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、肿瘤细胞和线粒体是必不可少的 或核DNA在某些条件下，导致动态平衡反应，如免疫控制 病原体和癌症。在某些情况下，例如核酸酶TREX1的丢失，过量的DNA会触发cGAS- 刺痛并导致慢性炎症(例如，艾卡迪·古铁埃S综合征或结构性活动性刺痛 等位基因，导致SAVI，一种遗传性血管病变)。当被其配体cGAMP激活时，STING从 ER→高尔基体→内体，通过Tbk1和irf3发出信号激活干扰素，然后被降解 通过溶酶体。这些贩运模式是斯汀功能的核心，但我们对许多 控制这些过程的基因。为了确定诱捕活动、贩运和退化的监管机构，我们 进行了两次全基因组CRISPR基因敲除筛选，以及邻近连接介导的蛋白质组学研究 分析，以及聚焦的CRISPR屏幕。对发现的几十个因素进行研究后，我们得出了两个有意义的结论 为这一提议提供基础的发现。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刺痛，这是刺痛的关键。 降解、自噬小体封闭和信号调节。由致病因子引起的这一途径的中断 ESCRT亚单位突变(在人类疾病中发现)在稳定状态下导致结构性刺痛信号。 其次，我们发现了一种新的内质网定位的STING与内体蛋白DNAJC13的相互作用，导致 限制刺激性内质网退出和激活。DNAJC13丢失或内质网-内吞体接触点中断 极大地促进了人们的活动。定义了一种控制自噬的刺痛泛素化模型 决议和限制刺激性ER退出的DNAJC13，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 刺伤和诱导ESCRT依赖的自噬，以及确定刺伤诱导的内体的影响 病毒感染上的微自噬。我们还将确定DNAJC13如何通过更改 诱捕人口贩子。我们将测试DNAJC13和ER-内体接触在限制刺痛激活中的作用 通过将STINER出口限制在TGN。为了定义刺痛相互作用的生化机制，我们将 使用深度突变扫描寻找负责与DNAJC13和DNAJC13相互作用的STING基序 DNAJC13对STING贩运的影响，以及控制STING本地化的其他方面的基序 和信号。最后，我们将研究基因突变如何参与人类调节刺痛的病理过程。 贩运、影响中枢神经系统细胞的炎症和死亡。更好地理解 打击贩运、降解和信号传递将帮助我们开发治疗方法，以抑制 自身免疫力或增强病原体和肿瘤免疫力。