Defining STING–sterol interactions with chemoproteomics

Stimulator of interferon genes (STING) is an intracellular pattern recognition receptor that plays a key role in responding to cytosolic DNA and cyclic dinucleotides. STING activity is tightly regulated to avoid aberrant STING activity, excessive type I IFN responses, and resultant autoinflammatory disease. As such understanding the molecular events regulating STING activity is critical. Recent work has revealed cellular cholesterol metabolism also functions to modulate STING activity, although the molecular events linking cholesterol homeostasis with STING remain incompletely understood. Here we pair genetic and chemoproteomic approaches to inform the mechanisms governing cholesterol modulation of STING activity. Using gain- and loss-of-function systems, we find that markedly increasing SCAP-SREBP2 processing and resultant cholesterol synthesis has little impact on STING activity. In contrast, we find that genetic deletion of <i>Srebf2</i> increased basal and ligand inducible type I IFN responses. Thus, STING can function in the absence of the SCAP-SREBP2 protein apparatus. Through activity-based protein profiling with three distinct sterol-mimetic probes, we provide direct evidence for STING-sterol binding. We also find that the mitochondrial protein VDAC1 co-purifies with STING and binds to sterol-mimetic probes. We also show that STING's subcellular localization is responsive to modulation of cellular sterol content. Our findings support a model where sterol synthesis in the ER regulates STING activity, aligning with recent studies indicating that cholesterol-mediated retention of STING in the endoplasmic reticulum occurs through cholesterol recognition amino acid consensus (CARC) motifs in STING.