UCLA

The cGAS-STING pathway has been increasingly recognized as an important pathway mediating Type I interferon inflammatory responses to mislocalized cytosolic dsDNA in a variety of disease pathologies. Recent investigations have shown surprising links between STING (STimulator of INterferon Genes) signaling and lipid metabolism. Previous studies from our lab and others have demonstrated that disruption of cholesterol biosynthetic programs in macrophages alters STING signaling. However, many of these results imply potentially contradictory models for how cholesterol metabolism influences STING signaling. The purpose of this thesis work is to uncover the molecular mechanism underlying these observations.We isolated macrophages with genetic knockouts of central lipid regulatory proteins to clarify their influence on STING signaling. We show that macrophages still form a competent STING response despite lacking SCAP or SREBP2, and genetic models of enhanced cholesterol biosynthesis actually drive down STING activity. We hypothesized that STING is instead regulated by binding directly to cholesterol. Using chemoproteomics approaches, we demonstrate that STING binds directly to three distinct sterol-mimetic photoactivated probes, and that this interaction can be competed by excess cholesterol. We identify co-immunoprecipitated probe-binding protein VDAC1 and show that STING-probe binding is not influenced by loss of VDAC1. Interaction with these probes is dependent on two cholesterol binding motifs we identify in STING. Using in silico ligand docking analysis, we visualize binding modes for cholesterol which neatly align with our proposed choelsterol-binding motifs. These motifs are present at a highly dynamic region of STING which undergoes a conformation change necessary for STING translocation and activation. We thus propose a model where cholesterol binding to STING at these motifs could sterically hinder this conformation change and thus repress STING activity by retention at the ER. In preliminary fluorescent microscopy data in both mouse and human cells, we confirm that STING localization in the ER is enhanced by addition of excess cholesterol, and that depletion of cholesterol spontaneously drives STING translocation even in the absence of activating ligand. Cholesterol binding directly to STING to influence its localization and activity represents a novel regulatory paradigm for STING signaling which could be exploited for future therapeutics.