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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 凋亡是一种保守的细胞途径，可导致半胱氨酸 - 天冬氨酸蛋白酶或胱天蛋白酶的激活。为了剖析execution子caspases -3，-6和-7在修复细胞凋亡中的非冗余作用，我们开发了一种正交蛋白酶，以选择性地激活人类细胞中的每种同工型。我们的方法在小分子控制下使用分裂的鸡蛋蚀刻病毒（TEV）蛋白酶，我们称为狙击手，caspase等位基因含有遗传编码的TEV裂解位点。广泛的生化和结构分析表明，炎症和引发蛋白胱天蛋白酶被募集到诱导caspase二聚化，自身蛋白质解析和蛋白酶激活的较大络合物中。相比之下，关于“ executioner caspase Interactome”的知之甚少，我们建议将Caspase -3，-7和-6的狙击手调节的处理与亲和力纯化和MS/MS分析相结合，以识别与各种激活状态处的执行器procaspases相互作用的新型蛋白质。 我们已经在HEK293细胞中生成了稳定的细胞系，该细胞共表达了狙击手，并且使用COOH末端3X-FLAG表位标签中的7个TEV编码的caspase等位基因（和对照）中的每一个。我们的策略使新型蛋白质与蛋白酶或裂解变体的新蛋白质进行了共培养。因此，可以鉴定出的蛋白质可能被视为促成或活性形式的功能调节剂。狙击手处理的动力学是快速，同步和位点选择性的，我们希望通过特定的裂解事件（即ProdoMain的裂解）绘制相互作用或丢失，即Prodomain的裂解，这些数据可能暗示（低分辨率）结合界面的电位位置。