Revealing the Mechanisms of Cleaved and Intact GSDMD Pore Formation and Membrane Rupture

ABSTRACT Humans are exposed to hundreds of millions of pathogens daily. The innate immune system is one of two immune arms and the first line of host defense. It quickly recognizes danger stimuli: damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). Sensor proteins including NLRP3 (nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat domain (LRR)-, and pyrin domain-containing protein 3) can detect PAMPs and DAMPs, resulting in the activation of supramolecular protein-signaling complexes called the NLRP3 inflammasome. Upon stimulation, NLRP3 interacts with the centrosomal NIMA-related kinase 7 (NEK7) and oligomerizes into a complex essential for recruiting the ASC adaptor protein, generating functionally active NLRP3 inflammasome. The assembled NLRP3 inflammasome specks recruit and activate caspase-1, a protease that cleaves pro-inflammatory cytokines interleukin (IL)-1β and IL-18 generating active cytokines. In addition to cytokine activation, caspase-1 cleaves the pore-forming protein gasdermin D (GSDMD), facilitating GSDMD-dependent pyroptotic cell death and secretion of IL-1β and IL-18 to release inflammatory signals. While imperative to the inflammatory response and host defense, aberrant hyperactivation or gain-of-function mutations in inflammasome proteins can cause constitutive cytokine activation and pyroptosis, leading to human diseases such as inflammatory bowel disease, cardiovascular disease, gout, and other rare autoinflammatory disorders. Moreover, inhibiting the inflammasome pathway is a therapeutic strategy for preventing lethal septic shock. Despite its therapeutic potential, many mechanistic details underlying the activation of inflammasomes remain unknown. Herein, I propose to elucidate the molecular mechanisms underlying the activation and regulation of the inflammasome executioner protein GSDMD. I aim to interrogate this complex system through a combination of biochemistry, biophysics, and cell biology. Completion of these independent aims will illuminate the mechanisms of how GSDMs are regulated and exert their pore- forming activity, which will not only provide new insights into pyroptosis but also afford new therapeutic strategies for treating inflammasome-related and pyroptosis-related diseases.