Wednesday, December 4, 2024
12/4/2024
DNA sensing by the cGAS-STING pathway is essential for recognizing pathogens, tumor cells, and mitochondrial or nuclear DNA under certain conditions, leading to homeostatic responses, such as immune control of pathogens and cancer. Under some conditions, such as loss of the nuclease TREX1, excess DNA triggers cGAS- STING and causes a chronic inflammation (e.g., Aicardi Goutierès Syndrome, or a constitutively active STING allele, causes SAVI, an inherited vasculopathy). Upon activation by its ligand, cGAMP, STING translocates from ER → Golgi → endosomes where it signals through TBK1 and IRF3 to activate interferons, and is then degraded via lysosomes. These trafficking patterns are central to STING’s function, and yet we lack knowledge of many of the genes regulating these processes. To identify regulators of STING activity, trafficking and degradation, we performed two genome-wide CRISPR knockout screens, as well as a proximity-ligation mediated proteomic analysis, and a focused CRISPR screen. Studying the dozens of factors found, we made two significant discoveries that provide the basis for this proposal. First, we found that ESCRT-dependent endosomal microautophagy requires recognition of ubiquitinated STING on endosomes, and is critical for STING degradation, autophagosome sealing and signaling regulation. Disruption of this pathway by a pathogenic ESCRT subunit mutant (found in a human disease) leads to constitutive STING signaling at steady state. Second, we uncovered a novel interaction of ER-localized STING with endosomal protein DNAJC13, leading to restriction of STING ER exit and activation. Loss of DNAJC13 or disruption of ER-endosome contact sites dramatically boosts STING activity. Having defined a model of STING ubiquitination controlling autophagy resolution, and DNAJC13 restricting STING ER exit, we propose to further our understanding of this pathway by studying the mechanisms underlying these processes, including identifying E3 ubiquitin ligases that modify STING and induce ESCRT-dependent autophagy, and determining the impact of STING-induced endosomal microautophagy on viral infections. We will also determine how DNAJC13 blocks STING activity by altering STING trafficking. We will test the roles of DNAJC13 and ER-endosomal contacts in limiting STING activation by restricting STING ER exit to the TGN. To define the biochemical mechanisms of STING interactions, we will use deep mutational scanning to find motifs on STING that are responsible for interactions with DNAJC13 and for the effects of DNAJC13 on STING trafficking, as well as motifs that control other aspects of STING localization and signaling. Finally, we will study how mutations in genes involved in human pathologies that regulate STING trafficking, impact inflammation and death of cells of the central nervous system. A better understanding of STING trafficking, degradation and signaling will help us develop therapeutic approaches to dampen autoimmunity or boost pathogen and tumor immunity.
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