Training in proteomics of novel kinase substrates for neurodegeneration

描述(申请人提供)：帕金森病(PD)是一种衰弱的神经退行性疾病，目前还没有有效的神经保护疗法。来自死后脑研究、氧化毒素模型和帕金森病遗传学的数据汇聚在一起，都表明线粒体激酶网络的改变与帕金森病的发病有关。线粒体PTEN诱导的激酶1(PINK1)突变发生在家族性和可能的散发性帕金森病中。这位候选人在培养和帕金森病发病的动物模型中神经元命运的信号调控方面是一位成熟而富有成效的研究人员。这位候选人发表的初步工作涉及线粒体靶向细胞外信号调节蛋白激酶2(ERK2)和减少PINK1信号转导促进多巴胺能神经元细胞死亡。野生型PINK1的过度表达在细胞死亡模型中提供了神经保护。因此，确定PINK1的下游靶点和氧化还原激活的ERK2的线粒体靶点，将代表着理解调控神经元生存和死亡的机制的重要步骤。使用强大的磷酸蛋白质组学和质谱学技术，可以完成对新的激酶底物的鉴定，如PINK1和上下文特定ERK2靶标的鉴定。这项建议利用了匹兹堡大学强大的细胞信号、蛋白质组/代谢组、药物和神经退化研究环境。候选人的近期目标是在匹兹堡蛋白质组核心设施主任比利·戴的指导下获得质谱学和磷酸蛋白质组学方面的培训。共同导师、自由基生物化学专家布鲁斯·弗里曼将提供基本氧化还原蛋白质组学方面的补充培训。除了短期课程、研讨会和技术讲习班外，还将从一项试验项目中获得实践经验，该项目涉及识别PINK1底物的ATP结合口袋突变和质谱分析。这一职业提升奖将为一位研究神经退行性疾病的知名内科科学家提供高度专业化的目标识别技术方面的基本交叉培训。巩固跨学科合作将使首席研究人员能够更快地将决定神经元能否成功适应致病压力的因素的知识转化为设计神经保护性PD疗法的新靶点。

DESCRIPTION (provided by applicant): Parkinson disease (PD) is a debilitating neurodegenerative disease for which there are no effective neuroprotective therapies. Converging data from post-mortem brain studies, oxidative toxin models, and PD genetics all implicate altered mitochondrial kinase networks in PD pathogenesis. Mutations in the mitochondrial PTEN-induced kinase 1 (PINK1) occur in familial and possibly sporadic PD. The candidate is an established and productive investigator in the signaling regulation of neuronal fate in culture and animal models of PD pathogenesis. The candidate's published and preliminary work implicate mitochondrial targeting of extracellular signal regulated protein kinase 2 (ERK2) and decreased PINK1 signaling in promoting dopaminergic neuronal cell death. Overexpression of wild type PINK1 confers neuroprotection in cell death models. Thus, identifying downstream targets of PINK1, and of mitochondrial targets of redox-activated ERK2, would represent important steps in understanding mechanisms that regulate neuronal survival and death. Identification of substrates for novel kinases such as PINK1, and of context specific ERK2 targets, can be accomplished using the powerful techniques of phospho-proteomics and mass spectroscopy. This proposal capitalizes on the strong cell signaling, proteomic/metabolomic, pharmaceutical, and neurodegeneration research environments at the University of Pittsburgh. The candidate's immediate goals are to obtain training in mass spectrometry and phospho-proteomics under the mentorship of Billy Day, Director of the Pittsburgh Proteomic Core Facility. Co-mentor Bruce Freeman, expert in free radical biochemistry, will provide complementary training in basic redox proteomics. In addition to short courses, seminars, and technical workshops, practical experience will be derived from a pilot project involving ATP-binding pocket mutagenesis and mass spectrometry to identify substrates of PINK1. Lay summary: This career enhancement award will provide an established physician-scientist studying neurodegenerative diseases with essential cross-training in highly specialized target identification technologies. Solidifying interdisciplinary collaborations will enable the principal investigator to more rapidly translate knowledge on factors that determine whether or not neurons can successfully adapt to disease promoting stresses into novel targets for designing neuroprotective PD therapies.