steric clash of the phosphorylated residue with P201 in the L2' loop. Another allosteric site was found on the "back" of caspase-6 that coordinates a zinc molecule that leads to inactivation. In total we have uncovered four independent allosteric sites in caspase-6, structurally characterized inhibition from these sites and demonstrated that each of these sites might be targeted for caspase-6 specific inhibition by synthetic or natural-product ligands.

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Vascular access dysfunction is a cause of morbidity and mortality in chronic kidney disease (CKD) patients that require hemodialysis. The major cause of vascular access failure is venous stenosis due to neointimal hyperplasia (NH). Vascular smooth muscle cells (VSMC) are critical for the development of NH lesions, as they have the ability to modulate their phenotype from a "contractile" to a "synthetic" phenotype in the presence of uremia, through the regulation of sensor genes for uremia danger signals and VSMC-specific differentiation genes. Recent research indicates that Caspase-1 (casp-1) activation plays an essential role in sensing metabolic danger signal-associated molecular patterns and initiating vascular inflammation. Carbamylated LDL, a uremic toxin that has been shown to be found in higher levels in patients with CKD and in CKD murine models when compared to controls, and could play a role in casp-1 activation. Therefore, the goal of this project is to examine the role of cLDL/CKD-driven casp-1 activation in VSMC and CKD-related NH. We have established a CKD mouse model and published on CKD-associated vascular remodeling. We exposed wild type and caspase-1 knockout mice to our CKD model, analyzed and quantified the NH lesion formed. We also examined in vitro and ex-vivo changes in VSMC-specific differentiation genes when exposed to uremic serum and cLDL, in the presence or absence of caspase-1 inhibitor. We found that CKD serum induces with casp-1 activation and phenotypic changes in VSMCs from a "contractile" to a "synthetic" phenotype, which are reversed with casp-1 inhibition. In an ex-vivo model using relative quantification we found that VSMC contractile markers α -Actin, Calponin, SM-22, and Smoothelin gene expression of CKD mouse carotid VSMC were higher in casp-1 knockout mice when compared to wild-type (1.40, 1.28, 1.22, 1.41 respectively). Also using an in-vivo model, relative quantification of α-actin decreased from 1.0 to 0.329 when VSMCs were exposed to uremic serum and but increased back to 0.588 when Caspase-1 inhibitor is added. The relative quantification of Calponin also decreased from 1.0 to 0.394 when exposed to uremic serum and increased back to 0.601 with caspase-1 inhibitor. We also found that caspase-1 deficiency significantly reversed CKD-related vascular remodeling in casp-1 knockout mice and reduced NH volume by 50% from 1,440,023in wild-type mice to 71,069 µm2 in casp-1 knockouts (p-value 0.002). This evidence provides evidence that casp-1 plays a critical role in NH formation. Furthermore our results provide a novel insight over the therapeutic potential of casp-1 inhibitors for CKD induced NH and other inflammation induced vascular remodeling. / Public Health

It is well established that cytosolic caspase-1 activation, mediated by inflammasome after pathogens-associated molecular patterns (PAMP) and metabolic danger-associated molecular patterns (DAMPs), mediates the initiation of inflammation in endothelial cells by its downstream targets such as Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and Sirtuin-1. However, it remains unknown whether proatherogenic lipids lysophosphatidylcholine (LPC) and reactive oxygen species (ROS) can promote nuclear localization of caspase-1. Using biochemical, bioinformatic, and immunologic approaches, we made the following findings: (1) DNA damage was found in atherosclerotic mice. (2) A nuclear exportation signal was mapped in the CARD domain of pro-caspase-1. LPC promotes nuclear localization of pro-caspase-1 in human aortic endothelial cells (HAECs), which may interrupt DNA damage and repair pathways. (3) Blockage of caspase-1 nuclear cytosol trafficking in HAEC activated by LPC may mediate inflammation and interrupt cell cycle regulation. (4) Pro-caspase-1 in the nucleus inhibits inflammation but promotes interferon pathways. Activation of caspase-1 in the nucleus promotes aging- and fos-related antigen 2 (FRA2) mediated DNA damage and apoptosis. (5) Inhibition of SUMOylation decreases pro-caspase-1 translocation into the cytosol from the nucleus. (6) Blockage of caspase-1 cytosol nuclear trafficking in HAEC activated by H2O2 may decrease caspase-1 activity and increase cell viability. Our results demonstrate, for the first time, that caspase-1 patrols in the cell, senses danger signals and interrupts the balance between DNA damage and DNA repair pathways. It is a novel insight that not only should we suppress the inflammation in the cytosol but also in the nucleus, which is important for the future development of therapeutics for cardiovascular diseases and other inflammatory diseases. / Biomedical Sciences

