Fate of cGAMP · Comprehensive Discovery of Cell-Autonomous Mechanisms Regulating cGAMP

The cGAS-STING pathway is a central component of the innate immunity that detects cytosolic double-stranded DNA (dsDNA). Upon sensing dsDNA, cGAS produces cGAMP, a second messenger that binds to and activates STING, thereby inducing various antiviral gene expression and promoting the production of proinflammatory cytokines and chemokines. While transient activation of this pathway is essential for host defense, persistent activation is deleterious and has been linked to autoimmune and autoinflammatory diseases. To prevent such chronic responses, cells employ regulatory mechanisms to timely terminate the cGAS-STING signaling, such as by trafficking activated STING to lysosomes for degradation.However, how cGAMP itself is regulated following STING activation remains poorly understood. Preliminary data from the host laboratory and others indicate that intracellular cGAMP levels are transient, suggesting the existence of additional, yet unidentified, regulatory mechanisms. The applicant hypothesizes that cells may employ active secretion, enzymatic degradation, or sequestration processes to eliminate residual cGAMP and thereby prevent sustained activation of innate immunity.To address this, the project will synthesize a series of fluorescent cGAMP analogs as proxies to identify regulatory mechanisms of cGAMP via live-cell super-resolution microscopy and FACS. Furthermore, systematic and unbiased CRISPR screens will be employed to discover genes involved in the cell-autonomous control of cGAMP, followed by biochemical and structural studies to elucidate the molecular mechanisms of cGAMP regulatory proteins.By uncovering how cells clear cGAMP after STING activation, this project will illuminate a critical yet unexplored aspect of innate immune regulation. Identifying the molecular regulators of cGAMP homeostasis may reveal new pathways contributing to autoimmunity and autoinflammation, providing novel opportunities for therapeutic intervention.