Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex
Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex
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DOI：
10.1021/bi980893w
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发表时间：
1998-07-28
期刊：
影响因子：
2.9
通讯作者：
Salvesen, GS
中科院分区：
文献类型：
--
作者：
Zhou, Q;Krebs, JF;Salvesen, GS
杆状病毒的抗凋亡蛋白 p35 被认为可以通过抑制半胱天冬酶来防止受感染昆虫细胞的自杀反应。 p35 在许多转基因动物或细胞系中的异位表达也具有抗细胞凋亡作用，从而产生了该蛋白是半胱天冬酶的一般抑制剂的假设。我们通过证明纯化的重组 p35 抑制人 caspase-1、-3、-6、-7、-8 和 -10 来验证这一假设，k(ass) 值为 1.2 x 10(3) 至 7 x 10(5) (M-1 s(-1))，Ki 值上限为 0.1 至 9 nM。对 12 种不相关的丝氨酸或半胱氨酸蛋白酶的抑制作用微不足道，这意味着 p35 是一种有效的 caspase 特异性抑制剂。假定的抑制环发生有利于 caspase-1 的突变，导致 caspase-3 抑制显着下降，但 caspase-1 抑制变化很小。 p35 与 caspase-3 的相互作用作为抑制机制的模型，揭示了经典的缓慢结合抑制，其中 caspase-3 二聚体的两个活性位点同等且独立地发挥作用。抑制是由酶和抑制剂之间形成复合物引起的，该复合物在非变性条件下可以被可视化，但通过 SDS 解离，得到在 Asp87（抑制剂的 P-1 残基）处裂解的 p35。复合物的形成需要底物结合裂隙未被占据。总而言之，这些数据表明 p35 是一种高度适合抑制 caspase 的活性位点定向抑制剂。
The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with k(ass) values from 1.2 x 10(3) to 7 x 10(5) (M-1 s(-1)), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction of p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P-1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.