Zinc is an essential cofactor of several proteins. The roles of zinc in these proteins are classified as catalytic, structural or regulatory. Zinc present in structural sites is considered to be a chemically inert, static structural element. On the contrary, previous studies on a C2H2 type zinc finger model compound and the C3H type HIV-1 NCp7 C-terminal zinc knuckle have shown that zinc at these sites can undergo coordination sphere expansion under the influence of a Pt based electrophile. The pentacoordination observed around zinc in these experiments raises an important question: are the structural zinc motifs found in the proteins susceptible to coordination sphere expansion? Through DFT modeling, the existence and nature of the five coordinate zinc species was investigated. mPW1PW91 was chosen as the DFT method by benchmarking against the experimental parameters of a molecule that closely resembles those to be modeled. The results suggest that the observed coordination sphere expansion is due to the flexible nature of thiolate and chloride ligands that are part of the structure. However, if certain conditions are not met, the same flexibility can lead to the destabilization of these rather fragile structures. Unlike the stable three or four coordinate catalytic and structural zinc sites, at regulatory sites, zinc is typically bound to one or two protein ligands. Zinc inhibition of caspases which are central to the process of apoptosis is one such scenario. Several of the caspases are inhibited by zinc at low micromolar range. Regulation of caspases is a strategy for drug development toward apoptosis related diseases; thus it is important to know the molecular details of zinc inhibition of caspases. Currently, it is speculated that zinc binds to the active site His and Cys residues of caspases thus competing with the substrate. However our studies on caspase-3, using enzyme kinetics and biophysical methods, imply more than one zinc binding sites. Contrary to current beliefs, more than 50% of the inhibition is achieved by zinc without affecting substrate binding. Using DFT models, an alternative inhibitory zinc binding site, which better fits our experimental observations, is predicted.

Doctor of Philosophy / Department of Biology / Rollie J. Clem / Apoptosis is an extremely conserved process among metazoans. This dissertation will describe apoptotic regulation in two orders of insects, Lepidoptera and Diptera. In the lepidopteran host Trichoplusia ni, we describe phenotypes of infection with the baculovirus AcMNPV lacking the caspase inhibitor gene P35. In the lepidopteran host Spodoptera frugiperda, infection with this mutant virus results in apoptosis, which dramatically hinders spread of the virus in the host. In T. ni, however, infection with this mutant virus is similar to wild-type with normal spread, but the end result of liquefaction does not occur. Experiments indicated that infection of T. ni cells with the P35 mutant virus (P35Δ) resulted in caspase activation, and the P35Δ virus lacked the ability to inhibit these active caspases. With the P35Δ virus a slower entry phenotype was observed, but when the P35Δ virus was grown in the presence of a caspase inhibitor the entry phenotype was rescued. This indicated that caspases have detrimental effects on budded virions, and illustrated that P35 is needed to make robust virions. With regards to Diptera, apoptosis-regulatory genes were annotated in the yellow fever mosquito, Aedes aegypti. The genes annotated included multiple caspases and caspase regulators. Phylogenetic relationships were determined among the caspases from Anopheles gambiae, Ae. aegypti and Drosophila melanogaster, expression patterns were determined for all the annotated genes in Ae. aegypti, and one of the genes, an IAP antagonist named IMP, was functionally characterized. Further characterization of the phylogenetic relationships of caspases from fifteen dipteran species was performed by obtaining gene models for caspases of recently sequenced genomes for twelve Drosophila species and three mosquito species. Furthermore, several Drosophila and mosquito species were found to contain caspase genes with substitutions in critical active site residues. These genes were proposed to encode caspase-like decoy molecules. While these have been found in humans and nematodes, this is the first report for these molecules in insects. One of the caspase-like decoy molecules was found to increase the activity of its paralog caspase.

