98 research outputs found
Déterminants moléculaires impliqués dans l'activation et l'activité de la caspase 7
Les caspases forment une famille de protĂ©ases Ă cystĂ©ine impliquĂ©es dans divers processus comme Fapoptose et lâinflammation. Durant l'apoptose, une forme de mort cellulaire programmĂ©e, les caspases initiatrices (caspases 8, 9 et 10) activent par protĂ©olyse les caspases exĂ©cutrices 3 et 7. Ces derniĂšres cliveront par la suite divers substrats cellulaires pour ainsi mener au dĂ©mantĂšlement contrĂŽlĂ© de la cellule qui est propre Ă l'apoptose. Durant mon doctorat, je me suis intĂ©ressĂ© Ă lâimplication de la caspase 7 durant l'apoptose, autant la façon dont elle est activĂ©e que la maniĂšre dont elle reconnait ses substrats. Dâabord, j'ai voulu Ă©tudier l'implication des sites de clivage du connecteur interdomaine (CID) de la caspase 7 dans son activation par les caspases initiatrices et les dĂ©terminants primaires sous-jacents impliquĂ©s dans cette activation. Cette Ă©tude a rĂ©vĂ©lĂ© l'importance de la localisation du site de clivage dans le CID de la caspase 7 pour une activation adĂ©quate par les caspases initiatrices. La mutagĂ©nĂšse de ces sites nous a permis de constater que la sĂ©quence des sites chez la caspase 7 Ă©tait presque optimale pour son activation par les caspases 8 et 9 et quâil contient des dĂ©terminants importants pour l'activitĂ© enzymatique de la caspase 7. Mes travaux soulignent Ă©galement lâimportance de la longueur du CID pour lâactivitĂ© prĂ©-clivage de la caspase 8, mais pas de la caspase 7. En somme, ces travaux ont permis dâĂ©claircir lâimportance des sites de clivage de caspase 7 pour sa reconnaissance par les caspases initiatrices et pour son activitĂ© catalytique. Ces travaux ont fait l'objet du premier article de cette thĂšse. Puis, je me suis intĂ©ressĂ© Ă la reconnaissance de certains substrats de la caspase 7 par des sites de reconnaissance situĂ©s Ă l'extĂ©rieur de la pochette de liaison du substrat, les exosites. J'ai identifiĂ© un exosite situĂ© dans le domaine N-terminal de la caspase 7 qui est principalement contenu dans une sĂ©quence polybasique de quatre rĂ©sidus lysine (K38-41). Cet exosite amĂ©liore le clivage des protĂ©ines poly(ADP ribose) polymĂ©rase 1 (PARP-1) et p23 sans toutefois changer lâactivitĂ© catalytique basale de la caspase 7. Il est transfĂ©rable sur la caspase 3 et est capable de lier PARP-1 seul. Cet exosite est Ă©galement utilisĂ© lors de l'apoptose pour favoriser le clivage de certains substrats par la caspase 7. Lâexistence de ce site explique les diffĂ©rentes activitĂ©s catalytiques des caspases 7 et 3 sur des substrats apoptotiques prĂ©cis. En somme, ces travaux constituent la premiĂšre dĂ©monstration de l'existence d'exosites chez les caspases. Mes travaux ont donc permis de mieux comprendre la caspase 7, autant les mĂ©canismes de son activation que ceux quâelle utilise pour choisir ces substrats apoptotiques
Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis
During apoptosis, hundreds of proteins are cleaved by caspases, most of them by the executioner caspase-3. However, caspase-7, which shares the same substrate primary sequence preference as caspase-3, is better at cleaving poly(ADP ribose) polymerase 1 (PARP) and Hsp90 cochaperone p23, despite a lower intrinsic activity. Here, we identified key lysine residues (K38KKK) within the N-terminal domain of caspase-7 as critical elements for the efficient proteolysis of these two substrates. Caspase-7âs N-terminal domain binds PARP and improves its cleavage by a chimeric caspase-3 by !30-fold. Cellular expression of caspase-7 lacking the critical lysine residues resulted in less-efficient PARP and p23 cleavage compared with cells expressing the wild-type peptidase. We further showed, using a series of caspase chimeras, the positioning of p23 on the enzyme providing us with a mechanistic insight into the binding of the exosite. In summary, we have uncovered a role for the N-terminal domain (NTD) and the N-terminal peptide of caspase-7 in promoting key substrate proteolysis
Dimerization and auto-processing induce caspase-11 protease activation within the non-canonical inflammasome
Caspase-11 is a cytosolic sensor and protease that drives innate immune responses to the bacterial cell wall component, LPS. Caspase-11 provides defence against cytosolic Gram-negative bacteria; however, excessive caspase-11 responses contribute to murine endotoxic shock. Upon sensing LPS, caspase-11 assembles a higher order structure called the non-canonical inflammasome that enables the activation of caspase-11 protease function, leading to gasdermin D cleavage and cell death. The mechanism by which caspase-11 acquires protease function is, however, poorly defined. Here, we show that caspase-11 dimerization is necessary and sufficient for eliciting basal caspase-11 protease function, such as the ability to auto-cleave. We further show that during non-canonical inflammasome signalling, caspase-11 self-cleaves at site (D285) within the linker connecting the large and small enzymatic subunits. Self-cleavage at the D285 site is required to generate the fully active caspase-11 protease (proposed here to be p32/p10) that mediates gasdermin D cleavage, macrophage death, and NLRP3-dependent IL-1ÎČ production. This study provides a detailed molecular mechanism by which LPS induces caspase-11-driven inflammation and cell death to provide host defence against cytosolic bacterial infection
