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.