IDENTIFICATION OF PROTEOLYSIS-DEPENDENT EXECUTIONER CASPASE PROTEIN COMPLEXES

蛋白水解依赖性执行器 Caspase 蛋白复合物的鉴定

• 批准号: 8363836
• 负责人: JAMES A WELLS
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363836
• 关键词: Affinity Chromatography; Alleles; Apoptosis; Aspartic Endopeptidases; Binding; Biochemical; Caspase; Cells; Cleaved cell; Complex; Cysteine; Data; Dimerization; Enzyme Precursors; Epitopes; Event; Funding; Grant; Human; Inflammatory; Kinetics; Location; Maps; Mass Spectrum Analysis; National Center for Research Resources; Pathway interactions; Peptide Hydrolases; Principal Investigator; Process; Protein Isoforms; Proteins; Proteolysis; Recruitment Activity; Research; Research Infrastructure; Resolution; Resources; Role; Site; Source; TEV protease; Tobacco; United States National Institutes of Health; Variant; Virus; caspase-3; cost; novel; protein complex; small molecule; stable cell line

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.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 细胞凋亡是一种保守的细胞途径，导致半胱氨酸-天冬氨酰蛋白酶或半胱天冬酶的激活。为了剖析刽子手 caspase-3、-6 和 -7 在协调细胞凋亡中的非冗余作用，我们开发了一种正交蛋白酶来选择性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 caspase 等位基因包含基因编码的 TEV 切割位点。广泛的生化和结构分析表明，炎症和起始半胱天冬酶被募集到更大的复合物中，诱导半胱天冬酶二聚化、自动蛋白水解和蛋白酶激活。相比之下，人们对“刽子手 caspase 相互作用组”知之甚少，我们建议将 SNIPer 调节的 caspase-3、-7 和 -6 处理与亲和纯化和 MS/MS 分析相结合，以鉴定在不同激活状态下与每个刽子手 caspase 前体相互作用的新蛋白质。 我们在 HEK293 细胞中生成了稳定的细胞系，该细胞系与 COOH 末端 3x-FLAG 表位标签共表达 SNIPer 和 7 个 TEV 编码的 caspase 等位基因（和对照）。我们的策略能够共纯化与酶原或切割变体相关的新型蛋白质。因此，鉴定出的蛋白质可以潜在地作为前体或活性形式的功能调节剂。 SNIPer 处理的动力学是快速、同步和位点选择性的，我们希望映射特定切割事件（即前结构域的切割）获得或失去的相互作用，并且这些数据可能暗示（在低分辨率下）结合界面的潜在位置。