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.

项目总结/摘要 衰老相关疾病与蛋白质稳态或蛋白质稳态的破坏有关。一个主要 蛋白质稳态的破坏者是细胞内病原体的感染，但人们对 这些感染可促进蛋白质稳态。我们的长期目标是剖析如何应对感染， 其他蛋白毒性应激源可以通过表征一种新的 我们在线虫C.称为细胞内病原体反应 或知识产权。我们以前的工作证明了编码淘汰蛋白组分的IPR基因是如何上调的， 环泛素连接酶促进改善的蛋白质稳定，包括增加的耐热性。的目的 我们的建议是确定这种多亚基泛素连接酶是如何组装的，以确定其靶标，并 阐明这些目标的命运以及它们如何影响耐热性。核心假设是， 细胞内感染和其它特异性蛋白毒性应激物诱导泛素连接酶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-盒蛋白FBXA-75或FBXA-158;并且该CUL-6- 158的氧化还原依赖性二聚化是可能的。 含有泛素连接酶四聚体（以产生总共八个亚基的酶复合物）导致其活化， 这种酶复合物使一个尚未确定的靶点泛素化，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已发表的关于 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，由CUL-6泛素连接酶介导的增加的耐热性依赖于CUL-6泛素连接酶。 溶酶体我们的工作是创新的，因为我们正在追求的知识产权，这是一个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， 蛋白质反应。 我们将用三个具体目标来测试我们的假设，包括目标1）确定二聚化， SKR-3、SKR-4、SKR-5、FBXA-75和FBXA-158在体外和体内的相互作用和功能;目的2） 鉴定CUL-6泛素连接酶的靶标;和 靶点，包括可能的溶酶体降解。预期结果是确定哪个SKR 蛋白质异源二聚化，SKR蛋白质与F-box蛋白质相互作用， 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， 将目标导向溶酶体。这项研究意义重大，因为它可能导致新的 用于与蛋白质稳态破坏相关的衰老相关疾病的治疗。