Dissecting the Molecular Mechanisms of NLRP3 Inflammasome Activation and Regulation.

Abstract This research proposal aims to elucidate the mechanisms underlying the activation and regulation of the NLRP3 inflammasome, focusing on conformational changes, post-translational modifications (PTMs) such as ubiquitination and lipidation, and novel regulatory factors. These regulatory mechanisms ensure precise activation and prevent excessive inflammation that could lead to tissue damage or chronic inflammatory diseases. By understanding these regulatory processes, we can gain valuable insights into NLRP3 biology and its role in various diseases, ultimately aiding in the development of targeted therapies for inflammatory and autoimmune conditions. Significant progress has already been made towards these aims, including high-resolution cryo-EM map and identification of novel regulators through mass spectrometry. My preliminary data have already revealed new conformational states of NLRP3, and identified specific ubiquitination and lipidation sites on NLRP3 that may be crucial for its activation. The K99 phase will focus on improving the resolution of my cryo-EM maps, verifying PTM sites, and determining the functional roles of these modifications. In the R00 phase, I plan to explore the roles of novel regulatory proteins in NLRP3 activation. Using mass spectrometry and protein interaction studies, I will identify and characterize new regulators that interact with NLRP3 in a temporal and spatial manner. TurboID technology will be employed to map the interactome of NLRP3 during different stages of activation, providing a comprehensive understanding of the dynamic changes in protein interactions. I will determine the cryo-EM structures of NLRP3 in complex with these identified regulators to reveal their mechanisms of action. My long-term interest lies in understanding immune responses following pathogen infection, both in health and disease. With the experience gained from a K99/R00 Award, I aim to establish an independent laboratory dedicated to basic and translational research on inflammasome activation, control, degradation, and its interaction with pathogens. This research will provide a solid foundation for developing novel therapeutic strategies to modulate inflammasome activity, offering new hope for patients suffering from chronic inflammatory and autoimmune diseases.