Martins L Miguel, Turk Benjamin E, Cowling Victoria, Borg Annabel, Jarrell Emily T, Cantley Lewis C, Downward Julian
Signal Transduction and Protein Isolation and Cloning Laboratories, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
J Biol Chem. 2003 Dec 5;278(49):49417-27. doi: 10.1074/jbc.M308659200. Epub 2003 Sep 25.
Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. We have used peptide libraries to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, we show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins.
凋亡抑制蛋白（IAPs）通过直接抑制半胱天冬酶来阻止细胞凋亡。丝氨酸蛋白酶HtrA2/Omi具有一个氨基末端IAP相互作用基序，类似于在促凋亡蛋白Reaper中发现的基序，该基序可将IAPs从半胱天冬酶上置换下来，从而增强半胱天冬酶的活性。HtrA2/Omi促进细胞死亡的特性不仅通过其对抗IAPs对半胱天冬酶抑制的能力来发挥，还通过其完整的丝氨酸蛋白酶活性来实现。我们利用肽库确定了HtrA2切割的最佳底物序列以及其PDZ结构域的优先结合序列。使用这些肽，我们发现HtrA2/Omi的PDZ结构域会抑制其蛋白水解活性，除非它与结合伴侣结合。对HtrA2/Omi进行热休克处理也会增加其蛋白酶活性。出乎意料的是，X连锁凋亡抑制蛋白（XIAP）与HtrA2/Omi的Reaper基序结合会导致蛋白水解活性显著增加，这表明IAPs有了新的作用。当HtrA2/Omi在凋亡刺激后从线粒体释放时，与IAPs的结合可能会使其功能从抑制半胱天冬酶转变为激活丝氨酸蛋白酶。因此，尽管IAPs过表达可以抑制半胱天冬酶激活，但它可能对依赖HtrA2/Omi的细胞死亡产生相反的影响。这一点，连同HtrA2/Omi降解IAPs的能力，可能会限制这些蛋白所能提供的整体细胞保护作用。
