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: 10665771
• 负责人: Emily R Troemel
• 金额: $ 32.86万
• 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/10665771
• 关键词: Affect; Aging; Autophagocytosis; Binding Proteins; Biochemical; Caenorhabditis elegans; Chronic; Co-Immunoprecipitations; Complex; Cullin Proteins; Cysteine; Data; Dimerization; Disease; Disulfides; Enzymes; F-Box Proteins; Genes; Genetic; Goals; Grant; Health; Heat Stress Disorders; Heat-Shock Response; Heterodimerization; Homo; In Vitro; Infection; Intestines; Knowledge; Ligase; Longevity; Lysosomes; Mass Spectrum Analysis; Mediating; Microsporidia; Mission; Multienzyme Complexes; Names; Nematoda; Organism; Outcome; Oxidation-Reduction; Pathway interactions; 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; biological adaptation to stress; burden of illness; candidate identification; dimer; disulfide bond; genetic analysis; improved; in vivo; innovation; mRNA Expression; multicatalytic endopeptidase complex; mutant; novel; novel strategies; pathogen; pharmacologic; 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.

项目概要/摘要 衰老相关疾病与蛋白质稳态的破坏有关。一个专业 蛋白质稳态的破坏者是细胞内病原体的感染，但人们对如何应对这些病原体知之甚少。 这些感染可能会促进蛋白质稳态。我们的长期目标是剖析对感染的反应和 其他蛋白毒性应激源可以通过表征新的特征来保护整体有机体健康和寿命 我们在线虫中发现的蛋白质稳态反应称为细胞内病原体反应 或知识产权。我们之前的工作证明了编码 cullin 成分的 IPR 基因的上调如何 环泛素连接酶促进改善蛋白质稳态，包括增加耐热性。此举的目的 建议是确定这种多亚基泛素连接酶是如何组装的，以确定其目标，并 阐明这些目标的命运以及它们如何影响耐热性。中心假设是 细胞内感染和其他特异性蛋白毒性应激源诱导泛素连接酶 mRNA 表达 亚基包括：1) Cullin CUL-6、2) RING 结构域蛋白 RCS-1、3) Skp 相关蛋白 SKR-3、4 或5、和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（以创建总共八个亚基的酶复合物）导致其激活， 并且这种酶复合物泛素化一个尚未识别的靶标，然后被溶酶体降解 调节蛋白质稳态。其基本原理是基于我们发布的有关 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学 数据表明，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，这是最近描述的蛋白质稳态 反应独立于典型的蛋白质稳态途径，如热休克反应和展开 蛋白质反应。 我们将通过三个具体目标来检验我们的假设，包括目标 1) 确定二聚化， SKR-3、SKR-4、SKR-5、FBXA-75 和 FBXA-158 的体外和体内相互作用和功能；目标2) 确定 CUL-6 泛素连接酶的靶标；目标 3) 描述这些物质的下游命运 目标，包括可能被溶酶体降解。预期结果是确定哪个 SKR 蛋白质异二聚化，哪个 SKR 蛋白与哪个 F-box 蛋白相互作用，哪个蛋白质是 这种泛素连接酶复合物，以及自噬因子和其他细胞成分参与其中 将靶标导向溶酶体。拟议的研究意义重大，因为它可能会带来新的结果 与蛋白质稳态破坏相关的衰老相关疾病的治疗。