Probing organismal proteostasis through the response to intracellular infection

通过对细胞内感染的反应探索机体蛋白质稳态

• 批准号: 10518300
• 负责人: Emily R Troemel
• 金额: $ 34.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10518300
• 关键词: Affect; Aging; Autophagocytosis; Binding Proteins; Biochemical; Caenorhabditis elegans; Chronic; Co-Immunoprecipitations; Complex; Cullin Proteins; Cysteine; Data; Dimerization; Disease; Enzymes; F-Box Proteins; Genes; Genetic; Goals; Grant; Health; Heat Stress Disorders; Heat-Shock Response; Heterodimerization; Homo; In Vitro; Infection; Intestines; Knowledge; Lead; Ligase; Light; Longevity; Lysosomes; Mass Spectrum Analysis; Mediating; Microsporidia; Mission; Multienzyme Complexes; Names; Nematoda; Organism; Outcome; Oxidation-Reduction; Oxides; Pathway interactions; Pharmacology; Proteins; Proteomics; Public Health; Publishing; RNA Viruses; Recombinant Proteins; Reporting; Research; Role; Stress; Structure; Tertiary Protein Structure; Testing; Tissues; Ubiquitin; Ubiquitin C; United States National Institutes of Health; Up-Regulation; Western Blotting; Work; Yeasts; base; biological adaptation to stress; burden of illness; dimer; disulfide bond; genetic analysis; improved; in vivo; innovation; mRNA Expression; multicatalytic endopeptidase complex; mutant; novel; novel strategies; pathogen; polyglutamine; protein aggregation; protein protein interaction; proteostasis; proteotoxicity; response; screening; stressor; transcriptomics; ubiquitin ligase; ubiquitin-protein ligase; yeast two hybrid system

Project Summary/Abstract Aging-related diseases are associated with disruptions in protein homeostasis, or proteostasis. A major disruptor of proteostasis is infection with intracellular pathogens, but it is poorly understood how responses to these infections may promote proteostasis. Our long-term goal is to dissect how responses to infection and other proteotoxic stressors can protect overall organismal health and lifespan through characterizing a novel proteostasis response we discovered in the nematode C. elegans called the Intracellular Pathogen Response or IPR. Our previous work demonstrated how upregulation of IPR genes that encode components of a cullin ring ubiquitin ligase promote improved proteostasis, including increased thermotolerance. The objective of this proposal is to determine how this multi-subunit ubiquitin ligase is assembled, to identify its target(s), and to elucidate the fate of those targets and how they impact thermotolerance. The central hypothesis is that intracellular infection and other specific proteotoxic stressors induce mRNA expression of ubiquitin ligase subunits including: 1) the Cullin CUL-6, 2) the RING domain protein RCS-1, 3) a Skp-Related Protein SKR-3, 4 or 5, and 4) F-Box Proteins FBXA-75 or FBXA-158; and that redox-dependent dimerization of this CUL-6- containing ubiquitin ligase tetramer (to create an enzyme complex of eight subunits total) leads to its activation, and that this enzyme complex ubiquitylates a yet-to-be identified target that is then degraded by the lysosome to regulate proteostasis. The rationale is based on our published genetic and biochemical data about the assembly and function of RCS-1/CUL-6/SKR-3,4,5/FBXA-75/158, and our unpublished in vitro and in vivo data about redox-dependent dimerization of SKR-3, together with our unpublished genetic and pharmacological data indicating that the increased thermotolerance mediated by the CUL-6 ubiquitin ligase is dependent on the lysosome. Our work is innovative because we are pursuing the IPR, which is a recently described proteostasis response acting independently of canonical proteostasis pathways like the heat shock response and unfolded protein responses. We will test our hypothesis with three specific aims including Aim 1) Determine the dimerization, interactions and function of SKR-3, SKR-4, SKR-5, FBXA-75 and FBXA-158, both in vitro and in vivo; Aim 2) Identify the target(s) of the CUL-6 ubiquitin ligase; and Aim 3) Characterize the downstream fate of these targets, including possible degradation by the lysosome. The expected outcome is to determine which SKR proteins heterodimerize, which SKR protein interacts with which F-box protein, which proteins are targeted by this ubiquitin ligase complex, and which autophagy factors and other cellular components are involved in directing targets to the lysosome. The proposed research is significant, because it could lead to new treatments for aging-related diseases associated with disruptions in proteostasis.

项目摘要/摘要 与衰老相关的疾病与蛋白质稳态或蛋白质的中断有关。专业 蛋白质的破坏因子是细胞内病原体的感染，但鲜为人知的反应如何 这些感染可能会促进蛋白质抑制。我们的长期目标是剖析对感染的反应和 其他蛋白质毒性应激源可以通过表征新颖 我们在线虫C.秀丽隐杆线虫中发现的蛋白抑制反应称为细胞内病原体反应 或IPR。我们以前的工作证明了如何上调cullin组件的IPR基因 环泛素连接酶可促进改善的蛋白质量，包括升高的耐热性。这个目的 建议是确定如何组装这种多亚基泛素连接酶，以识别其靶标和 阐明这些目标的命运及其如何影响热耐趋势。中心假设是 细胞内感染和其他特定蛋白毒性应激诱导泛素连接酶的mRNA表达 亚基包括：1）Cullin Cul-6，2）环域蛋白RCS-1，3）与SKP相关的蛋白SKR-3，4 或5，4）F-box蛋白FBXA-75或FBXA-158；以及该CUL-6-的氧化还原依赖性二聚化 含有泛素连接酶四聚体（创建八个亚基的酶复合物）导致其激活， 并且该酶复合物泛素化层呈尚未确定的靶标，然后被溶酶体降解 调节蛋白质的抑制作用。理由是基于我们发表的有关遗传和生化数据 RCS-1/CUL-6/SKR-3,4,5/FBXA-75/158的组装和功能，以及我们未发表的体外和体内数据 关于SKR-3的氧化还原依赖性二聚化，以及我们未发表的遗传和药理 数据表明由CUL-6泛素连接酶介导的耐热性增加取决于 溶酶体。我们的工作具有创新性，因为我们正在追求知识产权，这是最近描述的Proteostasis 响应独立于典型的蛋白质量途径（例如热冲击响应）并展开 蛋白质反应。 我们将以三个特定目的（包括目标1）确定二聚体化的三个特定目标来检验我们的假设。 SKR-3，SKR-4，SKR-5，FBXA-75和FBXA-158的相互作用和功能，包括体外和体内；目标2） 确定CUL-6泛素连接酶的靶标；目标3）特征这些命运的命运 靶标，包括溶酶体可能降解。预期的结果是确定哪个SKR 蛋白异二聚体，SKR蛋白与F-box蛋白相互作用，蛋白质是靶向的 这种泛素连接酶复合物，以及哪些自噬因素和其他细胞成分参与 将目标引导到溶酶体。拟议的研究很重要，因为它可能导致新的 与蛋白抑制性疾病相关的与衰老相关疾病的治疗方法。