Characterization of a neuronal ubiquitination machinery

描述（由申请人提供）：蛋白质泛素化已成为控制神经元回路的发育和功能的关键机制，并且其缺陷性调节已涉及多种神经变性疾病的发病机制，包括帕金森病、阿尔茨海默病、亨廷顿病和肌萎缩侧索硬化症。然而，目前对控制神经元中蛋白质泛素化的分子机制知之甚少。在泛素-蛋白酶体途径中，底物蛋白通过共价连接至泛素（一种76个氨基酸的多肽）而被标记为在蛋白酶体中降解。 泛素化过程涉及一个高度特异性的酶级联反应，其中泛素首先被E1泛素激活酶激活，然后转移到E2泛素缀合酶，最后通过E3泛素蛋白连接酶连接到底物。在这些酶中，E3连接酶是最重要的参与者，因为它决定了泛素介导的蛋白质降解的特异性。E3连接酶在神经退行性疾病中的重要性被最近的发现所强调，即E3连接酶parkin中的突变是帕金森氏病家族形式的原因。在寻找调节神经递质释放机制组分SNAP-25的神经元蛋白质时，申请人发现了一种称为Spring的新蛋白质。Spring是RING-B-box-coiled-coil（RBCC）蛋白家族的神经元特异性成员。 RBCC家族的重要性通过鉴定几种RBCC蛋白中的突变作为许多人类疾病的原因而得到强调，包括Opitz综合征，Mulibrey nanism和家族性地中海热。在该项目中，申请人将使用生物化学，蛋白质组学，分子生物学和细胞生物学方法的组合来测试Spring作为一种新型E3泛素蛋白连接酶来调节神经递质释放机制的周转的假设。此外，该项目还将表征Spring的神经元分布和突触定位，并探索这种新型蛋白质在阿尔茨海默病和帕金森病中的可能参与。成功完成拟议的研究将产生新的见解，控制神经元蛋白泛素化和神经递质释放的分子机制，并为我们理解和治疗许多神经系统疾病和精神疾病的最终目标提供基本信息。

DESCRIPTION (provided by applicant): Protein ubiquitination has emerged as a crucial mechanism for controlling development and function of neuronal circuits, and its defective regulation has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. However, very little is presently known about the molecular machinery that controls protein ubiquitination in neurons. In the ubiquitin-proteasome pathway, substrate proteins are marked for degradation in the proteasome by covalent linkage to ubiquitin, a 76-amino acid polypeptide. The ubiquitination process involves a highly specific enzyme cascade in which ubiquitin is first activated by an E1 ubiquitin-activating enzyme, then transferred to an E2 ubiquitin-conjugating enzyme, and finally ligated to the substrate by an E3 ubiquitin-protein ligase. Of these enzymes, E3 ligase is the most important player because it determines the specificity of ubiquitin-mediated protein degradation. The importance of E3 ligases in neurodegenerative disorders is highlighted by recent findings that mutations in the E3 ligase parkin are responsible for a familial form of Parkinson's disease. In a search for neuronal proteins that regulate the neurotransmitter release machinery component SNAP-25, the applicant has discovered a novel protein, called Spring. Spring is a neuron-specific member of the RING-B-box-coiled-coil (RBCC) protein family. The importance of the RBCC family is underscored by the identification of the mutations in several RBCC proteins as the causes for a number of human diseases, including Opitz syndrome, Mulibrey nanism, and familial Mediterranean fever. In this project, the applicant will use a combination of biochemical, proteomic, molecular biological, and cell biological approaches to test the hypothesis that Spring functions as a novel E3 ubiquitin-protein ligase to regulate the turnover of the neurotransmitter release machinery. In addition, this project will characterize neuronal distribution and synaptic localization of Spring, and explore the possible involvement of this novel protein in Alzheimer's disease and Parkinson's disease. Successful completion of proposed studies will yield novel insights into the molecular mechanisms that control neuronal protein ubiquitination and neurotransmitter release, and provide fundamental information towards our ultimate goal of understanding and treating numerous neurological diseases and psychiatric disorders.