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

衰老是生活中无处不在的过程。它影响大多数生物，特别是动物，但也影响许多单细胞生物。衰老的特征是与时间相关的功能衰退，最终导致有机体死亡。人们对衰老的分子原因仍知之甚少。对衰老研究贡献最大的有机体之一是萌芽酵母酿酒酵母。尽管它具有单细胞的性质和简单，但它表现出老化的表型，这与更复杂的生物有许多共同的特性。在酵母老化过程中，蛋白质聚集体随时间积累，这被认为是老化的标志，导致基本细胞功能崩溃。这可以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破坏性因素水平上升的综合影响。作为PQC的一种额外保护机制，聚集体在某些保护性的细胞位置聚合和隔离成大的包裹体已经进化。然而，酵母细胞聚合和形成包涵体的能力也随着年龄的增长而下降。这种情况的原因尚不清楚，可能是多个系统同时崩溃的结果。这项建议的第一个目标是更详细地研究老化过程中蛋白质聚集的过程，特别是关于蛋白质聚集如何在老化过程中功能下降的问题。第二个目标是调查相关的聚集蛋白如何作为酿酒酵母老化过程的潜在原因，这将通过设计新的菌株和允许从母细胞中去除蛋白质聚集的方法来进行研究。为了解决第一个目标，将使用一个错误折叠的、温度敏感的吡咯烷-5-羧酸还原酶(PR03)等位基因，即pro3-1。观察到在热应激过程中，Pro3-1能够有效地结合到1-2个包涵体中。然而，这种突变的蛋白质在衰老的细胞中不能有效地结合，而是形成大量的小聚集体，这表明特定的蛋白质质量控制途径失败。目标是使用用pro3-1-mRuby和Hsp104-GFP修饰的酵母基因敲除文库的高含量显微镜筛选来识别这些缺陷途径。HSP104是一种解聚酶，可以用来监测包涵体的形成和解聚。为了研究聚集蛋白质在衰老过程中的相关性，以及如果它们构成衰老的原因，聚集体将以一种工程的、人工的方式从母细胞中移除，进入子细胞。这将被研究如何影响母细胞的寿命。人工靶向将通过使用一种马达蛋白来实现。为了专门识别蛋白质聚集体，解聚酶Hsp104P将与这样的马达蛋白融合。

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.