Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran.
Int J Biol Macromol. 2017 Aug;101:283-289. doi: 10.1016/j.ijbiomac.2017.03.088. Epub 2017 Mar 18.
Contribution of individual BIR domains to Smac antagonism is investigated. Ammonium citrate was used to activate caspase-9 and pro-caspase-9 (D315, D330/A). However, the presence of citrate resulted in autoproteolysis of pro-caspase-9 and its inhibition by XIAP BIR3, which was not observed for apoptosome activated pro-caspase-9 indicating abnormal behavior of pro-caspase-9 in kosmotropic citrate salt. Thus, we used Apaf-1(residues 1-591) to activate caspase-9 through the formation of mini-apoptosome instead. Inhibition of apoptosome by XIAP BIR-1-2-3 was observed to be similar to that of BIR3 indicating that the cleavage of XIAP does not affect its potency. However, BIR1-2-3 was more prone to Smac antagonism due to simultaneous interaction of two BIR domains from XIAP with two N-terminal binding sites of Smac. Therefore, despite the role in caspase-9 activation, Apaf-1 does not influence caspase-9 inhibition by XIAP. In addition, caspase-3, -7 and -9 activity recovery by Smac protein and peptide were more efficient for BIR1-2-3 than for BIR1-2. Consequently, it can be proposed that the presence of multiple BIR domains for XIAP among different species along with dimeric nature of Smac are evolutionary designed to strengthen the antagonistic activity of Smac culminating in efficient induction of cell death.
研究了个体 BIR 结构域对 Smac 拮抗作用的贡献。使用柠檬酸铵激活 caspase-9 和前体 caspase-9（D315、D330/A）。然而，柠檬酸的存在导致前体 caspase-9 的自切割及其被 XIAP BIR3 抑制，而在凋亡小体激活的前体 caspase-9 中未观察到这种情况，表明前体 caspase-9 在亲水性柠檬酸盐中的异常行为。因此，我们使用 Apaf-1（残基 1-591）通过形成 mini-apoptosome 来激活 caspase-9。观察到 XIAP BIR-1-2-3 对凋亡小体的抑制作用与 BIR3 相似，表明 XIAP 的切割不影响其效力。然而，BIR1-2-3 更容易受到 Smac 的拮抗作用，因为 XIAP 的两个 BIR 结构域与 Smac 的两个 N 端结合位点同时相互作用。因此，尽管 Apaf-1 在 caspase-9 激活中起作用，但它不会影响 XIAP 对 caspase-9 的抑制作用。此外，Smac 蛋白和肽对 caspase-3、-7 和 -9 活性的恢复，对于 BIR1-2-3 比 BIR1-2 更有效。因此，可以提出，XIAP 在不同物种中存在多个 BIR 结构域以及 Smac 的二聚体性质是为了增强 Smac 的拮抗活性而进化设计的，最终导致细胞死亡的有效诱导。
