Roy N, Deveraux Q L, Takahashi R, Salvesen G S, Reed J C
The Burnham Institute, Program on Apoptosis and Cell Death Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
EMBO J. 1997 Dec 1;16(23):6914-25. doi: 10.1093/emboj/16.23.6914.
The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.
凋亡抑制蛋白（IAP）家族的蛋白质在进化过程中高度保守。然而，这些蛋白质干扰凋亡性细胞死亡的机制一直是个谜。最近，我们发现人类IAP家族蛋白之一XIAP能够结合并有效抑制特定的细胞死亡蛋白酶（半胱天冬酶），这些蛋白酶在凋亡相关的蛋白水解级联反应的远端起作用。在本研究中，我们调查了人类IAP家族其他三个已知成员，即c-IAP-1、c-IAP-2和NAIP。与XIAP类似，体外结合实验表明，c-IAP-1和c-IAP-2特异性结合终末效应细胞死亡蛋白酶半胱天冬酶-3和-7，但不结合近端蛋白酶半胱天冬酶-8、半胱天冬酶-1或-6。相反，NAIP未能与这些蛋白酶中的任何一种紧密结合。重组c-IAP-1和c-IAP-2在体外也抑制半胱天冬酶-3和-7的活性，估计的抑制常数Kis≤0.1微摩尔，而NAIP则没有。c-IAP-1和c-IAP-2含BIR结构域的区域足以抑制这些半胱天冬酶，尽管保留RING结构域的蛋白质活性稍强。利用一个无细胞系统，通过添加细胞色素c在胞质提取物中激活半胱天冬酶，c-IAP-1和c-IAP-2既抑制半胱天冬酶活性的产生，也抑制前体半胱天冬酶-3的蛋白水解加工。当通过基因转染过表达c-IAP-1和c-IAP-2，并使用抗癌药物依托泊苷诱导完整细胞凋亡时，也得到了类似的结果。与半胱天冬酶相互作用时未观察到c-IAP-1或c-IAP-2的裂解，这意味着其机制不同于杆状病毒p35蛋白，后者是半胱天冬酶的广谱自杀性失活剂。综上所述，这些发现表明，c-IAP-1和c-IAP-2通过抑制远端细胞死亡蛋白酶半胱天冬酶-3和-7发挥与XIAP类似的功能，而NAIP可能通过其他靶点抑制凋亡。