L'inflammasome est une voie de signalisation du système immunitaire inné impliquée dans la lutte contre les pathogènes et notamment dans la réponse aux infections bactérienne. L'activation de l'inflammasome entraine la sécrétion de cytokines pro-inflammatoires et une mort cellulaire caspase-1 dépendante. Des dérégulations de l'inflammasome conduisent aussi à des syndromes auto-inflammatoires graves ; il est donc essentiel de mieux comprendre sa régulation. Francisella tularensis est une bactérie intracellulaire facultative responsable de la tularémie. Son pouvoir pathogène est lié à sa capacité à s'échapper rapidement de son phagosome. Le système de surveillance du macrophage détecte la présence de F. tularensis via l'inflammasome AIM2. La détection de l'ADN bactérien induit la formation d'un large complexe composé de AIM2, le récepteur, d'ASC, l'adaptateur et de caspase-1, l'effecteur ; ce complexe forme un speck visible dans la cellule. Nous avons utilisé l'infection par F. tularensis de macrophages primaires murins pour étudier la régulation de l'inflammasome AIM2 dans un contexte physiologique. Nous avons ainsi identifié une boucle de rétrocontrôle, médiée par la caspase-1, qui régule négativement la formation/stabilité des specks AIM2. Nous avons étudié le rôle de facteurs vacuolaires et des espèces réactives de l'oxygène et de l'azote dans l'activation de l'inflammasome AIM2 lors de l'infection par Francisella. Nous avons ainsi mis en évidence le rôle clef des péroxynitrites dans cette activation. Nos résultats suggèrent que des décomposeurs catalytiques des péroxynitrites pourraient avoir un rôle thérapeutique dans les maladies liées à l'inflammasome / The inflammasome is an innate immune signaling pathway involved in the fight against pathogens. This pathway can also be activated by danger signals. Inflammasome activation induces the release of the pro-inflammatory cytokines IL-1b and IL-18 and cell death in a caspase-1 dependent manner. The inflammasome pathway is a key antibacterial pathway. Deregulation of the inflammasome pathway can lead to serious auto-inflammatory syndromes ; it is therefore critical to better understand inflammasome regulation. Francisella tularensis is a facultative intracellular bacterium responsible for tularemia. Its ability to cause disease is linked to its ability to rapidly escape from the phagosome into the host cytosol where it replicates. The macrophage surveillance system can detect F. tularensis presence in the cytosol through the AIM2 inflammasome. Recognition of DNA induces the formation of a large complex consisting of AIM2, the receptor; ASC, the adaptor and caspase-1, the effector; this complex is visible as a speck within the cell. We used F. tularensis infection of bone marrow derived macrophages to study the activation of the AIM2 inflammasome in a physiological context. We have identified a feedback loop, dependent on caspase-1, negatively regulating speck formation/stability. Then, we studied the role of vacuolar factors and reactive oxygen and nitrogen species in the AIM2 inflammasome activation during Francisella infection. We also described a key role for peroxynitrite in this activation. Our results suggest that catalytic decomposer of peroxynitrite may have a therapeutic potential in diseases linked to inflammasome

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar. Seu processo fisiopatológico é desencadeado pela expressão de fatores de virulência, que proporcionam sua invasão e sobrevivência nas células do hospedeiro, ativando o sistema imune inato e adaptativo da mucosa intestinal. Trabalhos recentes têm salientado a importância do sistema de secreção e da flagelina bacteriana como agonista de receptores da imuninade inata dos macrófagos, em especial alguns dos receptores do tipo NLR. Uma vez que esta espécie de E. coli também é capaz de expressar flagelina e fazer a montagem completa do flagelo e do sistema de secreção do tipo III, a nossa proposta foi avaliar o papel da flagelina e do sistema de secreção de EIEC na resposta imune dos macrófagos murinos. Para isso, utilizamos três cepas de EIEC: a cepa selvagem; a cepa mutante no gene responsável pela síntese da flagelina; e a cepa sem o plasmídio de virulência plnv, deficiente no sistema de secreção, para a infecção de macrófagos peritoniais de camundongos C57BI/6, caspase-1-/-, IPAF-/- e ASC-/-. Neste estudo foi possível observar que o escape bacteriano e a morte dos macrófagos infectados por EIEC, assim como a ativação da caspase-1 e posterior secreção de IL-1β é independente da flagelina bacteriana, mas dependente do sistema de secreção, além disso, a ativação da caspase-1 de macrófagos infectados por EIEC é dependente do receptor IPAF e parcialmente da proteína adaptadora ASC. Assim, no nosso modelo, a ativação da caspase-1 