Probing organismal proteostasis through the response to intracellular infection

项目摘要/摘要 维持蛋白质平衡，或蛋白质平衡，对健康至关重要。蛋白质平衡被扰乱在 几种与衰老有关的疾病，包括神经退行性疾病，如阿尔茨海默氏症、帕金森氏症和 亨廷顿氏病。护理这些疾病的患者正在消耗我们越来越多的 医疗保健预算。引人注目的是，仅照顾阿尔茨海默氏症患者一项就花费了226亿美元 一年，而且没有可用的治疗方法。因此，需要新的方法。其中一种方法涉及 利用宿主/病原体对细胞内微生物感染的反应。我们的长期目标是解剖 宿主细胞上调蛋白平衡途径以应对增加的负担的机制 细胞内感染和复制。弥合这一理解上的差距将为我们提供关于 蛋白平衡，并可能为神经退行性疾病提供新的治疗方法。我们的中心假设是 宿主可以感觉到细胞内感染的影响，并增加蛋白质平衡能力来应对这种情况 增加了负担。这里的目标是确定线虫线虫通过什么机制 一种天然的细胞内微生物感染后上调泛素连接酶组分 微孢子虫门。微孢子虫通常感染包括人类在内的所有动物，并可以复制到 很高的水平，但不会对宿主造成明显影响，这可能是由于宿主的补偿机制。 我们最近的发现表明，线虫上调泛素连接酶组分是为了响应 包括微孢子虫和病毒在内的多种细胞内感染(Bakowski等人，2014年)。在未发布的数据中，我们 已经分离出在F-box相关基因(fbxr-1)上有缺陷的突变株，该基因结构性地表达这些泛素 连接酶组分。Fbxr-1突变体提高了对病原菌的抵抗力，同时也大大增强了 耐热性和聚集蛋白水平降低，表明蛋白稳定能力得到改善。 我们假设线虫增加了Skp-cullin-F-box(SCF)泛素连接酶的转录 为了靶向错误折叠的蛋白质进行泛素化和破坏，增加对 蛋白毒素的侮辱。在具体目标1中，我们将确定库林在哪里，以及其上游的负面影响 调节剂FBXR-1起调节耐热性的作用。我们还将对 库林来检验这一假设，即它是SCF泛素连接酶多亚基成分的一部分。《特定目标2》 我们将使用遗传和生化方法鉴定这些SCF连接酶的其他成分。以特定的目标 3我们将确定FBXR-1在哪里调节蛋白质聚集体的水平。在具体目标4中，我们将执行 上位性和鉴定FBXR-1/Cullin途径的新成分，包括一个候选转录 因素。这种方法是创新的，因为它利用宿主的反应来强制细胞内感染 了解如何改善蛋白质平衡。这项拟议的研究具有重要意义，因为它可能导致 蛋白质平衡受损疾病的新治疗方法，如阿尔茨海默病。

Project Summary/Abstract Maintenance of protein homeostasis, or proteostasis, is critical for health. Proteostasis is perturbed in several aging-related diseases including neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases. Caring for patients with these diseases is consuming an increasing fraction of our health care budget. Strikingly, care for Alzheimer's patients alone is responsible for $226 billion in spending per year, and no treatments are available. Thus, new approaches are needed. One such approach involves leveraging the host/pathogen response to infection with intracellular microbes. Our long-term goal is to dissect the mechanisms by which host cells upregulate proteostasis pathways to cope with the increased burden of intracellular infection and replication. Closing this gap in our understanding will provide new insights about proteostasis, and could provide novel treatments for neurodegenerative diseases. Our central hypothesis is that hosts can sense the effects of intracellular infection and increase proteostasis capacity to cope with this increased burden. The objective here is to determine the mechanisms by which the nematode C. elegans upregulates ubiquitin ligase components in response to infection by a natural intracellular microbe that belongs to the microsporidia phylum. Microsporidia commonly infect all animals including humans, and can replicate to very high levels without causing overt effects on the host, likely due to host compensatory mechanisms. Our recent findings indicate that C. elegans upregulates ubiquitin ligase components in response to diverse intracellular infections including microsporidia and virus (Bakowski et al 2014). In unpublished data we have isolated mutants defective in an F-box-related gene (fbxr-1) that constitutively express these ubiquitin ligase components. fbxr-1 mutants have increased pathogen resistance, as well as greatly enhanced thermotolerance and reduced levels of aggregated proteins, indicating improved proteostasis capacity. We hypothesize that C. elegans increases transcription of Skp-Cullin-F-box (SCF) ubiquitin ligase components in order to target misfolded proteins for ubiquitylation and destruction, increasing tolerance of proteotoxic insults. In Specific Aim 1 we will determine where the Cullin, as well as its upstream negative regulator FBXR-1 act to regulate thermotolerance. We also will perform structure/function analysis on the Cullin to test the hypothesis that it is part of a multi-subunit SCF ubiquitin ligase component. In Specific Aim 2 we will identify these other SCF ligase components using genetic and biochemical approaches. In Specific Aim 3 we will determine where FBXR-1 regulates levels of protein aggregates. In Specific Aim 4 we will perform epistasis and characterize new components of the FBXR-1/Cullin pathway, including a candidate transcription factor. This approach is innovative because it leverages the host response to obligate intracellular infection to understand how proteostasis can be improved. The proposed research is significant because it could lead to new treatments for diseases of compromised proteostasis, such as Alzheimer's disease.