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

酿酒酵母衰老过程中错误折叠蛋白聚集过程及其相关性的研究

• 批准号: 396975076
• 负责人: Dr. Arthur Fischbach
• 金额: --
• 依托单位: Department of Microbiology and Immunology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/396975076?language=en
• 关键词: Investigations; process; relevance; aggregation; misfolded

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.

衰老是生命中普遍存在的过程。它影响大多数生物，特别是动物，但也影响许多单细胞生物。衰老的特征是时间依赖性功能下降，最终导致生物体死亡。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之一是芽殖酵母酿酒酵母。尽管它的单细胞性质和简单性，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，这被认为是老化的标志，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降低以及老化期间破坏剂水平升高的综合效应。作为PQC的额外保护机制，聚集体在某些保护性细胞位置处聚结和隔离成大的内含物已经发展。然而，酵母细胞聚集聚结和形成内含物的能力也随着老化而下降。为什么会出现这种情况仍然是未知的，可能是多个系统同时崩溃的结果。本提案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体的聚结如何在衰老过程中功能下降的问题。第二个目的是研究相关的聚集蛋白是如何作为一个潜在的原因，在S。酿酒酵母，这将通过工程新的菌株和方法，允许从母细胞中去除蛋白质聚集体进行研究。为了解决第一个目标，将采用吡咯啉-5-羧酸还原酶（PRO3）的错误折叠的温度敏感等位基因，即pro3-1。观察到pro3-1能够在热强制降解期间有效地掺入1-2个包涵体中。然而，这种突变蛋白在老化细胞中不能有效地聚结，而是形成大量的小聚集体，表明特定蛋白质质量控制途径的失败。目标将是使用用pro3-1-mRuby和Hsp 104-GFP修饰的酵母敲除文库的高含量显微镜筛选来鉴定这些缺陷途径。Hsp 104是一种解聚酶，可用于监测夹杂物的形成和分解。为了研究衰老过程中聚集蛋白的相关性，以及它们是否构成衰老的原因，将以工程化的人工方式将聚集体从母细胞中移除到子细胞中。将研究这如何影响母细胞的寿命。人工靶向将通过使用马达蛋白来实现。为了特异性识别蛋白质聚集体，解聚酶Hsp 104 p将与这样的马达蛋白融合。