A designed redox-controlled caspase.
A designed redox-controlled caspase.
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设计的氧化还原控制的半胱天冬酶。
DOI：
10.1002/pro.673
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Hardy,JeanneA
中科院分区：
文献类型：
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作者：
Witkowski,WitoldA;Hardy,JeanneA
半胱天冬酶是一类功能强大的半胱氨酸蛋白酶。在健康或癌细胞中引入活化的半胱天冬酶会导致诱导细胞凋亡。在这项研究中，我们设计并表征了一种 caspase-7，它可以在氧化细胞外条件下失活，然后在还原细胞内条件下重新激活。当两个底物结合环通过工程化的二硫键锁定在一起时，该版本的 caspase-7 会变构失活。当二硫键被还原时，蛋白质恢复其全部功能。通过免疫印迹和质谱可以很容易地观察到 caspase-7 的非活性锁环版本。晶体学上观察到的酶的还原和重新激活形式是第一个 caspase-7 结构，其中底物结合凹槽即使在没有活性位点配体的情况下也是正确排序的。在重新激活的结构中，催化二元半胱氨酸-组氨酸以能够支持催化的方向相距 3.5 Å。这种氧化还原控制的 caspase-7 版本特别适合与氧化还原触发的运载体配合进行靶向细胞死亡。
Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase‐7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase‐7 is allosterically inactivated when two of the substrate‐binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop‐locked version of caspase‐7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase‐7 structure in which the substrate‐binding groove is properly ordered even in the absence of an active‐site ligand. In the reactivated structure, the catalytic‐dyad cysteine–histidine are positioned 3.5 Å apart in an orientation that is capable of supporting catalysis. This redox‐controlled version of caspase‐7 is particularly well suited for targeted cell death in concert with redox‐triggered delivery vehicles.