dos macrófagos infectados por EIEC parece estar envolvida com o processamento e secreção de IL-1β e, possivelmente na secreção de IL-18, mas não na morte celular. No modelo de infecção in vivo, o sistema de secreção bacteriano foi importante para a sobrevivência bacteriana no hospedeiro, assim como para a indução de uma resposta inflamatória no local da infecção. Ainda, a caspase-1 parece ter um papel importante para o controle da infecção in vivo por EIEC, podendo assim contribuir para uma resposta imune protetora do hospedeiro. / Enteroinvasive Escherichia coli (EIEC) is one of the etiologic agents responsible for bacillary dysentery. The pathophysiological process induced by this bacteria is triggered by the expression of virulence factors that provide the invasion and survival in host cells, resulting in activation of innate and adaptive immune system present on intestinal mucosa. Recent studies have emphasized the importance of the secretion system and bacterial flagellin as agonist of innate immune receptors present in macrophage, especially NLR (Nod like receptors). Then, our proposal was evaluate the role of flagellin (f1iC) and secretion system of EIEC in the induction of immune response of murine macrophages using the EIEC strains wild type (WT), mutant flagellin gene (f1iC), and a strain deficient in secretion system (DSS) for infection of peritoneal macrophages of C57Bl/6, caspase-1-/-, IPAF-/- and ASC-/-- mice. In this study we observed that the bacterial escape and death of infected macrophages with EIEC, the caspase-1 activation and subsequent IL-1β secretion is independent of bacterial flagellin, but dependent of secretion system, moreover, the caspase-1 activation in infected macrophages is IPAF-dependent and partially dependent of the adapter protein ASC. Thus, in our model, the caspase-1 activation in EIEC infected macrophages seems to be involved with the processing and secretion of IL-1β and possibly with the secretion of IL-18, but not involved with cell death. In the infection model in vivo, bacterial secretion system was important for bacterial survival in the host, as well as for the inflammatory response induction at the infection site. In addition, caspase-1 seems to have an important role to the control of in vivo infection by EIEC and can contribute to a protective immune response of the host.

Les RTKs sont impliqués dans le dialogue au sein des tissus par la régulation de nombreuses réponses cellulaires dont la survie, la prolifération ou la mobilité. Dans les cancers, ces récepteurs sont fréquemment dérégulés notamment par des mutations activatrices. Ainsi, la suractivation des RTKs induit la transformation cellulaire et la tumorigenèse en favorisant par exemple la survie cellulaire. Depuis le début des années 2000, le développement de molécules inhibitrices de l’activité tyrosine kinase (TKI) et d’anticorps bloquant l’interaction ligand/récepteur ont montré que les RTKs représentent des cibles thérapeutiques majeures dans le traitement des cancers.MET est un RTK exprimé par les cellules épithéliales, dont le ligand est l’Hepatocyte Growth Factor/Scatter Factor (HGF/SF). En plus de son rôle pro-survie, MET peut également favoriser l’apoptose en absence de ligand et sous l’effet d’un stress. MET est alors clivé par les caspases et libère dans le cytosol un fragment de 40 kDa nommé p40MET. Ce fragment active la voie intrinsèque de l’apoptose en causant la perméabilisation des mitochondries. Cependant, les mécanismes moléculaires responsables de cette perméabilisation et l’impact physiologique de la fonction pro-apoptotique de MET étaient encore inconnus.Mon travail de thèse a permis de démontrer que le fragment p40MET se localise dans la région des MAMs, constituant l’interface entre le réticulum endoplasmique et les mitochondries, où il favorise un transfert de calcium entre les deux organites. Ce transfert déclenche une surcharge de calcium dans les mitochondries, responsable de leur perméabilisation. De plus, nous avons développé une lignée de souris transgéniques dans lesquelles MET est muté sur l’un des sites caspases. Ces souris sont incapables de produire le fragment p40MET pro-apoptotique. Ce modèle nous a permis de démontrer l'importance du clivage de MET dans l’amplification de l’apoptose in vivo. Ainsi, nos travaux apportent les premières preuves de la fonction de MET en tant que récepteur à dépendance au sein d’un organisme et décrivent un nouveau mécanisme de signalisation pro-apoptotique par la dérégulation des flux calciques.Ces dernières années, la découverte de mutations touchant les RTKs dans les cancers a augmenté de façon exponentielle. Toutefois, pour une grande majorité de mutations, leurs conséquences fonctionnelles sont totalement inconnues. Ainsi, en parallèle de mon principal sujet de thèse