Characterization of a neuronal ubiquitination machinery

描述(申请人提供)：蛋白质泛素化已成为控制神经元回路发育和功能的关键机制，其调节缺陷与多种神经退行性疾病的发病机制有关，包括帕金森氏病、阿尔茨海默病、亨廷顿病和肌萎缩侧索硬化症。然而，目前对神经元中控制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，底物蛋白通过与泛素(一种由76个氨基酸组成的多肽)的共价连接而在蛋白酶体中被标记为降解。泛素化过程涉及一个高度特异的酶级联过程，在这个过程中，泛素首先被E1泛素激活酶激活，然后转移到E2泛素结合酶，最后通过E3泛素蛋白连接酶连接到底物上。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。最近的发现突显了E3连接酶在神经退行性疾病中的重要性，即E3连接酶Parkin的突变是导致一种家族性帕金森病的原因。在寻找调节神经递质释放机制组件SNAP-25的神经元蛋白质时，申请人发现了一种新的蛋白质，称为Spring。Spring是环-B-盒-螺旋线圈(RBCC)蛋白家族中神经元特异性的成员。几种RBCC蛋白的突变被确认为许多人类疾病的原因，包括Opitz综合征、多布雷综合征和家族性地中海热，突显了RBCC家族的重要性。在这个项目中，申请者将使用生化、蛋白质组学、分子生物学和细胞生物学的组合方法来验证Spring作为一种新的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，该项目还将描述Spring的神经元分布和突触定位，并探索这种新蛋白在阿尔茨海默病和帕金森病中的可能参与。建议研究的成功完成将为控制神经元蛋白泛素化和神经递质释放的分子机制提供新的见解，并为我们理解和治疗无数神经疾病和精神障碍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.