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)的磷酸化增强这种神经蛋白的毒性的机制。利用酵母系统，我们试图扩展我们的发现，即一个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。其具体目的是：1)通过在呼吸缺陷细胞或过氧化酶体缺陷细胞中表达1-syn并用ROS敏感的染料染色来确定ROS是来自线粒体还是来自过氧化酶体。2)确定1-syn在S129位的磷酸化增强1-syn导致细胞死亡的机制。双杂交筛选将被用来寻找与有毒的磷酸化形式的WT 1-syn相互作用的蛋白质。3)描述当激酶/磷酸酶平衡向激酶倾斜时细胞是如何死亡的。尽管我们发现的保护细胞免受1-syn诱导的ROS的必要磷酸酶不能被删除，但它的非必要调节亚基可以被删除。4)我们发现了两个酵母信号基因，它们与WT 1-syn和A30P呈现合成致死作用，而不是A53T。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、囊泡运输和细胞凋亡。我们假设这两个信号蛋白与有毒的磷酸化形式的1-syn(WT或A30P)结合，从而保护细胞免受1-syn的积累。这一假说将通过监测各种1-SYN和信号蛋白之间的直接结合以及监测信号基因缺失时细胞如何死亡来检验。了解激酶/磷酸酶和信号分子如何调节1-syn的磷酸化状态，从而导致其毒性可能导致新的神经保护疗法，可以延缓甚至预防帕金森病的发生。这种疗法将以这样一种方式调节激酶-磷酸酶平衡，以使1-syn进入去磷酸化状态。项目叙事 帕金森氏病相关蛋白α-突触核蛋白的简单化学修饰(磷酸化)显著增加了α-突触核蛋白的毒性。我们的目标是确定调节α-突触核蛋白化学修饰的酶和蛋白质。这些信息可能会导致新的药物，其功能是抑制α-突触核蛋白的化学修饰。

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.