Interplay between alpha-synuclein induced toxicity in Parkinson’s disease and cellular proteostasis in yeast

蛋白质动态平衡是维持细胞正常功能的重要因素。它是通过蛋白质合成和处理之间的微妙平衡来实现的。蛋白平衡缺陷与各种神经退行性疾病有关，包括帕金森氏病(PD)。帕金森病的特征是α-突触核蛋白聚集成蛋白质包涵体，表明细胞蛋白平衡显著失调。在S129，α-突触核蛋白的积累伴随着高度的磷酸化，这表明这种翻译后修饰与致病性和α-突触核蛋白的转换有关。在之前的资助期间，我们已经利用酵母工具箱来模拟PD的细胞基本机制，表征了必要的蛋白稳定途径与α-突触核蛋白毒性、聚集和周转之间的相互作用。α-突触核蛋白在酵母中的表达会导致毒性，这被认为是与神经元相似的显著生长抑制和包涵体形成。对酵母菌株集合的全基因组筛选发现了α-突触核蛋白毒性和聚集的多个调节器。最突出的类别与蛋白质动态平衡有关，包括蛋白质合成和泛素依赖的降解。我们发现α-突触核蛋白的表达降低了26S蛋白酶体亚基的丰度，并改变了泛素结合物。蛋白平衡功能障碍与α-突触核蛋白的转换有关，并依赖于S129的磷酸化。这项研究的目的是了解酵母中α-突触核蛋白毒性和细胞蛋白稳定之间的复杂相互作用，以及S129磷酸化在这一过程中的作用。主要目标将通过两个具体目标进行评估：(I)我们将确定，α-突触核蛋白和磷酸化缺陷的S129A变体如何影响酵母中的蛋白质动态，哪些蛋白质改变了它们的稳定性，以及对细胞蛋白质平衡的影响。主要焦点将放在这一提议的初步实验中确定的蛋白酶体伴侣Rpn14/PAAF1上。(Ii)我们将破译α-突触核蛋白和S129A表达对26S蛋白酶体稳态的影响。我们的目的是阐明这些蛋白如何通过蛋白吞噬作用影响26S蛋白酶体的降解，并在不同的胁迫条件下将它们分选到保护性蛋白酶体储存颗粒中。我们的目标是获得受α-突触核蛋白积累和聚集影响的蛋白平衡网络的关键组成部分的综合图景，这将有助于我们理解衰老和疾病的分子机制。

Protein homeostasis is an essential factor for maintenance of normal cellular function. It is achieved by a delicate balance between protein synthesis and disposal. Deficits in proteostasis are linked to various neurodegenerative diseases, including Parkinson’s disease (PD). Characteristic for PD is the aggregation of alpha-synuclein into protein inclusions, indicating significant dysfunction in cellular proteostasis. Accumulation of alpha-synuclein is accompanied with highly elevated phosphorylation at S129, suggesting that this posttranslational modification is linked to pathogenicity and alpha-synuclein turnover. In the previous funding period, we have characterized the interplay between essential proteostasis pathways and alpha-synuclein toxicity, aggregation and turnover using the toolbox of yeast to model cellular basic mechanisms of PD. Expression of alpha-synuclein in yeast leads to toxicity, which is recognized as significant growth reduction and inclusion formation similar to neurons. Genome-wide screens with yeast strain collections identified multiple modulators of alpha-synuclein toxicity and aggregation. The most prominent categories were connected with protein homeostasis, including protein synthesis and ubiquitin-dependent degradation. We found that expression of alpha-synuclein reduces the abundance of 26S proteasome subunits and alters the ubiquitin conjugates. The proteostasis dysfunction correlates with alpha-synuclein turnover and depends on S129 phosphorylation. The aim of this research project is to understand the complex interplay between alpha-synuclein toxicity and cellular proteostasis in yeast, as well as the role of S129 phosphorylation in this process. The main goal will be assessed by two specific objectives: (i) We will determine, how alpha-synuclein and the phosphorylation deficient S129A variant affect protein dynamics in yeast, which proteins change their stability and what are the consequences for the cellular proteostasis. The main focus will be on the proteasomal chaperone Rpn14/PAAF1 identified in preliminary experiments for this proposal. (ii) We will decipher the impact of alpha-synuclein and S129A expression on the 26S proteasome homeostasis. We aim to elucidate, how these proteins affect the degradation of the 26S proteasomes by proteaphagy and their sorting into the protective proteasome storage granules under different stress conditions. Our goal is to obtain an integrative picture of the key components of the proteostasis network affected by alpha-synuclein accumulation and aggregation that will contribute to our understanding of the molecular mechanisms in aging and disease.