Global Identification of Mammalian Synaptic Caspase Targets

细胞凋亡，或程序性细胞死亡，是所有多细胞生物体的一个特征，并具有 在发育、动态平衡和疾病中的关键作用。这一深入研究的过程 它的特点是需要一系列已知的蛋白水解酶的激活 作为半胱氨酸依赖的天冬氨酸导向的蛋白酶。半胱氨酸天冬氨酸酶 大量特异的靶蛋白，编排完整有序的转化 细胞分裂成碎片，可被吞噬细胞从体内清除。近期 有证据表明，半胱氨酸天冬氨酸酶在大脑中的非凋亡作用，包括调节 突触的可塑性。最近发现半胱氨酸天冬氨酸氨基转移酶的激活先于缠绕 阿尔茨海默病小鼠模型的形成。然而，令人惊讶的是，几乎没有 已知靶向神经元突触蛋白的全球范围 Caspase、caspase的特定切割部位以及caspase-2的作用机制。 介导的卵裂改变了神经元的功能。这项提案将N-终端标签 从复杂蛋白质含量中正向选择切割蛋白的方法 突触体或脑裂解物，允许系统地识别caspase 突触小体制备中的靶点以及离散脑区和 神经细胞凋亡诱导的模型。该分析不仅将确定 突触中caspase的蛋白质底物，但也将揭示 蛋白水解性切割，提供了即时的分子洞察功能 乳沟事件。从这个项目中获得的信息不仅会刺激 进一步研究caspase裂解突触靶标的功能作用，但将 也为人类神经疾病提供了直接的候选生物标记物， 包括创伤、中风和神经退行性疾病。

Apoptosis, or programmed cell death, is a feature of all multicellular organisms and has critical roles in development, homeostasis, and disease. This intensely studied process is characterized by and requires the activation of a family of proteolytic enzymes known as caspases (cysteine-dependent aspartate-directed proteases). Caspases cleave a large number of specific target proteins, orchestrating the orderly transformation of intact cells into fragments that may be removed from the body by phagocytic cells. Recent evidence points to non-apoptotic roles for caspases in the brain, including the regulation of synaptic plasticity. Caspase activation was recently shown to precede tangle formation in a mouse model of Alzheimer's disease. However, surprisingly little is known about the global scope of neuronal synaptic proteins targeted by caspases, the specific sites of cleavage, and mechanisms by which caspase- mediated cleavages alter neuronal function. This proposal will N-terminus labeling approach to positively select cleaved proteins from the complex protein content of synaptosomes or brain lysates, allowing systematic identification caspase targets in synaptosomal preparations as well as in discrete brain regions and models of neuronal apoptotic induction. The analysis will not only identify the protein substrates of caspases in synapses, but will also reveal the precise site of proteolytic cleavage, providing immediate molecular insight into the function of the cleavage event. The information derived from this project will not only stimulate further research on the functional roles of caspase cleavage of synaptic targets, but will also provide immediate candidate biomarkers for human neurological disorders, including trauma, stroke, and neurodegenerative diseases.