nous avons évalué la pertinence biologique et clinique des mutations de RTK identifiées par séquençage haut débit à partir d’échantillons de patients. Le séquençage de tissus sains, de tumeurs colorectales et de métastases hépatiques de 30 patients a permis d'identifier de nombreuses mutations somatiques. Parmi elles, certaines affectent le domaine kinase des récepteurs et sont présentes à la fois dans les tumeurs et les métastases. L’analyse fonctionnelle que j’ai menée sur 7 de ces mutations révèle qu’elles ne provoquent ni la suractivation de la kinase ni la transformation des fibroblastes NIH3T3. Au contraire, deux mutations de RTKs provoquent une inhibition drastique de leur activité kinase. Ces résultats démontrent que ces variants de RTK ne sont pas des cibles appropriées pour l’utilisation de TKI à des fins thérapeutiques et démontre l’intérêt de coupler la recherche de variants à des études fonctionnelles [...] / RTKs are involved in tissue dialogue by regulating many cellular mechanisms such as survival, proliferation or mobility. In cancers, these receptors are frequently deregulated, as a result of various molecular alterations leading to their activation. RTKs overactivation induces cell transformation and tumorigenesis notably by promoting survival. Since the early 2000s, the development of tyrosine kinase inhibitors (TKI) demonstrated that RTKs represent major therapeutic targets in cancer treatment.MET receptor and its ligand the Hepatocyte Growth Factor/Scatter Factor (HGF/SF) are known to promote survival of many epithelial structures during embryogenesis and later during adulthood. Besides pro-survival role of the ligand-activated MET, the receptor is also able to promote apoptosis, which has led to classify it within the dependence receptor family. Indeed, in absence of its ligand and under stress conditions, MET is cleaved by caspases leading to the production of an intracellular fragment of nearly 40 kDa named p40MET able to amplify apoptosis. This fragment activates the intrinsic pathway of apoptosis by causing mitochondrial permeabilization. However, the molecular mechanisms involved in this permeabilization and the physiological impact of the pro-apoptotic function of MET were still unknown.My PhD work has evidenced p40MET localization at the MAM microdomain and characterized a calcium transfer from the endoplasmic reticulum to the mitochondria triggered by p40MET. This calcium transfer triggers a calcium overload in mitochondria leading to their membrane permeabilization and apoptosis. In addition, we engineered a knock-in mouse model expressing mutated MET at the C-terminal caspase site. These mice are unable to produce the pro-apoptotic p40MET fragment. This model allowed us to assess the importance of MET cleavage in physiological apoptosis in vivo. Altogether, our work brings the first evidence for MET function as a dependence receptor in an organism and demonstrates a new signaling mechanism involved in apoptosis amplification by p40MET through calcium flux deregulation. This process may be relevant in the physio-pathology of organs where MET is expressed.In recent years, the discovery of mutations affecting RTKs in cancers has increased exponentially. However, for a large majority of mutations, their functional consequences are totally unknown. Thus, in parallel of my main thesis topic, we evaluated the biological and clinical relevance of RTKs mutations identified by high throughput sequencing from patient samples. Sequencing of healthy tissues, colorectal tumours and liver metastases of 30 patients has identified many somatic mutations. Some of them affect the receptor kinase domain and are present in both tumors and metastases. Functional analysis of 7 of these mutations shows that they do not cause neither kinase overactivation nor transformation of NIH3T3 fibroblasts. On the contrary, two RTK mutations cause drastic inhibition of the corresponding kinase activity. These findings indicate that these RTK variants are not suitable targets for TKI. Therefore, it appears important to set up reliable functional assays to interpret identified variants and classify them as pathogenic or neutral.In conclusion, my work opens up new perspectives on therapeutic strategies targeting RTKs in cancers. First of all, the pro-apoptotic capacities of some RTKs are undoubtedly a brake to tumorigenesis, and their stimulations could reinforce the effectiveness of anti-cancer therapies. On the other hand, we have shown that RTKs mutations in the kinase domain do not necessarily lead to overactivation of the receptor suggesting that they are probably not involved in tumorigenesis and that treatment with TKIs targeting them would be ineffective. This functional information could notably influence the choice of a suitable targeted therapy.