Mechanisms of protection against alpha-synuclein-induced oxidative stress

描述（由申请人提供）：该项目的长期目标是了解α -突触核蛋白（1-syn）磷酸化增强该神经蛋白毒性的机制。利用酵母系统，我们寻求扩展我们的发现，即高度保守的磷酸酶和两个高度保守的信号蛋白可以防止1-syn诱导的ROS和细胞死亡。具体目的是：1)通过在呼吸缺陷细胞或过氧化物酶体缺陷细胞中表达1-syn，并用ROS敏感染料染色，确定ROS是来自线粒体还是来自过氧化物酶体。2)确定S129位点1-syn磷酸化增强1-syn致细胞死亡能力的机制。将采用双杂交筛选来寻找与WT 1-syn的毒性磷酸化形式相互作用的蛋白质。3)描述当激酶/磷酸酶平衡向有利于激酶的方向转变时细胞是如何死亡的。虽然我们发现的保护细胞免受1-syn诱导的ROS的必需磷酸酶不能被删除，但它的非必需调节亚基可以被删除。实验将使用gfp标记的1-syn来确定当激酶/磷酸酶平衡通过敲除磷酸酶调节基因而向有利于激酶的方向倾斜时，1-syn包涵形成、蛋白酶体功能障碍或线粒体功能障碍是否会导致细胞死亡。4)我们发现了两个酵母信号基因与WT 1-syn和A30P有合成致死相互作用，而与A53T没有。这些信号基因也存在于人类神经元中，它们调节细胞周期、细胞内信号、分化、离子通道、囊泡运输和凋亡。我们假设这两种信号蛋白与毒性磷酸化形式的1-syn （WT或A30P）结合，从而保护细胞免受1-syn的积累。这一假设将通过监测各种1-syns与信号蛋白之间的直接结合以及监测当信号基因被删除时细胞如何死亡来验证。了解激酶/磷酸酶和信号分子如何调节1-syn磷酸化状态及其毒性，可能会导致新的神经保护疗法，从而延缓甚至预防PD的发作。这样的治疗方法可以调节激酶-磷酸酶平衡，使1-syn进入去磷酸化状态。项目的叙述

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanism by which phosphorylation of alpha-synuclein (1-syn) enhances the toxicity of this neuroprotein. Employing a yeast system, we seek to extend our findings that a highly conserved phosphatase and two highly conserved signaling proteins prevent 1-syn-induced ROS and cell death. The specific aims are to: 1) Determine whether ROS originate from mitochondria or peroxisomes by expressing 1-syn in respiratory-deficient cells or peroxisome-deficient cells and staining with an ROS-sensitive dye. 2) Determine the mechanism by which phosphorylation of 1-syn at S129 enhances 1-syn's ability to cause cell death. A two-hybrid screen will be employed to find proteins that interact with the toxic phosphorylated form of WT 1-syn. 3) Characterize how cells die when the kinase/phosphatase balance shifts in favor of kinases. Although the essential phosphatase that we discovered that protects cells from 1-syn-induced ROS cannot be deleted, its non-essential regulatory subunits can be. Experiments will use GFP-tagged 1-syns to determine whether 1-syn inclusion formation, proteasome dysfunction, or mitochondrial dysfunction causes cell death when the kinase/phosphatase balance is tipped in favor of the kinases by knocking out phosphatase regulatory genes. 4) We discovered two yeast signaling genes that exhibit synthetic lethal interactions with WT 1-syn and A30P but not A53T. These signaling genes are also present in human neurons where they regulate the cell cycle, intracellular signaling, differentiation, ion-channels, vesicle trafficking, and apoptosis. We hypothesize that these two signaling proteins bind to toxic phosphorylated forms of 1- syn (WT or A30P), and this protects cells from the build up of 1-syn. This hypothesis will be tested by monitoring for direct binding between the various 1-syns and the signaling protein and by monitoring how cells die when the signaling genes are deleted. Understanding how kinases/phosphatases and signaling molecules regulate 1-syn phosphorylation state and hence its toxicity could lead to novel neuroprotective therapeutics that could delay or even prevent the onset of PD. Such therapeutics would modulate the kinase-phosphatase equilibrium in such a way as to drive 1-syn into a dephosphorylated state.7. Project Narrative A simple chemical modification (phosphorylation) of the Parkinson's disease-related protein alpha- synuclein dramatically increases the toxicity of alpha-synuclein. Our goal is to determine the enzymes and proteins that regulate the chemical modification of alpha-synuclein. This information could lead to novel drugs that function to inhibit the chemical modification of alpha-synuclein.