Investigations on the process and relevance of aggregation of misfolded proteins during aging in Saccharomyces Cerevisiae

衰老是生命中无处不在的过程。它影响大多数生物，特别是动物，但也影响许多单细胞生物。衰老的特征是一种随时间变化的功能衰退，最终导致有机体的死亡。人们对衰老的分子原因仍然知之甚少。对衰老研究贡献最大的生物体之一是出芽酵母酿酒酵母。尽管它的单细胞性质和简单性，但它表现出衰老表型，与更复杂的生物体共享许多特性。在酵母的老化过程中，蛋白质聚集体随着时间的推移而积累，这被认为是衰老的标志，导致基本细胞功能的崩溃。这可归因于蛋白质质量控制系统（PQC）去除聚集蛋白质的能力下降以及老化过程中有害物质水平升高的综合影响。作为PQC的另一种保护机制，聚集体在细胞的某些保护性位置聚合和隔离成大包裹体。然而，酵母细胞聚集并形成包涵体的能力也随着年龄的增长而下降。为什么会出现这种情况仍然未知，可能是多个系统同时崩溃的结果。本研究的第一个目标是更详细地研究衰老过程中蛋白质聚集的过程，特别是关于蛋白质聚集的聚结如何在衰老过程中功能下降的问题。第二个目标是研究相关的聚集蛋白是如何作为酿酒酵母衰老过程的潜在原因的，这将通过工程新菌株和允许从母细胞中去除蛋白质聚集物的方法进行研究。为了解决第一个目标，将使用错误折叠的吡咯-5-羧酸还原酶（PRO3）的温度敏感等位基因，即PRO3 -1。结果表明，在热胁迫下，pro3-1能够有效地融入1-2个包涵体中。然而，这种突变蛋白在衰老细胞中不能有效地结合，而是形成大量的小聚集体，表明特定的蛋白质质量控制途径失败。我们的目标是利用pro3-1-mRuby和Hsp104-GFP修饰的酵母敲除文库的高含量显微镜筛选来识别这些缺陷通路。Hsp104是一种分解酶，可用于监测包涵体的形成和分解。为了研究在衰老过程中聚集的蛋白质的相关性，以及它们是否构成衰老的原因，将通过工程的、人工的方式将聚集的蛋白质从母细胞移到子细胞中。将研究这如何影响母细胞的寿命。利用马达蛋白实现人工靶向。为了特异性识别蛋白质聚集体，解聚酶Hsp104p将被融合到这样一个马达蛋白上。

Aging is a ubiquitous process in life. It affects most living organisms, in particular animals but also many unicellular organisms. Aging is characterized by a time-dependent functional decline leading ultimately to death of the organism. The molecular causes of aging are still weakly understood. One of the organisms that contributed the most to aging research is the budding yeast Saccharomyces cerevisiae. Despite its unicellular nature and simplicity, it exhibits an aging phenotype, which shares many properties with more complex organisms. During aging of yeast, protein aggregates accumulate with time, which is considered a hallmark of aging, giving rise to a collapse of essential cellular functions. This can be attributed to a combined effect of a decreased capacity of the protein quality control system (PQC) to remove aggregated proteins together with elevated levels of damaging agents during aging. As an additional protection mechanism to the PQC, the coalescence and sequestration of aggregates into large inclusions at certain protective cellular positions has evolved. However, the ability of yeast cells for aggregate coalescence and to form inclusions declines with aging, as well. Why this is the case is still unknown and could be the results of a simultaneous collapse of multiple systems.The first objective of this proposal is to investigate the process of protein aggregation during aging in more detail, especially regarding the question of how the coalescence of protein aggregates declines in its function during aging. The second objective is to investigate how relevant aggregated proteins are as a potential cause of the aging process in S. cerevisiae, which will be investigated by engineering new strains and approaches allowing the removal of protein aggregates from mother cells. To address the first objective, a misfolding, temperature sensitive allele of Pyrroline-5-carboxylate reductase (PRO3), i.e. pro3-1 will be employed. It was observed that pro3-1 is able to be efficiently incorporated into 1-2 inclusion bodies during heat stress. However, this mutant protein does not efficiently coalesce in aging cells and instead forms a large number of small aggregates, indicating a failure of specific protein quality control pathways. The goal will be to identify these defective pathways using a high content microscopy screen of the yeast knockout library modified with pro3-1-mRuby and Hsp104-GFP. Hsp104 is a disaggregase and can be used to monitor inclusion formation and disassembly. To investigate the relevance of aggregated proteins during aging and if they constitute a cause of aging, aggregates will be removed from the mother cell in an engineered, artificial way into the daughter cell. It will be investigated how this influences the lifespan of the mother cell. The artificial targeting will be achieved by using a motor protein. To specifically recognize protein aggregates, the disaggregase Hsp104p will be fused to such a motor protein.