Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes
Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes
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DOI：
10.1016/j.molcel.2006.06.021
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发表时间：
2006-08-18
期刊：
影响因子：
16
通讯作者：
Bogyo, Matthew
中科院分区：
文献类型：
--
作者：
Berger, Alicia B.;Witte, Martin D.;Bogyo, Matthew
半胱氨酸蛋白酶是一种半胱氨酸蛋白酶，是细胞凋亡的关键效应因子。目前，在不同的细胞凋亡过程中，缺乏可用于监测特定caspase调控的工具。我们描述了高选择性抑制物和活性部位探针的发展及其在直接监测执行者(caspase-3和-7)和启动子(caspase-8和-9)caspase活性方面的应用。具体地说，这些试剂被用来分析caspase在刺激无细胞提取物和完整细胞凋亡时的激活动力学。这些研究发现了一个全长的caspase-7中间体，它在该途径的早期被催化激活，其进一步的处理是由成熟的执行者caspase而不是启动者caspase介导的。与处理过的caspase-7相比，这种形式也显示出明显的抑制物敏感性。我们的数据表明，caspase-7的激活通过一种以前未确定的中间体进行，该中间体是在没有切割完整的酶原的情况下形成的。
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.