SENSITIVE GLOBAL PROFILING OF PROTEOLYSIS IN APOPTOSIS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 人类基因组中编码的大约600种蛋白酶参与了多种生物过程。一些作为与蛋白质催化剂相关的非特异性降解酶，在许多位点彻底切割许多蛋白质底物。相比之下，其他几个功能作为选择性的翻译后修饰剂，只切割少数蛋白质底物，通常只在一个或几个位点。细胞凋亡是受有限的细胞内蛋白水解调节的过程的一个重要例子。这种遗传程序化和非炎症形式的细胞死亡是体内平衡和组织更新的核心组成部分。由于化学治疗剂通常通过诱导细胞凋亡来杀死细胞，因此从治疗观点来看，该过程也是高度相关的。不幸的是，蛋白酶信号在复杂的生物样品，如凋亡细胞的全面表征往往是有限的可用的蛋白质组学方法。我们已经建立了一种新的方法，全球测序的蛋白水解切割位点在复杂的生化混合物，是基于使用的工程肽连接酶的选择性生物素化的游离蛋白N-末端和积极的富集相应的N-末端肽。在与UCSF质谱设施的合作，我们正在应用这种方法来研究细胞凋亡中的蛋白水解，使用各种癌细胞系和细胞毒性诱导剂作为模型系统。我们的目标是在全球范围内表征细胞凋亡中的蛋白水解如何随时间、细胞凋亡诱导剂和细胞类型而变化。UCSF质谱设施在提供质谱仪器、软件和这项工作成功所必需的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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.