Training in proteomics of novel kinase substrates for neurodegeneration

描述（由申请人提供）：帕金森病（PD）是一种使人衰弱的神经退行性疾病，目前尚无有效的神经保护疗法。来自死后脑研究、氧化毒素模型和PD遗传学的聚合数据都暗示了PD发病机制中线粒体激酶网络的改变。线粒体PTEN诱导的激酶1（PINK 1）突变发生在家族性和可能散发性PD中。候选人是一个既定的和富有成效的研究者在神经元命运的信号调节的文化和动物模型的PD发病机制。候选人的已发表和初步工作涉及线粒体靶向细胞外信号调节蛋白激酶2（ERK 2）和减少PINK 1信号传导促进多巴胺能神经元细胞死亡。野生型PINK 1的过表达在细胞死亡模型中赋予神经保护作用。因此，确定PINK 1的下游靶点，以及氧化还原激活的ERK 2的线粒体靶点，将代表理解调节神经元存活和死亡的机制的重要步骤。新型激酶（如PINK 1）的底物和背景特异性ERK 2靶点的鉴定可以使用磷酸化蛋白质组学和质谱学的强大技术来完成。该提案利用了匹兹堡大学强大的细胞信号传导，蛋白质组学/代谢组学，制药和神经退行性变研究环境。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和磷酸蛋白质组学的培训。共同导师布鲁斯弗里曼，在自由基生物化学专家，将提供基本的氧化还原蛋白质组学的补充培训。除了短期课程，研讨会和技术研讨会，实践经验将来自一个试点项目，涉及ATP结合口袋诱变和质谱鉴定底物的PINK 1。简单总结：这个职业提升奖将提供一个既定的医生，科学家研究神经退行性疾病与必要的交叉培训，在高度专业化的目标识别技术。巩固跨学科合作将使主要研究者能够更快地将决定神经元是否能够成功适应疾病促进压力的因素的知识转化为设计神经保护性PD疗法的新目标。

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.