Elucidating the Structural Mechanisms of NAIP Receptors in Bacterial Detection and Inflammasome Activation

Project Summary Inflammasomes are mega-Dalton protein complexes that initiate inflammation responses and play important roles in the innate immune system. Upon activation, inflammasomes recruit and activate the effector protein caspase-1. Caspase-1 in turn cleaves the Gasdermin D (GSDMD) protein to release its N-terminal domain, which inserts into the cell membrane to punch holes on the cell surface. As a result, the host cell will undergo pyroptotic cell death (pyroptosis) and release the cell contents into the extracellular environment. Pro-inflammatory cytokines IL-1 and IL-18, both activated by caspase-1, will also be released to activate the downstream inflammation reactions. NAIP is a family of cytosolic immunological receptors that activate the NAIP/NLRC4 inflammasomes in response to Gram-negative bacterial infections. There are seven NAIP proteins in mouse, each sense a specific ligand such as Flagellin, Needle protein or Inner Rod protein in the type III secretion system. In previous studies, we have shown active NAIPs activate NLRC4 through the nucleated polymerization mechanism. However, it is still largely unknown about how NAIPs remain inactive in the resting cells, and how is the ligand specificity is determined among different NAIPs. Unlike in mouse, humans only have one NAIP and it was shown to be activated by all three bacterial ligands. This leads to many questions including what is the structural basis of the broad detection of bacterial ligands by human NAIP and what does it tell us about the human inflammasomal bacterial detection mechanism? To address these questions, we propose to combine electron cryo-microscopy (cryoEM) with biochemical and functional studies to elucidate the structural mechanisms underlying the signal transduction by NAIP proteins. Dysregulation of NAIP/NLRC4 inflammasomes causes macrophage activation syndrome (MAS) and autoinflammation, and mutations of NAIP protein are highly correlated with spinal muscular atrophy (SMA). The successful execution of this work will broadly advance the development of effective therapies to treat infectious diseases, autoinflammatory diseases, SMA, and cancer through targeting the inflammasome pathway.