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: 10348290
• 负责人: Shitao Li
• 金额: $ 22.8万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10348290
• 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; Structure; Systemic Lupus Erythematosus; Testing; Therapeutic; 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.

项目总结 来自病原体或寄主的过量DNA会激活胞浆DNA传感器，即环状GMP-AMP合成酶 产生第二信使的环状GMP-AMP(CGAMP)。CGAMP触发信号级联 导致I型干扰素(干扰素)的表达。已经认为cGAS被隔离在细胞质中，并且 不能访问通常限制在细胞室中的宿主DNA，例如细胞核。最近的 核cGAS的发现极大地改变了这一模式。结构研究表明cGAS与 核小体的H_2A-H_2B杂二聚体和结合作用将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作用， 微核中的染色质碎片激活cGAS。目前，我们不知道cGAS是否还在 微核中核小体的固定化以及cGAS是如何被激活的 染色质对DNA损伤的影响。DNA损伤反应(DDR)中的内源性缺陷 自身炎症性疾病，如共济失调-毛细血管扩张(A-T)。因此，迫切需要澄清 DNA损伤时控制cGAS激活的机制。 我们的目标是确定K63连接的泛素化在DNA损伤诱导的cGAS激活和 阐明K63连接的多泛素调节cGAS活性的潜在机制。我们的 初步数据发现，cGAS在DNA损伤和抑制时受到K63连接的泛素化的影响 K63泛素化阻断DNA损伤诱导的cGAS信号通路及干扰素刺激 基因表达。根据现有文献和我们的初步数据，我们假设K63与 多聚泛素促进微核中cGAS的激活。我们将考察两个目标：第一，我们将确定 K63连接泛素化在cGAS激活中的作用。第二，我们将确定通过哪些机制 K63连接的多泛素促进DNA损伤诱导的cGAS激活。 微核中的染色质碎片对cGAS有很强的免疫刺激作用，但cGAS是如何 克服组蛋白的隔离并与其他DNA结合蛋白竞争访问DNA尚未 待定。这一探索性的R21应用表明，K63连接的泛素化促进了cGAS DNA损伤后的激活。总之，本研究阐明了核cGAs的调控机制。 并为开发新的感染和自身免疫治疗策略提供了基础 疾病。