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的特征， 在发育、体内平衡和疾病中的关键作用。这个经过深入研究的过程 其特征在于并需要激活已知的蛋白水解酶家族 如半胱氨酸蛋白酶（半胱氨酸依赖性谷氨酸定向蛋白酶）。半胱天冬酶切割a 大量的特定靶蛋白，精心安排了完整的 细胞分裂成碎片，这些碎片可以被吞噬细胞从体内清除。最近 有证据表明半胱天冬酶在大脑中的非凋亡作用，包括调节 突触可塑性。最近发现半胱天冬酶激活先于缠结 在阿尔茨海默病的小鼠模型中形成。然而，令人惊讶的是， 已知的神经元突触蛋白的全球范围内的目标， 半胱天冬酶，切割的特定位点，以及半胱天冬酶- 介导的裂解改变神经元功能。该提案将N-末端标记 从复合蛋白质内容物中阳性选择切割蛋白质的方法 突触体或脑裂解物，允许系统识别半胱天冬酶 突触体制备物以及离散脑区域中的靶点， 神经元凋亡诱导模型。该分析不仅将确定 突触中半胱天冬酶的蛋白质底物，但也将揭示 蛋白水解裂解，提供直接的分子洞察的功能， 卵裂事件。从这个项目中获得的信息不仅会刺激 进一步研究caspase切割突触靶点的功能作用， 还提供了人类神经障碍的直接候选生物标志物， 包括创伤、中风和神经变性疾病。

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.