Mathematical modelling of activation-induced heterogeneity in TNF, IL6, NOS2, and IL1ÎČ expression reveals cell state transitions underpinning macrophage responses to LPS
Background: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we aimed to develop mathematical models to explore theoretical cellular states underpinning the empirically observed responses of macrophages following lipopolysaccharide (LPS) challenge. Methods: We obtained empirical data following primary and secondary responses to LPS in two in vitro cellular models (bone marrow-derived macrophages or BMDMs, and RAW 264.7 cells) and single-cell protein measurements for four key inflammatory mediators: TNF, IL-6, pro-IL-1ÎČ, and NOS2, and used mathematical modelling to understand heterogeneity. Results: For these four factors, we showed that macrophage community AIH is dependent on LPS dose and that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical three-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a four-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS. Conclusions: Overall, we provide a mathematical model for studying macrophage ecology and community dynamics that can be used to elucidate the role of phenotypically negative macrophage populations in AIH and, primary and secondary responses to LPS
Great balls of fire : activation and signalling of inflammatory caspases
Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through diverse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling
Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria
The human non-canonical inflammasome controls caspase-4 activation and gasdermin-D-dependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of non-canonical inflammasome signaling
Response Inhibition and Error Monitoring during a Visual Go/No-Go Task in Inuit Children Exposed to Lead, Polychlorinated Biphenyls, and Methylmercury
Background: Lead (Pb) and polychlorinated biphenyls (PCBs) are neurotoxic contaminants that have been related to impairment in response inhibition
"It's Like the Pieces of a Puzzle That You Know": Research Interviews With People Who Inject Drugs Using the VidaviewTM Life Story Board
Bei dem Life Story Board (LSB) handelt es sich um ein visuelles Tool, das in therapeutischen Kontexten zum Einsatz kommt, um die Lebenswelt zu ko-konsturieren, die die persönlichen, relationalen und zeitlichen Aspekte individueller gelebter Erfahrung umfasst. In unserer Studie zu Drogennutzung und Schadensreduzierung interviewten wir Menschen, die Drogen injizieren unter Einsatz des LSB, um herauszufinden, ob sich hieraus Potenziale fĂŒr eine verbesserte qualitative Forschung ergeben könnten. In unserem Forschungsteam arbeiteten neben Akademiker*innen auch frĂŒhere oder aktuelle Drogenkonsument*innen mit. Interviews wurden von jeweils zwei Personen gefĂŒhrt: eine agierte als Interviewer*in, die andere war fĂŒr das LSB zustĂ€ndig.Entlang der Ergebnisse war nachvollziehbar, dass Interviewende und Interviewte in unterschiedlicher Weise mit dem LSB interagierten: WĂ€hrend die Interviewer*innen es nutzten, um sich im Leitfaden zu orientieren, half es den Befragten, die eigene Lebensgeschichte mittels einer Vielzahl an emotionalen und kognitiven ĂuĂerungen zu validieren oder zu unterstreichen. Das LSB erlaubte, sich an spezifische Situationen oder VorfĂ€lle zu erinnern, Perspektiven hinzuzugewinnen und der eigenen Geschichte zusĂ€tzlichen Sinn zu verleihen. Insoweit arbeiteten Interviewte und Interviewende unter jeweils unterschiedlichen Vorzeichen mittels des LSB gemeinsam an einer (Re-)PrĂ€sentation der jeweiligen Lebensgeschichte.The Life Story Board (LSB) is a visual tool used in therapeutic circumstances to co-construct a lifescape that represents the personal, relational and temporal aspects of a person's lived experiences. We conducted a study of the drug use and harm reduction experiences of people who inject drugs through research interviews using the LSB to determine whether it has the potential to enhance qualitative research. Our team included community researchers who were current or former drug users and academic researchers. Interviews were conducted by two community researchers: an interviewer and a storyboarder who populated the LSB.Results showed that interviewers and participants interacted with the LSB in different ways. The board functioned to situate the interviewers in the interview schedule, whereas participants often used the board as a way to validate or reinforce their life story. Participants expressed a variety of emotional and cognitive responses to the board. Overall, the LSB helped participants focus on their life story to recall specific occasions or incidents and enabled them to gain perspective and make greater sense of their lives. Both participants and interviewers engaged with the LSB in nuanced ways that enabled them to work together to represent the participant's life story
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