Takahashi A, Musy P Y, Martins L M, Poirier G G, Moyer R W, Earnshaw W C
Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, United Kingdom.
J Biol Chem. 1996 Dec 20;271(51):32487-90. doi: 10.1074/jbc.271.51.32487.
CrmA, a poxvirus gene product with a serpin-like structure, blocks a variety of apoptotic death events in cultured cells. Based on the ability of CrmA to inhibit the interleukin-1beta converting enzyme in vitro, it has been speculated that interleukin-1beta converting enzyme-related proteases (caspases) essential for apoptosis are the cellular targets of CrmA. Here we found that rabbitpox virus CrmA/SPI-2 inhibits the cleavage of lamin A mediated by a caspase in our cell-free system of apoptosis. In the presence of CrmA/SPI-2, nuclear apoptosis in vitro was blocked at an intermediate stage after collapse of the chromatin against the nuclear periphery and before nuclear shrinkage and disintegration into apoptotic body-like fragments. Using N-(acetyltyrosinylvalinyl-Nepsilon-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone, which derivatizes the active forms of caspases, we could show that one of five caspases active in the extracts is inhibited both by CrmA/SPI-2 and by a peptide spanning the lamin A apoptotic cleavage site. These results reveal that CrmA/SPI-2 can inhibit a caspase responsible both for lamin A cleavage and for the nuclear disintegration characteristic of apoptosis.
CrmA是一种具有丝氨酸蛋白酶抑制剂样结构的痘病毒基因产物，可阻断培养细胞中的多种凋亡死亡事件。基于CrmA在体外抑制白细胞介素-1β转换酶的能力，推测凋亡所必需的白细胞介素-1β转换酶相关蛋白酶（半胱天冬酶）是CrmA的细胞靶点。在此，我们发现兔痘病毒CrmA/SPI-2在我们的无细胞凋亡系统中可抑制半胱天冬酶介导的核纤层蛋白A的切割。在存在CrmA/SPI-2的情况下，体外核凋亡在染色质靠向核周边塌陷后、核收缩并解体成凋亡小体样片段之前的中间阶段被阻断。使用N-（乙酰基酪氨酰缬氨酰-Nε-生物素基赖氨酰）天冬氨酸[（2,6-二甲基苯甲酰）氧基]甲基酮对半胱天冬酶的活性形式进行衍生化，我们可以证明提取物中五种有活性的半胱天冬酶之一既被CrmA/SPI-2抑制，也被跨越核纤层蛋白A凋亡切割位点的肽抑制。这些结果表明，CrmA/SPI-2可抑制一种对半胱天冬酶介导的核纤层蛋白A切割以及凋亡特征性的核解体均起作用的半胱天冬酶。
