Abstract
Our immune system is under constant attack of invading pathogens. The initiation of an appropriate immune response that leads to clearance of these microorganisms and infected cells is dependent on the ability of the immune system to discern between harmful invaders and the body’s own constituents. The intracellular Nod Like
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Receptors (NLRs) play an essential role in the specific recognition of microbial products, such as components of the bacterial cell wall and viral RNA, but also recognize danger signals released by infected cells, such as intracellular ATP or mitochondrial proteins. Activation of NLRs by recognition of these ligands results in the upregulation of inflammatory cytokine production and the proteolytic activation of caspase-1. Activated caspase-1 in turn is required for the secretion of the inflammatory cytokines and induces a specific inflammatory cell death called pyroptosis. Ligand recognition by NLRs is thought to induce a series of conformational changes that abrogate the auto-inhibited state of the NLR and finally lead to their multimerization into a large complex called inflammasome. The inflammasome presumably serves as a platform for the recruitment of downstream signaling partners. For a long time it has remained unknown whether or not NLRs directly interact with their ligands, and what the inflammasome looks like. This thesis presents the structural and functional investigation of the activation mechanism of a subset of NLRs, particularly NAIP5 and NLRC4 which are known to specifically respond to the bacterial tail protein flagellin. In vitro structural and functional studies on NLRs have long been hampered by the difficulty to purify them at sufficient amounts due to their inherent tendency to aggregate upon overexpression. We developed a novel method that lead to reduced NLR aggregation upon overexpression and enabled us to perform cellular activation assays as well as to purify small amounts of NAIP5 and NLRC4. We show that NAIP5 directly interacts with a highly conserved region within flagellin and subsequently induces the formation of a caspase-1 activating, hetero-oligomeric NAIP5/NLRC4 inflammasome. Investigation of the purified complex by electron microscopy provided the first insights into the multimeric inflammasome structure, which appeared to be far larger than was thus far expected. Finally, by further structural analysis of an extended helical NAIP5/NLRC4 complex, we confirm that the NLR needs to undergo conformational rearrangement in order to proceed from its inactive state to an inflammasome-incorporated state where it can exert its function. In summary, our results provide the first proof for direct NLR-ligand interaction and suggest a model for the NLR activation mechanism that leads to (hetero-)oligomeric inflammasome formation
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