Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis
Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis
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DOI：
10.1007/s10495-007-0053-0
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发表时间：
2007-07-01
期刊：
影响因子：
7.2
通讯作者：
Ohkawa, Kiyoshi
中科院分区：
文献类型：
--
作者：
Asakura, Tadashi;Sasagawa, Atsuko;Ohkawa, Kiyoshi
用合成的DXR和GSH的戊二醛偶联物(GSH-DXR)处理细胞后，caspase-3和-9通过典型的DNA碎片被有效激活后，细胞色素c从线粒体释放到胞浆中。这种细胞凋亡受JNK信号通路的调控。在本实验中，GSH-DXR与GST P1-1的变构结合导致其酶活性消失，并激活了JNK的激酶活性，而没有解离JNK-GST P1-1复合体。活性中心区(W38H和C47S)突变的重组GST P1-1分子在与野生型JNK结合时失去GST活性，突变的GST P1-1分子不能抑制JNK的活性。据报道，JNK信号是由GST P1-1通过与C末端的相互作用来调节的。我们证实GST P1-1缺失突变体(Delta 194-209)和C末端的定点突变体(R201A)不能结合和抑制JNK。这些结果表明，不仅GST P1-1的C末端区域与JNK分子结合，而且GST P1-1的活性中心区域在JNK酶活性的调节中起着重要作用。这些结果表明，构象改变后GSH-DXR结合变构抑制GST P1-1活性可能激活JNK，并通过线粒体途径诱导细胞凋亡。
Treatment of cells with a synthetic conjugate of DXR with GSH via glutaraldehyde (GSH-DXR) caused cytochrome c to be released from the mitochondria to the cytosol following potent activation of caspase-3 and -9 by typical DNA fragmentation. This apoptosis was regulated by the JNK-signaling pathway. In the present experiment, binding of GSH-DXR to GST P1-1 allosterically led to the disappearance of its enzyme activity and activated the kinase activity of JNK without dissociation of the JNK-GST P1-1 complex. The recombinant GST P1-1 molecule with a mutation in the active center region (W38H and C47S) lost its GST activity when bound to JNK to the same degree as the wild-type, with the mutated GST P1-1 molecule failing to inhibit the activity of JNK. It has been reported that JNK-signaling is regulated by GST P1-1 via interaction with the C-terminus. We confirmed that GST P1-1 deletion mutant (Delta 194-209) and a site-directed mutant (R201A) in the C-terminal region failed to bind and inhibit JNK. These results indicated that not only binding of the C-terminal region of GST P1-1 to the JNK molecule, but also the active center region of GST P1-1 play important roles in the regulation of JNK enzyme activity. The findings suggested that allosteric inhibition of GST P1-1 activity by the binding of GSH-DXR following conformational change may activate JNK and induce apoptosis via the mitochondrial pathway in the cells.