Vozdek Roman, Wang Bingying, Li Kathy H, Pramstaller Peter P, Hicks Andrew A, Ma Dengke K
Institute for Biomedicine, Eurac Research, Affiliated institute of the University of Lübeck, Bolzano, 39100, Italy.
Cardiovascular Research Institute and Department of Physiology, University of California San Francisco, San Francisco, CA, 94158, USA.
Open Res Eur. 2023 Sep 15;2:23. doi: 10.12688/openreseurope.14235.2. eCollection 2022.
Parkin, which when mutated leads to early-onset Parkinson's disease, acts as an E3 ubiquitin ligase. How Parkin is regulated for selective protein and organelle targeting is not well understood. Here, we used protein interactor and genetic screens in ( to identify new regulators of Parkin abundance and showed their impact on autophagy-lysosomal dynamics and alpha-Synuclein processing. We generated a transgene encoding mCherry-tagged Parkin - Parkinson's Disease Related 1 (PDR-1). We performed protein interactor screen using Co-immunoprecipitation followed by mass spectrometry analysis to identify putative interacting partners of PDR-1. Ribonucleic acid interference (RNAi) screen and an unbiased mutagenesis screen were used to identify genes regulating PDR-1 abundance. Confocal microscopy was used for the identification of the subcellular localization of PDR-1 and alpha-Synuclein processing. We show that the transgene rescues the mitochondrial phenotype of mutants and that the expressed PDR-1 reporter is localized in the cytosol with enriched compartmentalization in the autophagy-lysosomal system. We determined that the transgenic overexpression of the PDR-1 reporter, due to inactivated small interfering RNA (siRNA) generation pathway, disrupts autophagy-lysosomal dynamics. From the RNAi screen of putative PDR-1 interactors we found that the inactivated Adenine Nucleotide Translocator , or hybrid ubiquitin genes encoding a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively, induced PDR-1 abundance and affected lysosomal dynamics. In addition, we demonstrate that the abundant PDR-1 plays a role in alpha-Synuclein processing. These data show that the abundant reporter of Parkin affects the autophagy-lysosomal system together with alpha-Synuclein processing which can help in understanding the pathology in Parkin-related diseases.
帕金森蛋白（Parkin）发生突变时会导致早发性帕金森病，它作为一种E3泛素连接酶发挥作用。目前对于Parkin如何被调控以实现对特定蛋白质和细胞器的靶向作用还了解甚少。在此，我们在[具体实验对象]中进行了蛋白质相互作用因子和基因筛选，以鉴定调控Parkin丰度的新因子，并展示了它们对自噬 - 溶酶体动力学及α - 突触核蛋白加工的影响。我们构建了一个编码mCherry标记的帕金森病相关蛋白1（PDR - 1）的转基因。我们通过免疫共沉淀结合质谱分析进行蛋白质相互作用因子筛选，以鉴定PDR - 1的假定相互作用伙伴。利用RNA干扰（RNAi）筛选和无偏向性诱变筛选来鉴定调控PDR - 1丰度的基因。共聚焦显微镜用于鉴定PDR - 1和α - 突触核蛋白加工的亚细胞定位。我们发现该转基因可挽救[具体突变体]的线粒体表型，且表达的PDR - 1报告基因定位于细胞质中，在自噬 - 溶酶体系统中呈现富集的区室化分布。我们确定，由于小干扰RNA（siRNA）生成途径失活，PDR - 1报告基因的转基因过表达会破坏自噬 - 溶酶体动力学。从对PDR - 1假定相互作用因子的RNAi筛选中，我们发现失活的腺嘌呤核苷酸转位酶，或分别编码与核糖体蛋白L40和S27a融合的单拷贝泛素的杂交泛素基因，可诱导PDR - 1丰度并影响溶酶体动力学。此外，我们证明大量存在的PDR - 1在α - 突触核蛋白加工中发挥作用。这些数据表明，Parkin的大量报告基因会连同α - 突触核蛋白加工一起影响自噬 - 溶酶体系统，这有助于理解与Parkin相关疾病的病理机制。
