IDENTIFICATION OF PROTEOLYSIS-DEPENDENT EXECUTIONER CASPASE PROTEIN COMPLEXES

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。 细胞凋亡是一种保守的细胞途径，其导致半胱氨酸-天冬酰蛋白酶或半胱天冬酶的活化。为了剖析执行者半胱天冬酶-3、-6和-7在协调细胞凋亡中的非冗余作用，我们开发了一种正交蛋白酶来选择性地激活人类细胞中的每种亚型。我们的方法使用小分子控制下的分裂烟草蚀刻病毒（TEV）蛋白酶，我们称之为SNIPer，其中半胱天冬酶等位基因包含遗传编码的TEV切割位点。广泛的生物化学和结构分析表明，炎症和引发剂半胱天冬酶被招募到更大的复合物，诱导半胱天冬酶二聚化，自动蛋白水解和蛋白酶活化。相比之下，很少有人知道的“刽子手caspase相互作用组”，我们建议结合联合收割机SNIPer调节的处理caspase-3，-7和-6的亲和纯化和MS/MS分析，以确定新的蛋白质（S），相互作用的刽子手procaspases在不同的激活状态。 我们已经在HEK 293细胞中产生了稳定的细胞系，其共表达SNIPer和具有COOH末端3x-FLAG表位标签的7个TEV编码的半胱天冬酶等位基因（和对照）中的每一个。我们的策略使新的蛋白质，无论是与酶原或切割的变体的共纯化。因此，被鉴定的蛋白质可以潜在地作为前体或活性形式的功能调节剂进行分组。SNIPer加工的动力学是快速的、同步的和位点选择性的，并且我们希望通过特定的切割事件来绘制相互作用的图谱，即，前结构域的切割，并且这些数据可以暗示（在低分辨率下）结合界面的潜在位置。

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. Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the non-redundant roles of the executioner caspases-3, -6 and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-Tobacco Etch Virus (TEV) protease under small-molecule control, that we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. Extensive biochemical and structural analyses indicate that the inflammatory and initiator caspases are recruited to larger complexes that induce caspase dimerization, auto-proteolysis, and protease activation. In contrast, little is known about the 'executioner caspase interactome,' and we propose to combine SNIPer-regulated processing of caspase-3, -7 and -6 with affinity purification and MS/MS analysis to identify novel protein(s) that interact with each of the executioner procaspases at various states of activation. We have generated stable cell lines in HEK293 cells that co-express the SNIPer and each of 7 TEV-encoded caspase alleles (and controls) with a COOH-terminal 3x-FLAG epitope tag. Our strategy enables the co-purification of novel proteins that associate with either the proenzyme or cleaved variants. Proteins that are identified, therefore, can be potentially binned as functional modulators of the pro- or active forms. The kinetics of SNIPer processing is rapid, synchronous and site-selective, and we hope to map interactions are either gained or lost by a specific cleavage event, i.e., cleavage of the prodomain, and these data may imply (at low resolution) the potential location of the binding interface.