Factors regulating strength and duration of STING signaling

调节 STING 信号强度和持续时间的因素

• 批准号: 10367563
• 负责人: Nir Hacohen
• 金额: $ 42.36万
• 依托单位: BROAD INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10367563
• 关键词: Affect; Alleles; Autoimmunity; Autophagocytosis; Autophagosome; Biochemical; Biology; CRISPR screen; Cell Death; Cell model; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Development; Endosomes; Factor Analysis; Genes; Goals; Golgi Apparatus; Hereditary Spastic Paraplegia; Human; Human Pathology; IRF3 gene; Immune; Immune system; Immunity; Inflammation; Inherited; Interferons; Knock-out; Knowledge; Ligands; Ligation; Lysosomes; Malignant Neoplasms; Mediating; Mitochondria; Modeling; Mutate; Mutation; Neuraxis; Nuclear; Optical Methods; Pathogenicity; Pathway interactions; Pattern; Process; Proteins; Proteomics; Regulation; Resolution; Role; Sensory; Signal Transduction; Site; Stimulator of Interferon Genes; TBK1 gene; TREX1 gene; Testing; Therapeutic; Tumor Immunity; Ubiquitination; Vaccines; Vascular Diseases; Virus; Virus Diseases; Work; disease-causing mutation; genome-wide; human disease; immune activation; immune health; improved; mutant; mutation screening; neoplastic cell; novel; nuclease; pathogen; pseudotoxoplasmosis syndrome; recruit; response; seal; sensor; trafficking; tumor; ubiquitin-protein ligase

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.

cGAS-STING 通路的 DNA 传感对于识别病原体、肿瘤细胞和线粒体至关重要 或核DNA在某些条件下，导致稳态反应，例如免疫控制 病原体和癌症。在某些条件下，例如核酸酶 TREX1 丢失，过量 DNA 会触发 cGAS- STING 并引起慢性炎症（例如，Aicardi Goutierès 综合征，或持续活跃的 STING） 等位基因，导致 SAVI（一种遗传性血管病）。被其配体 cGAMP 激活后，STING 从 ER → 高尔基体 → 内体，通过 TBK1 和 IRF3 发出信号以激活干扰素，然后被降解 通过溶酶体。这些贩运模式是 STING 功能的核心，但我们对许多方面缺乏了解 调节这些过程的基因。为了确定 STING 活动、贩运和降解的监管者，我们 进行了两次全基因组 CRISPR 敲除筛选，以及邻近连接介导的蛋白质组学 分析和重点 CRISPR 筛选。通过研究发现的数十个因素，我们得出了两个重要的结论： 这些发现为本提案提供了基础。首先，我们发现ESCRT依赖的内体 微自噬需要识别内体上泛素化的 STING，这对 STING 至关重要 降解、自噬体密封和信号调节。致病性物质破坏该途径 ESCRT 亚基突变体（在人类疾病中发现）导致稳定状态下的组成型 STING 信号传导。 其次，我们发现了内质网定位的 STING 与内体蛋白 DNAJC13 的一种新的相互作用，从而导致 限制 STING ER 退出和激活。 DNAJC13 丢失或 ER 内体接触位点破坏 显着提高 STING 活性。定义了 STING 泛素化控制自噬的模型 分辨率，以及 DNAJC13 限制 STING ER 退出，我们建议通过以下方式进一步了解该途径 研究这些过程的潜在机制，包括识别修饰 E3 泛素连接酶 STING 并诱导 ESCRT 依赖性自噬，并确定 STING 诱导的内体的影响 微自噬对病毒感染的影响。我们还将确定 DNAJC13 如何通过改变来阻止 STING 活性 STING 贩运。我们将测试 DNAJC13 和 ER 内体接触在限制 STING 激活中的作用 通过限制 STING ER 退出 TGN。为了定义 STING 相互作用的生化机制，我们将 使用深度突变扫描来寻找 STING 上负责与 DNAJC13 相互作用的基序 了解 DNAJC13 对 STING 运输的影响，以及控制 STING 定位其他方面的基序 和信号。最后，我们将研究涉及人类病理的基因突变如何调节 STING 贩运，影响中枢神经系统细胞的炎症和死亡。更好地理解 STING 运输、降解和信号传导将帮助我们开发抑制 STING 的治疗方法 自身免疫或增强病原体和肿瘤免疫力。