Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 1417653761, Iran.
Biochem Biophys Res Commun. 2011 Apr 1;407(1):158-62. doi: 10.1016/j.bbrc.2011.02.130. Epub 2011 Mar 1.
The importance of the ATP binding site of human Neuronal Apoptosis Inhibitory Protein (NAIP) on its ability in prevention of intrinsic apoptotic pathway was investigated. Thus, ATP binding lysine 476 of NAIP, which is located at the Nucleotide Binding Oligomerization Domain (NOD) was mutated to threonine and the effect of this mutation on autoproteolysis of procaspase-9 and the cleavage of procaspase-3 by apoptosome was investigated. Formation of apoptosome was induced by the addition of cytochrome c and dATP to lysates of HeLa cells transfected with pcDNA-NAIP or pcDNA-NAIP (K476T). Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3. However, K476T variant of NAIP did not block autocleavage of procaspase-9 efficiently. Furthermore, cleavage pattern of procaspase-9 was altered in the presence of mutant NAIP. Interestingly no effect on the procaspase-3 cleavage by apoptosome was observed. The presence of NOD domain by itself had no effect on autocleavage of procaspase-9 yet slightly reduced the cleavage of procaspase-3 by apoptosome. Pull down experiment showed direct interaction of the NOD domain of NAIP with the CARD-NOD domain of Apoptotic Protease Activating Factor 1 (APAF-1). The physical association of these domains was confirmed by pull-down assays. These observations taken with previous findings indicate that the integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein.
研究了人神经元凋亡抑制蛋白（NAIP）ATP 结合位点对其阻止内在凋亡途径的能力的重要性。因此，突变了位于核苷酸结合寡聚结构域（NOD）的 NAIP 的 ATP 结合赖氨酸 476 为苏氨酸，并研究了该突变对 procaspase-9 自身切割和凋亡小体切割 procaspase-3 的影响。通过向转染了 pcDNA-NAIP 或 pcDNA-NAIP（K476T）的 HeLa 细胞裂解液中添加细胞色素 c 和 dATP 来诱导凋亡小体的形成。全长野生型 NAIP 可阻止 procaspase-9 到 caspase-9 和 procaspase-3 到 caspase-3 的切割。然而，K476T 变体的 NAIP 不能有效地阻止 procaspase-9 的自身切割。此外，在存在突变 NAIP 的情况下，procaspase-9 的切割模式发生改变。有趣的是，凋亡小体对 procaspase-3 的切割没有影响。NOD 结构域本身的存在对 procaspase-9 的自身切割没有影响，但略微降低了凋亡小体对 procaspase-3 的切割。下拉实验显示 NAIP 的 NOD 结构域与凋亡蛋白酶激活因子 1（APAF-1）的 CARD-NOD 结构域直接相互作用。通过下拉实验证实了这些结构域的物理关联。这些观察结果与以前的发现表明，NOD 结构域的完整性对于 NAIP 蛋白有效抑制 procaspase-9 和 procaspase-3 的切割至关重要。
