TARGETS OF A MECHANISM-BASED PROBE AGAINST CYSTEINE PROTEASES

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 使用LC-MS/MS，我们已经鉴定了针对在细胞凋亡过程中上调的半胱氨酸蛋白酶的基于机制的探针的靶标为组织蛋白酶B，这暗示了其在细胞死亡中的活性。 细胞通过一个称为细胞凋亡的程序来控制自己的死亡，这是所有后生动物发育和体内平衡所不可或缺的。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，组织蛋白酶蛋白酶可以在细胞凋亡期间从溶酶体释放，在那里它们可以参与细胞死亡。我们在这里报告的开发活动为基础的探针，在优化的条件下，报告组织蛋白酶B活性仅在凋亡细胞通过阅读释放的组织蛋白酶B从溶酶体。组织蛋白酶B缺失小鼠细胞凋亡的生物化学表征显示半胱天冬酶的延迟和次优活化。我们的数据进一步支持了组织蛋白酶B在胞质溶胶中作为细胞死亡前馈回路的正调节因子的作用，并为未来的研究提供了化学工具。 这项工作已发表在Chem. Biol.

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.