Proteomics of widespread, reversible protein assemblies in aging and aggregation

描述（由申请人提供）：蛋白质必须组织成复合物，根据细胞需要组装和分解，同时避免灾难性聚集。这种聚集对重要的人类疾病至关重要，如阿尔茨海默氏症、帕金森氏症和亨廷顿氏病，甚至可能是衰老本身。这种竞争性更新建立在资助GM076536（用于单细胞中全基因组蛋白质和RNA定位的细胞芯片）的第一个资助周期中的观察基础上：数百种不同的、正常的胞质蛋白质种类在酵母S的静止细胞中形成代谢物诱导的、可逆的、宏观点状病灶。啤酒。这些宏观组合是功能性的还是灾难性的？这个问题的答案对于我们了解静止细胞和基于聚集的疾病非常重要。如果这些组件是功能性的或包含功能性蛋白质，这代表了静止细胞中出乎意料的高水平组织，对人类细胞具有重要的影响，人类细胞在其生命的大部分时间都处于静止状态。如果这些组装代表广泛的聚集，则这些结构可能是衰老和聚集相关疾病的重要因素。更一般地说，蛋白质聚集可能是一种新的调节机制吗？这项资助提出了质谱蛋白质组学，遗传学，细胞生物学和成像实验，以评估这些假设，并更好地了解这种广泛的，可逆的蛋白质聚集的机制基础。实验在具体目标我集中于确定动力学，调节和机制的焦点形成酵母细胞。酵母中很少有人类不存在的基本调节形式，初步测试表明这些病灶也在人类细胞中形成。因此，在目标II中，我们的目标是更广泛地确定这些病灶及其调控逻辑是否在人类细胞中保守，因此可能被进化选择。最后，初步证据表明，病灶形成增加复制和时间老化的细胞。目标III中的实验将定义病灶形成和聚集对细胞寿命的贡献，并确定聚集和衰老之间的因果关系。这些将提供具体的证据，在蛋白质的基础上，无论是在静止细胞中形成功能蛋白质组装或灾难性的聚集。这项工作将定义蛋白质焦点和聚集体在多大程度上控制静止细胞的存活并影响细胞寿命，并且是更好地理解聚集疾病和衰老的基础力量的一步。 公共卫生相关性：蛋白质必须组织成复合物，根据细胞的需要组装和分解，同时避免灾难性的聚集。这种聚集对重要的人类疾病至关重要，如阿尔茨海默氏症、帕金森氏症和亨廷顿氏病，甚至可能是衰老本身。这项资助提出了质谱蛋白质组学，细胞生物学和成像实验，以了解在静止细胞中观察到的广泛，可逆的蛋白质聚集的机制基础。这项工作将增加我们对蛋白质聚集的机制和环境的理解，从而将朝着更好地理解聚集性疾病和衰老的潜在力量迈出一步。

DESCRIPTION (provided by applicant): Proteins must organize into complexes that assemble and disassemble depending on cellular needs, while simultaneously avoiding catastrophic aggregation. Such aggregation is critical for important human diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, and perhaps even aging itself. This competitive renewal builds from an observation made in the first funding cycle of grant GM076536 (Cell chips for genome- wide protein and RNA localization in single cells): hundreds of distinct, normally cytosolic protein species form metabolite-induced, reversible, macroscopic punctate foci in quiescent cells of the yeast S. cerevisae. Are these macroscopic assemblies functional or catastrophic? The answers to this question are important for our understanding of quiescent cells and of aggregation-based disease. If these assemblies are functional or contain functional proteins, this represents unexpectedly high levels of organization in quiescent cells, with important ramifications for human cells, which are in the quiescent state for the majority of their lives. If these assemblies represent widespread aggregation, such structures are likely important factors for aging and aggregation-related disease. More generally, might protein aggregation be a novel regulatory mechanism? This grant proposes mass spectrometry proteomics, genetics, cell biology, and imaging experiments to evaluate these hypotheses and to better understand the mechanistic basis for this widespread, reversible protein aggregation. Experiments in Specific Aim I focus on determining kinetics, regulation, and mechanisms of foci formation in yeast cells. Only rarely is there a basic form of regulation in yeast that doesn't exist in humans, and initial tests suggest these foci also form in human cells. Thus, in Aim II, we aim to determine more broadly if these foci, and their regulatory logic, are conserved in human cells and hence likely selected for evolutionarily. Lastly, initial evidence suggests foci formation increases both in replicatively and chronologically aged cells. Experiments in Aim III will define the contribution of foci formation and aggregation towards cellular lifespan, and determine the causal relationship between aggregation and aging. These will provide concrete evidence, on a protein-by-protein basis, either for forming functional protein assemblies in quiescent cells or for catastrophic aggregation. This work will define the extent to which protein foci and aggregates govern survival in quiescent cells and impact cellular lifespan, and is a step towards better understanding of the forces underlying aggregation diseases and aging. PUBLIC HEALTH RELEVANCE: Proteins must organize into complexes that assemble and disassemble depending on cellular needs, while simultaneously avoiding catastrophic aggregation. Such aggregation is critical for important human diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases, and perhaps even aging itself. This grant proposes mass spectrometry proteomics, cell biology and imaging experiments to understand the mechanistic basis for widespread, reversible protein aggregation observed in quiescent cells. This work will increase our understanding of the mechanisms and circumstances under which proteins aggregate, and thus will be a step towards better understanding of the forces underlying aggregation diseases and aging.