Probing organismal proteostasis through the response to intracellular infection

项目摘要/摘要 与衰老相关的疾病与蛋白质稳态的破坏有关。一位少校 蛋白平衡的破坏者是细胞内病原体的感染，但人们对此知之甚少 这些感染可能会促进蛋白平衡。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描述一种新的 我们在线虫体内发现的蛋白平衡反应称为细胞内病原体反应 或知识产权。我们之前的工作展示了编码扑杀的组成部分的IPR基因是如何上调的 环泛素连接酶可促进蛋白稳定性的改善，包括提高耐热性。这样做的目的是 建议是确定这种多亚基泛素连接酶是如何组装的，确定其靶标(S)，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诱导泛素连接酶基因表达 亚基包括：1)cullin cul-6，2)环结构域蛋白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-依赖于氧化还原的二聚- 含有泛素连接酶四聚体(以创建总共八个亚基的酶复合体)导致其激活， 这种酶复合体泛素化一个尚未确定的靶标，然后被溶酶体降解 来调节蛋白平衡。其理论基础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内外数据 关于SKR-3的氧化还原依赖的二聚作用，以及我们未发表的遗传学和药理学 数据表明，CuL-6泛素连接酶介导的耐热性增加依赖于 溶酶体。我们的工作是创新的，因为我们正在追求知识产权，这是最近描述的一种蛋白质稳定剂 不依赖于典型的蛋白平衡途径的反应，如热休克反应 蛋白质反应。 我们将用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在体内外的相互作用和功能；目的2) 确定CuL-6泛素连接酶的靶标(S)；并目的3)表征这些泛素连接酶的下游命运 靶标，包括溶酶体可能的降解。预期的结果是确定哪个SKR 蛋白质异二聚化，哪种SKR蛋白与哪种F-box蛋白相互作用，哪些蛋白是靶向 这种泛素连接酶复合体，以及自噬因子和其他细胞成分参与其中 将目标定向到溶酶体。这项拟议的研究意义重大，因为它可能带来新的 与蛋白质代谢紊乱相关的衰老相关疾病的治疗。

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.