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: 10490901
• 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/10490901
• 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-STING途径的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，如免疫控制， 病原体和癌症。在某些条件下，例如核酸酶TREX 1的缺失，过量的DNA触发cGAS-1。 刺痛并引起慢性炎症（例如，Aparthodi Goutierès综合征，或组成性活性STING 等位基因，导致SAVI，一种遗传性血管病）。在被其配体cGAMP激活后，STING从 ER →高尔基体→内体，在那里它通过TBK 1和IRF 3发出信号以激活干扰素，然后降解 通过溶酶体。这些贩运模式是STING功能的核心，但我们缺乏对许多 调节这些过程的基因。为了确定STING活动、贩运和退化的监管机构，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研究。 分析，以及聚焦的CRISPR筛选。通过研究发现的几十个因素，我们得出了两个重要的结论， 这些发现为这一提议提供了基础。首先，我们发现ESCRT依赖的内体 微自噬需要识别核内体上的泛素化STING，并且对于STING至关重要。 降解、自噬体封闭和信号调节。病原体干扰这一途径， ESCRT亚基突变体（在人类疾病中发现）导致稳态的组成型STING信号传导。 第二，我们发现了ER定位的STING与内体蛋白DNAJC 13的新相互作用，导致 限制STING ER退出和激活。DNAJC 13缺失或ER-内体接触位点破坏 显著提高STING活动。定义了STING泛素化控制自噬的模型， 决议和DNAJC 13限制STING ER退出，我们建议通过以下方式进一步了解这一途径： 研究这些过程的机制，包括鉴定E3泛素连接酶， STING和诱导ESCRT依赖性自噬，并确定STING诱导的内体 微自噬对病毒感染的影响我们还将确定DNAJC 13如何通过改变STING活性来阻断STING活性。 STING贩卖。我们将测试DNAJC 13和ER-内体接触在限制STING激活中的作用。 通过限制STING ER出口到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DNAJC 13相互作用的基序， DNAJC 13对STING运输的影响，以及控制STING定位的其他方面的基序 和信号。最后，我们将研究基因突变如何参与调节STING的人类病理学 运输，影响中枢神经系统细胞的炎症和死亡。更好地了解 STING的运输、降解和信号传导将帮助我们开发治疗方法， 自身免疫或增强病原体和肿瘤免疫。