Elucidating the roles of CMPK2 in mitochondrial homeostasis and antiviral immunity

阐明 CMPK2 在线粒体稳态和抗病毒免疫中的作用

• 批准号: 10313146
• 负责人: Sylvia Torres-Odio
• 金额: $ 3.7万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313146
• 关键词: Elucidating; roles; CMPK2; mitochondrial; homeostasis

PROJECT SUMMARY Cytidine/uridine monophosphate kinase 2 (CMPK2) is an interferon-regulated enzyme that was originally reported to catalyze the ATP-dependent phosphorylation of dCMP and dUMP to diphosphate forms in vitro. Due to a putative mitochondrial targeting sequence, CMPK2 was postulated to function in the mitochondrial deoxyribonucleotide salvage pathway necessary for the synthesis and maintenance of mitochondrial DNA (mtDNA). However, more recent data have revealed that CMPK2 prefers ribonucleotide diphosphate substrates in vitro and functions to restrict HIV and other RNA viruses in cell based assays. Beyond these seemingly contrasting findings, no other studies have addressed the cellular localization and tissue expression patterns of CMPK2 or utilized genetic knockouts to determine true biological activity. Therefore, the overall objective of this proposal is to close these knowledge gaps and mechanistically advance understanding of CMPK2 in mitochondrial function, tissue homeostasis, and antiviral innate immunity using a diverse toolkit of cell and animal models. The central hypothesis is that by maintaining mitochondrial homeostasis, CMPK2 boosts cell-intrinsic innate immunity and limits runaway inflammation triggered by mitochondrial stressors and viral infection. In support of this hypothesis, ongoing studies have revealed that CMPK2 localizes strongly to mitochondria and that ectopic overexpression of CMPK2 is sufficient to protect cells from RNA virus infection. Moreover, after systemic challenge with innate immune agonists, CMPK2 is markedly upregulated in the lungs and liver, and CMPK2 knockout mice exhibit elevated expression of proinflammatory cytokines and type I interferon (IFN-I) responses after Toll-like receptor (TLR) stimulation. To gain additional insight into how CMPK2 functions in mitochondria and antiviral immunity, two related, but independent aims are proposed. Aim 1 will elucidate the molecular mechanisms by which CMPK2 maintains mitochondrial homeostasis at rest and during stress. Here, CMPK2 knockout cells, novel lines reconstituted with mutant CMPK2 vectors lacking nucleotide kinase activity or mitochondrial targeting, and whole body CMPK2 knockout mice will be utilized. Aim 2 will determine that the mitochondrial activity of CMPK2 restricts coronavirus replication and maintains mitochondrial function during infection. Here, an intranasal mouse hepatitis virus challenge protocol that models acute respiratory distress syndrome and closely mirrors coronavirus pneumonia in humans will be employed. This research will fundamentally advance our understanding of how CMPK2 functions in mitochondrial homeostasis and antiviral innate immunity at both the cellular and organismal levels. Moreover, it may have a positive impact on public health by revealing novel CMPK2-centered strategies to maintain mitochondrial homeostasis, boost antiviral immunity, and limit damaging inflammation during coronavirus infection.

项目概要 胞苷/尿苷单磷酸激酶 2 (CMPK2) 是一种干扰素调节酶，最初是 据报道在体外催化 dCMP 和 dUMP 的 ATP 依赖性磷酸化为二磷酸形式。到期的 根据假定的线粒体靶向序列，CMPK2 被假定在线粒体中发挥作用 线粒体 DNA 合成和维持所必需的脱氧核糖核苷酸挽救途径 （线粒体DNA）。然而，最近的数据表明 CMPK2 更喜欢核糖核苷酸二磷酸底物 体外，并在基于细胞的测定中发挥限制 HIV 和其他 RNA 病毒的作用。除了这些看似 相比之下，没有其他研究涉及细胞定位和组织表达模式 CMPK2 或利用基因敲除来确定真正的生物活性。因此，本次活动的总体目标 建议是缩小这些知识差距并机械地促进对 CMPK2 的理解 使用多种细胞和动物工具包研究线粒体功能、组织稳态和抗病毒先天免疫 模型。核心假设是，通过维持线粒体稳态，CMPK2 增强细胞内在 先天免疫并限制线粒体应激和病毒感染引发的失控炎症。在 为了支持这一假设，正在进行的研究表明 CMPK2 强烈定位于线粒体并且 CMPK2 的异位过度表达足以保护细胞免受 RNA 病毒感染。而且，之后 先天免疫激动剂的全身性挑战，CMPK2 在肺和肝脏中显着上调，并且 CMPK2 敲除小鼠表现出促炎细胞因子和 I 型干扰素 (IFN-I) 表达升高 Toll 样受体 (TLR) 刺激后的反应。为了进一步了解 CMPK2 如何在 线粒体和抗病毒免疫是两个相关但独立的目标。目标 1 将阐明 CMPK2 在休息和应激期间维持线粒体稳态的分子机制。这里， CMPK2 敲除细胞，用缺乏核苷酸激酶活性的突变 CMPK2 载体重建的新细胞系 或线粒体靶向，并且将使用全身 CMPK2 敲除小鼠。目标 2 将确定 CMPK2 的线粒体活性限制冠状病毒复制并维持线粒体功能 感染。这里是模拟急性呼吸窘迫的鼻内小鼠肝炎病毒攻击方案 将采用与人类冠状病毒肺炎密切相关的综合症。这项研究将 从根本上增进我们对 CMPK2 如何在线粒体稳态和抗病毒中发挥作用的理解 细胞和有机体水平的先天免疫。此外，它还可能对公众产生积极影响 通过揭示以 CMPK2 为中心的新策略来维持线粒体稳态、增强抗病毒作用，从而促进健康 免疫力，并限制冠状病毒感染期间的破坏性炎症。