TARGETS OF A MECHANISM-BASED PROBE AGAINST CYSTEINE PROTEASES

基于机制的半胱氨酸蛋白酶探针的靶标

• 批准号: 8361536
• 负责人: Tom Muir
• 金额: $ 0.13万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361536
• 关键词: Apoptosis; Apoptotic; Biochemical; Caspase; Cathepsins; Cathepsins B; Cell Death; Cells; Cessation of life; Chemicals; Cysteine Protease; Cytosol; Data; Development; Funding; Future; Grant; Homeostasis; Investigation; Knockout Mice; Lysosomes; Measurement; National Center for Research Resources; Peptide Hydrolases; Principal Investigator; Publishing; Reading; Reporting; Research; Research Infrastructure; Resources; Role; Source; United States National Institutes of Health; Work; base; cost; feeding; macromolecule; programs; tool

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. Using LC-MS/MS, we have identified the target of a mechanism-based probe against cysteine proteases upregulated during apopotosis as Cathepsin B, implicating its activity in cell death. Cells control their own death through a program termed apoptosis, which is indispensable for development and homeostasis in all metazoans. Lysosomal cysteine proteases are not normally thought of as participating in apoptosis; however, recent reports have shown that the cathepsin proteases can be released from the lysosome during apoptosis, where they can participate in cell death. We report here the development of an activity-based probe that, under optimized conditions, reports on cathepsin B activity only in apoptotic cells by reading out the release of cathepsin B from the lysosomes. Biochemical characterization of apoptosis in cells from cathepsin B null mice shows delayed and suboptimal activation of caspases. Our data further supports a role for cathepsin B in the cytosol as a positive regulator of a cell death feed-forward loop and provides a chemical tool for future investigations. This work has been published: Pratt MR, Sekedat MD, Chiang KP, Muir TW Direct measurement of cathepsin B activity in the cytosol of apoptotic cells by an activity-based probe. Chem Biol. 2009 Sep 25;16(9):1001-12

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。 使用LC-MS/MS，我们已经确定了基于机制的探针的靶标，该探针针对凋亡过程中上调的半胱氨酸蛋白酶作为组织蛋白酶B，这暗示了其在细胞死亡中的活性。 细胞通过称为凋亡的程序来控制自己的死亡，这对于所有后生动物中的发展和稳态都是必不可少的。通常认为溶酶体半胱氨酸蛋白酶参与凋亡。但是，最近的报道表明，在凋亡过程中可以从溶酶体中释放组织蛋白酶蛋白酶，在那里它们可以参与细胞死亡。我们在这里报告了基于活性的探针的开发，该探针在优化的条件下，仅通过读取从溶酶体中释放组织蛋白酶B的释放来报道组织蛋白酶B活性。来自组织蛋白酶b的细胞中细胞凋亡的生化表征显示caspase的延迟和次优地激活。我们的数据进一步支持了在细胞质中，组织蛋白酶B作为细胞死亡进料前环的积极调节剂的作用，并为将来的研究提供了化学工具。 这项工作已发表： Pratt MR，Sekedat MD，Chiang KP，Muir TW 通过基于活性的探针，直接测量凋亡细胞的细胞质中的组织蛋白酶B活性。化学生物。 2009年9月25日； 16（9）：1001-12