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.

通过CGAS插入途径进行DNA感测对于识别病原体，肿瘤细胞和线粒体至关重要 或在某些条件下核DNA，导致稳态反应，例如免疫控制 病原体和癌症。在某些情况下，例如核酸酶丢失TREX1，多余的DNA触发CGAS- 刺痛并引起慢性炎症（例如AicardiGoutierès综合征或组成症的活性刺激 等位基因，导致萨维（Savi），一种遗传性的血管病）。在其配体激活后，cgamp，刺激的刺激 er→高尔基体→内体，其中它通过TBK1和IRF3发出信号以激活干扰素，然后降解 通过溶酶体。这些贩运模式对于Sting的功能至关重要，但我们缺乏许多知识 控制这些过程的基因。为了确定刺痛活动，贩运和退化的监管机构，我们 进行了两个全基因组CRISPR基因敲除屏幕，以及一个接近介导的蛋白质组学 分析和集中的CRISPR屏幕。研究发现的数十个因素，我们做了两个重要的 为该提案提供基础的发现。首先，我们发现依赖于ESCRT的内体 微自噬需要识别内体上的泛素刺，对于刺痛至关重要 降解，自噬体密封和信号调节。通过致病性破坏这一途径 ESCRT亚基突变体（在人类疾病中发现）导致稳态的刺激信号传导。 其次，我们发现了ER定位的刺激与内体蛋白DNAJC13的新型相互作用，导致 限制刺痛的出口和激活。 DNAJC13的损失或ER粘体接触位点的破坏 急剧增强了刺痛活动。定义了控制自噬的刺激泛素化模型 解决方案和DNAJC13限制了刺痛的出口，我们建议通过 研究这些过程的基础机制，包括鉴定E3泛素连接酶 刺激和诱导的ESCRT依赖性自噬，并确定刺激诱导的内体的影响 病毒感染的微自噬。我们还将确定DNAJC13如何通过改变来阻断刺激活性 刺痛。我们将测试DNAJC13和ER-粘体接触在限制刺激激活中的作用 通过将刺痛出口限制为TGN。为了定义刺激相互作用的生化机制，我们将 使用深度突变扫描来查找有关刺激的基序，这些基序是导致与DNAJC13相互作用和 对于DNAJC13对刺激贩运的影响，以及控制刺激性定位其他方面的图案 和信号。最后，我们将研究如何调节刺痛的人类病理中涉及的基因突变 中枢神经系统细胞的贩运，影响注射和死亡。更好地理解 刺痛贩运，退化和信号传导将有助于我们开发该死的治疗方法 自身免疫性或增强病原体和肿瘤免疫学。