Phosphorylation mimetic motif of cryptochrome proteins blocks IRF3 activation

隐花色素蛋白的磷酸化模拟基序可阻断 IRF3 激活

• 批准号: 10583785
• 负责人: Shitao Li
• 金额: $ 22.85万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-15 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10583785
• 关键词: Adaptor Signaling Protein; Alanine; Asthma; Attenuated; Autoimmune Diseases; Binding; C-terminal; Circadian Rhythms; Communicable Diseases; Consensus; Data; Dimerization; Docking; Exhibits; Genes; Glutamic Acid; Goals; Host Defense; Human; IRF3 gene; Immune; Immune System Diseases; Influenza A virus; Innate Immune Response; Innate Immune System; Interferon Type I; Knock-out; Knowledge; Literature; Mediating; Molecular; Mutation; Natural Immunity; Nucleic Acids; Pathology; Pattern; Pattern recognition receptor; Periodicity; Phosphorylation; Phosphorylation Site; Pilot Projects; Polyubiquitin; Production; Proteins; Proteomics; Rheumatoid Arthritis; Role; Signal Pathway; Signal Transduction; Site; Stimulator of Interferon Genes; Symptoms; TBK1 gene; TLR3 gene; TRAF2 gene; Testing; Tretinoin; Ubiquitination; Virus Diseases; autoinflammatory diseases; circadian; cryptochrome; cryptochrome 2; human disease; hydrophilicity; in vivo; mimetics; novel; novel therapeutics; pathogen; recruit

Project summary The innate immune system employs germline-encoded pattern recognition receptors (PRRs) to detect common pathogen-associated molecular patterns. However, only several PRR signaling pathways, including RIG-I- MAVS, cGAS-STING, and TLR3/4-TRIF, activate interferon regulatory factor 3 (IRF3). Recent studies showed that the innate immune adaptors (MAVS, STING, and TRIF) are phosphorylated at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site) by TBK1. The phosphorylation of the pLxIS motif in innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production. However, whether and how the host or pathogen utilizes this motif to attenuate or block IRF3 signaling is unknown. Our overall objective in this application is to determine the role of the cryptochrome (CRY) proteins in IRF3 signaling pathways and host defense to viral infection. Our preliminary proteomic data showed that cryptochrome (CRY) proteins, CRY1 and CRY2, interacted with IRF3. Interestingly, CRY1 and CRY2 have two and one conserved pLxIE motifs, respectively. As glutamic acid (E) is a phosphorylation mimic, this pLxIE motif might be a constitutively phosphorylated pLxIS. Our pilot studies showed that mutation of E to alanine (A) in the pLxIE abolished the binding to IRF3, suggesting that the pLxIE motif is also a docking site for IRF3. Furthermore, deficiency of both CRY proteins also augmented IRF3 signaling and type I IFN production. However, how CRY proteins inhibit innate immune responses is not well elucidated. Based on the existing literature and our preliminary data, we hypothesize that CRY proteins are decoy adaptors for IRF3 via the pLxIE motif and the binding blocks interaction between IRF3 and innate immune adaptors, thereby inhibiting IRF3 phosphorylation and activation. Aim 1 will determine the role of CRY1/2 in the RIG-I-mediated IRF3 signaling pathway. Aim 2 will dissect the molecular mechanisms of how CRY1/2 suppresses IRF3-mediated innate immunity. Many human immune diseases exhibit circadian rhythmicity in their symptoms and pathology, including asthma, rheumatoid arthritis, and other disorders of the immune system. Our study will bridge the gaps in understanding the role of the circadian proteins CRY1/2 in host innate immune response. This novel mechanism proposed in our application will advance our understanding of the crosstalk between innate immunity and circadian.

项目概要 先天免疫系统利用种系编码的模式识别受体 (PRR) 来检测常见的 病原体相关的分子模式。然而，只有几种 PRR 信号通路，包括 RIG-I- MAVS、cGAS-STING 和 TLR3/4-TRIF 可激活干扰素调节因子 3 (IRF3)。最近的研究表明 先天免疫接头（MAVS、STING 和 TRIF）在各自的 C 端被磷酸化 TBK1 的共有基序，pLxIS（p，亲水残基；x，任何残基；S，磷酸化位点）。这 先天免疫接头蛋白中 pLxIS 基序的磷酸化是必需且保守的 选择性招募 IRF3 来激活 I 型 IFN 产生的机制。然而，主机是否以及如何 或病原体利用该基序来减弱或阻断 IRF3 信号传导尚不清楚。我们在此方面的总体目标 应用是确定隐花色素 (CRY) 蛋白在 IRF3 信号通路和宿主中的作用 防御病毒感染。我们的初步蛋白质组数据表明，隐花色素 (CRY) 蛋白 CRY1 和CRY2，与IRF3相互作用。有趣的是，CRY1 和 CRY2 有两个和一个保守的 pLxIE 基序， 分别。由于谷氨酸 (E) 是磷酸化模拟物，因此该 pLxIE 基序可能是一个组成型 磷酸化 pLxIS。我们的初步研究表明，pLxIE 中 E 突变为丙氨酸 (A) 消除了 与 IRF3 结合，表明 pLxIE 基序也是 IRF3 的对接位点。此外，两者都缺乏 CRY 蛋白还增强了 IRF3 信号传导和 I 型 IFN 的产生。然而，CRY 蛋白如何抑制 先天免疫反应尚未得到很好的阐明。根据现有文献和我们的初步数据，我们 假设 CRY 蛋白是 IRF3 的诱饵接头，通过 pLxIE 基序和结合块 IRF3 与先天免疫接头之间的相互作用，从而抑制 IRF3 磷酸化和激活。 目标 1 将确定 CRY1/2 在 RIG-I 介导的 IRF3 信号通路中的作用。目标 2 将剖析 CRY1/2 如何抑制 IRF3 介导的先天免疫的分子机制。 许多人类免疫疾病的症状和病理表现出昼夜节律，包括 哮喘、类风湿性关节炎和其他免疫系统疾病。我们的研究将弥补以下方面的差距 了解昼夜节律蛋白 CRY1/2 在宿主先天免疫反应中的作用。这部小说 我们的应用中提出的机制将增进我们对先天之间串扰的理解 免疫力和昼夜节律。