IDENTIFICATION OF PROTEOLYSIS-DEPENDENT EXECUTIONER CASPASE PROTEIN COMPLEXES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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，导致半胱氨酸天冬氨酸蛋白酶或半胱氨酸蛋白酶的激活。为了分析执行者caspase-3、-6和-7在协调细胞凋亡中的非冗余作用，我们开发了一种有选择性地激活人类细胞中每一种异构体的正交酶。我们的方法使用了一种受小分子控制的裂解烟草蚀刻病毒(TEV)蛋白酶，我们称之为狙击手，caspase等位基因包含基因编码的TEV裂解位点。广泛的生化和结构分析表明，炎性和启动型caspase被招募到更大的复合体中，诱导caspase二聚化、自身蛋白分解和蛋白酶激活。相反，人们对‘执行者caspase互动组’知之甚少，我们建议将狙击手调控的caspase-3、-7和-6的处理与亲和纯化和MS/MS分析相结合，以确定在不同激活状态下与每个执行者半胱氨酸酶相互作用的新蛋白(S)。 我们已经在HEK293细胞中建立了稳定的细胞系，它们共同表达狙击手和7个TEV编码的caspase等位基因(和对照)，并带有COOH末端3x-FLAG表位标签。我们的策略使得与酶原或裂解变异体相关的新蛋白的共同纯化成为可能。因此，被鉴定的蛋白质可以潜在地被归类为前体或活性形式的功能调节器。狙击手处理的动力学是快速、同步和位点选择性的，我们希望映射相互作用是通过一个特定的切割事件获得或丢失，即原结构域的切割，这些数据可能暗示(在低分辨率)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.