Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer
Cytokine response modifier a inhibition of initiator caspases results in covalent complex formation and dissociation of the caspase tetramer
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DOI：
10.1074/jbc.m605151200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Gettins, Peter G. W.
中科院分区：
文献类型：
--
作者：
Dobo, Jozsef;Swanson, Richard;Gettins, Peter G. W.
活性半胱天冬酶通常由两个催化结构域组成，每个结构域包含一个大亚基（p20）和一个小亚基（p10），因此完全活性的半胱天冬酶具有组织（p20-p10）（2）。牛痘丝氨酸蛋白酶抑制剂crmA通过抑制内源性半胱天冬酶抑制宿主细胞凋亡和炎症。我们报告的机制crmA用于抑制半胱天冬酶1，6和8。非变性PAGE显示形成紧密的crmA-caspase复合物。基质辅助激光解吸电离飞行时间质谱法提供了共价crmA-p20硫酯键的证据。分离的复合物的SDS-PAGE显示p10亚基从起始物半胱天冬酶1和8中几乎完全丧失，但从执行物半胱天冬酶-6中没有。这通过测序和蛋白质印迹法对胱天蛋白酶-1进行了证实。尺寸排阻色谱法表明与半胱天冬酶1和8的复合物的尺寸类似于60 kDa，与crmA中心点p20种类一致，表明p20-p10界面和可能的p10-p10界面已经被破坏。相反，crmA中心点caspase-6复合物表现为crmA（2）中心点（p20-p10）（2）和crmA中心点（p20-p10）的平衡混合物。复合物脱酰速率都很慢，表明共价硫代酰基中间体的有效动力学捕获。这些结果表明crmA通过其下调细胞凋亡和炎症的新丝氨酸蛋白酶抑制剂抑制机制。这涉及（i）与起始剂半胱天冬酶8或1快速形成共价复合物，（ii）非常缓慢的脱酰作用，和（iii）起始剂半胱天冬酶p10亚基的损失，但不包括执行剂半胱天冬酶的损失，使得通过起始剂半胱天冬酶的脱酰作用形成的任何游离p20不能重新结合成活性异源四聚体，从而导致细胞凋亡和炎症的不可逆抑制。
Active caspases are generally composed of two catalytic domains, each containing a large (p20) and a small (p10) subunit so that a fully active caspase has the organization (p20-p10)(2). The cowpox serpin crmA suppresses host apoptosis and inflammation by inhibiting endogenous caspases. We report on the mechanism crmA uses to inhibit caspases 1, 6, and 8. Native PAGE showed formation of tight crmA-caspase complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry provided evidence for a covalent crmA-p20 thioester linkage. SDS-PAGE of isolated complexes showed near complete loss of the p10 subunit from initiator caspases 1 and 8 but not from the executioner caspase-6. This was confirmed for caspase-1 by sequencing and Western blotting. Size exclusion chromatography indicated a size of similar to 60 kDa for complexes with caspases 1 and 8, consistent with a crmA center dot p20 species, suggesting that the p20-p10 interface and possibly the p10-p10 interface had been disrupted. In contrast, crmA center dot caspase-6 complex behaved as an equilibrium mixture of crmA(2)center dot(p20-p10)(2) and crmA center dot(p20-p10). Complex deacylation rates were all slow, suggesting effective kinetic trapping of the covalent thioacyl intermediate. These results suggest a novel serpin inhibition mechanism through which crmA down-regulates apoptosis and inflammation. This involves (i) rapid formation of covalent complex with initiator caspases 8 or 1, (ii) very slow deacylation, and (iii) loss of the caspase p10 subunit for initiator but not for executioner caspases, so that any free p20 formed by deacylation of initiator caspases cannot reassociate to active heterotetramer, thus resulting in irreversible inhibition of apoptosis and inflammation.