TARGETS OF A MECHANISM-BASED PROBE AGAINST CYSTEINE PROTEASES

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 使用 LC-MS/MS，我们确定了一种基于机制的探针的靶标，该探针针对细胞凋亡过程中上调的半胱氨酸蛋白酶，即组织蛋白酶 B，这表明其在细胞死亡中的活性。 细胞通过一种称为细胞凋亡的程序控制自身死亡，这对于所有后生动物的发育和体内平衡都是不可或缺的。通常认为溶酶体半胱氨酸蛋白酶不参与细胞凋亡；然而，最近的报告表明，组织蛋白酶可以在细胞凋亡过程中从溶酶体中释放出来，参与细胞死亡。我们在这里报告了一种基于活性的探针的开发，在优化条件下，通过读出溶酶体中组织蛋白酶 B 的释放，仅报告凋亡细胞中的组织蛋白酶 B 活性。组织蛋白酶 B 缺失小鼠细胞凋亡的生化特征显示半胱天冬酶的激活延迟且未达到最佳状态。我们的数据进一步支持了组织蛋白酶 B 在细胞质中作为细胞死亡前馈循环的正调节因子的作用，并为未来的研究提供了化学工具。 该工作已发表在《Chem》上。生物。

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.