K63-linked ubiquitination regulates cGAS activation upon DNA damage

K63 连接的泛素化在 DNA 损伤时调节 cGAS 激活

• 批准号: 10495239
• 负责人: Shitao Li
• 金额: $ 19万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10495239
• 关键词: Antiviral Agents; Ataxia Telangiectasia; Ataxia Telangiectasia Patients; Autoimmune Diseases; Binding; Cell Nucleus; Chromatin; Communicable Diseases; Cyclic GMP; Cytosol; DNA; DNA Binding; DNA Damage; DNA Viruses; DNA-Binding Proteins; Data; Defect; Disease; Fibroblasts; Foundations; Gene Expression; Goals; Histones; Host Defense; Immobilization; Interferon Type I; Interferons; Knock-out; Ligation; Link; Literature; Mediating; Modeling; Natural Immunity; Nuclear; Nucleic Acids; Nucleosomes; Play; Polyubiquitin; Proteomics; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Systemic Lupus Erythematosus; Testing; Ubiquitination; Virus Diseases; autoinflammatory; base; cell injury; novel therapeutic intervention; pathogen; protein complex; response; sensor; ubiquitin-protein ligase; viral DNA

Project summary Excessive DNA either from pathogen or host activates the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS) that produces the second messenger, cyclic GMP-AMP (cGAMP). cGAMP triggers a signal cascade leading to type I interferon (IFN) expression. It has been thought that cGAS is sequestered in the cytosol and cannot access host DNA normally restricted in cellular compartments, such as the nucleus. The recent discovery of nuclear cGAS has dramatically changed this model. Structural studies showed cGAS binds to the H2A-H2B heterodimer of the nucleosome and the binding immobilizes cGAS on the chromatin; thus, cGAS cannot access the nearby DNA. However, the micronuclei caused by DNA damage are immunostimulatory, and the chromatin fragments in micronuclei activate cGAS. Currently, we don’t know whether cGAS is still immobilized on the nucleosomes in the micronuclei and how cGAS is activated by the interaction with chromatin upon DNA damage. Endogenous defects in the DNA damage response (DDR) induce autoinflammatory diseases, such as Ataxia-Telangiectasia (A-T). Thus, there is a critical need to elucidate the mechanisms controlling cGAS activation upon DNA damage. Our objective is to define the role of K63-linked ubiquitination in DNA damage-induced cGAS activation and elucidate the underlying mechanisms by which K63-linked polyubiquitin regulates cGAS activity. Our preliminary data found that cGAS was subject to K63-linked ubiquitination upon DNA damage and inhibition K63-linked ubiquitination abrogated DNA damage-induced cGAS signaling pathway and interferon-stimulated gene expression. Based on the existing literature and our preliminary data, we hypothesize that K63-linked polyubiquitin facilitates cGAS activation in the micronuclei. We will examine two aims: First, we will determine the role of K63-linked ubiquitination in cGAS activation. Second, we will determine the mechanisms by which K63-linked polyubiquitin facilitates DNA damage-induced cGAS activation. The chromatin fragments in the micronuclei are a strong immunostimulatory agent for cGAS, but how cGAS overcome the histone sequestration and competes with other DNA binding proteins to access the DNA has yet to be determined. This exploratory R21 application proposes that K63-linked ubiquitination facilitates cGAS activation upon DNA damage. Overall, this study elucidates the regulatory mechanisms of nuclear cGAS activation and provides foundations for developing novel therapeutic strategies for infectious and autoimmune diseases.

项目摘要 来自病原体或宿主的过量DNA激活胞质DNA传感器，环GMP-AMP合酶 环磷酸腺苷（cGAMP）是产生第二信使环磷酸腺苷（cGAS）的一种酶。cGAMP触发信号级联 导致I型干扰素（IFN）表达。已经认为cGAS被隔离在细胞溶质中， 无法进入通常限制在细胞隔室（例如细胞核）中的宿主DNA。近期 核CGAS的发现极大地改变了这一模型。结构研究表明，CGAS结合到 核小体的H2 A-H2 B异二聚体和结合将cGAS固定在染色质上;因此，cGAS 无法获取附近的DNA然而，DNA损伤引起的微核具有免疫刺激性， 微核中的染色质片段激活cGAS。目前，我们不知道cGAS是否仍然是 固定在微核中的核小体上以及cGAS如何通过与 染色质的DNA损伤。DNA损伤反应（DDR）中的内源性缺陷诱导 自身炎性疾病，如共济失调-毛细血管扩张症（A-T）。因此，迫切需要阐明 控制DNA损伤后cGAS活化的机制。 我们的目标是确定K63连接的遍在蛋白化在DNA损伤诱导的cGAS激活中的作用， 阐明K63连接的多聚泛素调节cGAS活性的潜在机制。我们 初步数据发现，cGAS在DNA损伤和抑制后经历K63连接的泛素化 K63连接的泛素化废除了DNA损伤诱导的cGAS信号通路和干扰素刺激的 基因表达。根据现有文献和我们的初步数据，我们假设K63相关的 多聚泛素促进微核中的cGAS活化。我们将研究两个目标：首先，我们将确定 K63连接的泛素化在cGAS激活中的作用。第二，我们将确定机制， K63连接的多聚泛素促进DNA损伤诱导的cGAS活化。 微核中的染色质片段是cGAS的强免疫刺激剂，但cGAS如何 克服组蛋白隔离并与其他DNA结合蛋白竞争进入DNA的能力， 有待确定。这个探索性的R21应用提出K63连接的泛素化促进cGAS DNA损伤后激活。总的来说，这项研究阐明了核cGAS的调控机制， 激活并为开发感染性和自身免疫性疾病的新治疗策略提供基础。 疾病