SENSITIVE GLOBAL PROFILING OF PROTEOLYSIS IN APOPTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 人类基因组中编码的大约600种蛋白水解酶参与了多种生物过程。有些作为与蛋白质分解代谢相关的非特异性降解酶，在许多位置彻底裂解许多蛋白质底物。相比之下，其他几个则作为选择性翻译后修饰因子发挥作用，通常只在一个或几个位点切割少数蛋白质底物。细胞凋亡是一个受细胞内有限蛋白分解调节的过程的重要例子。这种遗传程序性和非炎症形式的细胞死亡是动态平衡和组织更新的核心组成部分。由于化疗药物通常通过诱导细胞凋亡来杀死细胞，从治疗的角度来看，这一过程也是高度相关的。不幸的是，在复杂的生物样本中，如凋亡细胞中的蛋白水解酶信号的综合特征常常受到现有蛋白质组学方法的限制。我们建立了一种新的对复杂生化混合物中蛋白水解性裂解位点进行全局测序的新方法，该方法基于工程多肽连接酶对游离蛋白N末端的选择性生物素化和相应N末端多肽的正浓缩。与加州大学旧金山分校的质谱学设备合作，我们正在使用多种癌细胞株和细胞毒诱导剂作为模型系统，将这种方法应用于研究细胞凋亡中的蛋白分解。我们的目标是在全球范围内表征细胞凋亡中的蛋白分解如何随时间、凋亡诱导剂和细胞类型的变化而变化。加州大学旧金山分校的质谱学设施在提供这项工作成功所必需的质谱仪、软件和专业知识方面发挥了关键作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 600 or so proteases encoded in the human genome are involved in a diversity of biological processes. Some function as nonspecific degradative enzymes associated with protein catabolism, exhaustively cleaving many protein substrates at many sites. In contrast, several others function as selective post-translational modifiers, cleaving only a few protein substrates, usually at only one or a few sites. Apoptosis is an important example of a process regulated by limited intracellular proteolysis. This genetically programmed and non-inflammatory form of cell death is a central component of homeostasis and tissue turnover. Since chemotherapeutics typically kill cells by induction of apoptosis, this process is also highly relevant from a therapeutic standpoint. Unfortunately, comprehensive characterization of protease signaling in complex biological samples such as apoptotic cells is often limited by available proteomic methods. We have established a novel method for global sequencing of proteolytic cleavage sites in complex biochemical mixtures that is based on use of an engineered peptide ligase for selective biotinylation of free protein N-termini and positive enrichment of corresponding N-terminal peptides. In collaboration with the UCSF Mass Spectrometry Facility, we are applying this method to the study of proteolysis in apoptosis, using a variety of cancer cell lines and cytotoxic inducers as model systems. Our goal is to globally characterize how proteolysis in apoptosis varies as a function of time, apoptotic inducer, and cell type. The UCSF Mass Spectrometry Facility has been pivotal in providing the mass spectrometry instrumentation, software, and expertise essential for the success of this work.