Institute of Biochemistry and Biophysics, University of Tehran, Enghelab Ave, P.O. Box 13145-1384, Tehran, Iran.
Int J Biochem Cell Biol. 2010 Jun;42(6):958-64. doi: 10.1016/j.biocel.2010.02.008. Epub 2010 Feb 18.
Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.
研究了全长 NAIP 及其 BIR3 结构域抑制内在凋亡途径蛋白酶的能力。重组 NAIP-BIR3（NBIR3）和全长蛋白均可显著降低内源性效应子半胱天冬酶的活性。Western blot 实验表明，全长 NAIP 和其 BIR3 结构域抑制凋亡小体激活的 caspase-9 对半胱天冬酶-3 的切割。此外，全长 NAIP 抑制凋亡小体复合物中半胱天冬酶-9 的自身切割，表明与其他凋亡抑制蛋白（IAPs）不同，人 NAIP 是半胱天冬酶-9 的抑制剂。此外，BIR3 结构域对单链 caspase-9（人 caspase-9，D315、D330/A 点突变，消除了 caspase-9 的蛋白水解加工，但不消除其催化活性）的抑制表明该结构域是与 caspase-9 相互作用的部分。一致地，NBIR3 而不是 X 连锁凋亡抑制蛋白（XIAP）-BIR3 结构域在凋亡小体复合物中单链 capsase-9 的下拉实验证实，该蛋白可以在凋亡小体形成之前与前体半胱天冬酶-9 结合，然后进行自身切割。相互作用研究表明，NBIR3 的 C338W 变体而非野生型蛋白与 SMAC-肽和 SMAC 蛋白均有相互作用。这些数据表明，将 C338 突变为 Trp 足以容纳 NAIP-BIR3 与 SMAC-肽和蛋白的相互作用。总之，这些结果表明，NAIP 的进化是为了在凋亡小体形成的起始阶段防止凋亡，而这种抑制不能被 SMAC 型蛋白拮抗。
