Berger Alicia B, Witte Martin D, Denault Jean-Bernard, Sadaghiani Amir Masoud, Sexton Kelly M B, Salvesen Guy S, Bogyo Matthew
Department of Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305, USA.
Mol Cell. 2006 Aug;23(4):509-21. doi: 10.1016/j.molcel.2006.06.021.
Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.
半胱天冬酶是半胱氨酸蛋白酶，是凋亡性细胞死亡中的关键效应分子。目前，缺乏可用于在不同凋亡程序背景下监测特定半胱天冬酶调控的工具。我们描述了高选择性抑制剂和活性位点探针的开发及其在直接监测执行性（半胱天冬酶 -3 和 -7）和起始性（半胱天冬酶 -8 和 -9）半胱天冬酶活性方面的应用。具体而言，这些试剂用于剖析无细胞提取物和完整细胞中凋亡刺激后半胱天冬酶激活的动力学。这些研究鉴定出一种全长半胱天冬酶 -7 中间体，它在该途径早期被催化激活，其进一步加工由成熟的执行性半胱天冬酶而非起始性半胱天冬酶介导。与加工后的半胱天冬酶 -7 相比，这种形式还表现出明显不同的抑制剂敏感性。我们的数据表明，半胱天冬酶 -7 的激活通过一种以前未被表征的中间体进行，该中间体在完整酶原未裂解的情况下形成。
