Proteomics of widespread, reversible protein assemblies in aging and aggregation

描述（由申请人提供）：蛋白质必须组织成根据细胞需要组装和拆卸的复合物，同时避免灾难性聚集。这种聚集对于重要的人类疾病，如阿尔茨海默氏症、帕金森氏症和亨廷顿氏症，甚至衰老本身都是至关重要的。这种竞争性更新建立在GM076536（单细胞基因组蛋白和RNA定位细胞芯片）第一个资助周期的观察基础上：数百种不同的、正常的细胞质蛋白在酵母酵母静止细胞中形成代谢物诱导的、可逆的、宏观的点状灶。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理解静止细胞和聚集性疾病是很重要的。如果这些组合是功能性的或含有功能性蛋白质，这代表了静止细胞中意想不到的高水平组织，这对人类细胞具有重要的影响，因为人类细胞大部分时间都处于静止状态。如果这些组合代表广泛的聚集，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？该基金提出质谱蛋白质组学、遗传学、细胞生物学和成像实验来评估这些假设，并更好地了解这种广泛的、可逆的蛋白质聚集的机制基础。《特异性目的1》的实验集中于确定酵母细胞中病灶形成的动力学、调控和机制。酵母中很少有人类不存在的基本调节形式，初步测试表明，这些灶也会在人类细胞中形成。因此，在Aim II中，我们的目标是更广泛地确定这些焦点及其调控逻辑是否在人类细胞中保守，并因此可能被进化选择。最后，初步证据表明，病灶形成在复制性和年岁性衰老细胞中都有所增加。Aim III中的实验将定义病灶形成和聚集对细胞寿命的贡献，并确定聚集与衰老之间的因果关系。这将提供具体的证据，以蛋白质为基础，在静止细胞中形成功能性蛋白质组装或灾难性聚集。这项工作将确定蛋白质焦点和聚集体在多大程度上控制静止细胞的存活和影响细胞寿命，并且是朝着更好地理解聚集性疾病和衰老背后的力量迈出的一步。

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.