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

蛋白质稳态是维持正常细胞功能的重要因素。它是通过蛋白质合成和处理之间的微妙平衡来实现的。蛋白质稳态的缺陷与各种神经退行性疾病有关，包括帕金森病（PD）。 PD的特征是α-突触核蛋白聚集成蛋白质内含物，表明细胞蛋白质稳态的显著功能障碍。α-突触核蛋白的积累伴随着S129处高度升高的磷酸化，表明这种翻译后修饰与致病性和α-突触核蛋白周转有关。在上一个资助期，我们已经使用酵母的工具箱来模拟PD的细胞基本机制，表征了基本蛋白质抑制途径与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导致毒性，这被认为是与神经元类似的显著生长减少和包涵体形成。用酵母菌株收集物进行的全基因组筛选鉴定了α-突触核蛋白毒性和聚集的多种调节剂。最突出的类别与蛋白质稳态有关，包括蛋白质合成和泛素依赖性降解。我们发现α-突触核蛋白的表达减少了26 S蛋白酶体亚基的丰度，并改变了泛素结合物。蛋白质稳态功能障碍与α-突触核蛋白周转相关，并依赖于S129磷酸化。该研究项目的目的是了解α-突触核蛋白毒性和酵母细胞蛋白质稳态之间复杂的相互作用，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S129 A变体如何影响酵母中的蛋白质动力学，哪些蛋白质改变了它们的稳定性以及对细胞蛋白质稳态的影响。主要的重点将是蛋白酶体伴侣Rpn 14/PAAF 1在初步实验中确定的这一建议。(ii)我们将破译α-突触核蛋白和S129 A表达对26 S蛋白酶体稳态的影响。我们的目的是阐明，这些蛋白质如何影响26 S蛋白酶体的降解，通过蛋白吞噬和他们的排序到保护性蛋白酶体储存颗粒在不同的压力条件下。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质稳态网络的关键组成部分的综合图片，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.