Elucidating Factors Involved in Inflammasome Regulation - PROJECT SUMMARY/ABSTRACT Inflammasomes are large multiprotein oligomers that assemble in innate immune cells upon detecting danger signals from pathogens or cell dysfunction. Inflammasome formation results in the maturation of proinflammatory cytokines, leading to an inflammatory response as a defense mechanism. However, aberrant inflammasome activity is associated with the perpetuation of inflammation, which causes life-threatening diseases including autoimmune and autoinflammatory disorders, atherosclerosis, and cancer. Therefore, understanding the regulatory mechanisms of inflammasome assembly and activity is key to designing anti-inflammatory therapies. When detecting danger signals, cytosolic pattern recognition receptors prompt inflammasome formation by self- association. The protein AIM2 is a well-known cytosolic sensor that polymerizes upon binding to pathogenic DNA and recruits the inflammasome adaptor ASC, which massively self-associates, forming a filamentous speck. ASC carries two oligomerization domains, PYD and CARD, of the Death Domain family that interact with PYD- containing sensors and the CARD of procaspase-1 via homotypic binding. Thus, ASC acts as a molecular glue connecting the sensor and procaspase. ASC speck formation is required for procaspase activation resulting in the maturation of proinflammatory cytokines. ASC specks are formed several hours after infection, which provides a time window to interfere with inflammasome assembly. However, the factors regulating inflammasome formation and growth are poorly understood. Our laboratory has pioneered structural studies at atomic resolution of the adaptor ASC. We identified ASC-sensor binding interfaces leading to structural models of the assembly. Our biophysical studies on ASC isoforms have shown differential polymerization kinetics and interdomain dynamics which agree with their various capabilities to elicit inflammasome activity observed in cells. Using single-molecule techniques, we have provided mechanistic insight into the assembly of AIM2 on DNA such as oligomer size, shape and growth rates, movement of oligomers on DNA, and directions of oligomer growth. These studies are important for the mechanistic understanding of inflammasome growth. In addition, our recent structural studies on the ASC isoforms have led us to design peptides capable of decreasing inflammasome activity in cells. Overall, our laboratory is well-positioned to study inflammasome regulation at the molecular level. In this proposal, we will determine the effects of sensor and adaptor isoforms on inflammasome assembly, as well as regulatory factors connecting ASC with the effector procaspase-1. We will develop peptides with increased stability and enhanced capability to reduce inflammasome activity in cells, with the potential to serve as scaffolds for drug design. The combination of multiple biophysical and biochemical tools, including NMR spectroscopy, single-molecule techniques, computational methods, and cell-based assays will be key to the successful completion of this proposal.
