TARGETS OF A MECHANISM-BASED PROBE AGAINST CYSTEINE PROTEASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 利用LC-MS/MS，我们已经确定了针对在细胞凋亡过程中上调的半胱氨酸蛋白酶的一个基于机制的探针的靶标为组织蛋白酶B，这表明它在细胞死亡中具有活性。 细胞通过一种称为细胞凋亡的程序控制自己的死亡，这对所有后生动物的发育和动态平衡都是不可或缺的。溶酶体半胱氨酸蛋白酶通常不被认为参与细胞凋亡；然而，最近的报道表明，组织蛋白酶在细胞凋亡过程中可以从溶酶体中释放出来，在那里它们可以参与细胞死亡。我们在这里报道了一种基于活性的探针的开发，在优化的条件下，它只通过从溶酶体中读出组织蛋白酶B的释放来报告组织蛋白酶B在凋亡细胞中的活性。组织蛋白酶B缺失小鼠细胞凋亡的生化特征表明，caspase的激活延迟且不理想。我们的数据进一步支持组织蛋白酶B在胞浆中作为细胞死亡前馈循环的正向调节器的作用，并为未来的研究提供了一个化学工具。这项工作已在《化学》杂志上发表。比奥尔。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 in Chem. Biol.