Yu H-M, Xu J, Li C, Zhou C, Zhang F, Han D, Zhang G-Y
Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou 221002, Jiangsu, People's Republic of China.
Neuroscience. 2008 Sep 9;155(4):1120-32. doi: 10.1016/j.neuroscience.2008.03.061. Epub 2008 Apr 4.
S-nitrosylation, as a post-translational protein modification, recently has been paid more and more attention in stroke research. S-nitrosylation regulates protein function by the mechanisms of covalent attachment that control the addition or the removal of nitric oxide (NO) from a cysteine thiol. The derivation of NO is established by the demonstration that, in cerebral neurons, NO mainly generates from neuronal nitric oxide synthase (nNOS) during the early stages of reperfusion. In the past researches, we demonstrate that global ischemia-reperfusion facilitates the activation of glutamate receptor 6 (GluR6) -mediated c-Jun N-terminal kinase (JNK) signaling pathway. The objective of this study is primarily to determine, during the early stages of reperfusion in rat four-vessel occlusion (4-VO) ischemic model, whether nNOS-derived NO affects the GluR6-mediated JNK signaling route via S-nitrosylation which is performed mainly by the biotin switch assay. Here, we show that administration of 7-nitroindazole, an inhibitor of nNOS, or ketamine, an antagonist of N-methyl-d-aspartate receptor (NMDAR), diminishes the increased S-nitrosylation of GluR6 induced by cerebral ischemia-reperfusion. In contrast, 2-amion-5,6-dihydro-6-methyl-4H-1,3-thiazine, an inhibitor of inducible NO synthase does not affect S-nitrosylation of GluR6. Moreover, treatment with sodium nitroprusside (SNP), an exogenous NO donor, increases the S-nitrosylation and phosphorylation of nNOS, leading to the attenuation of the increased S-nitrosylation of GluR6 and the assembling of GluR6* postsynaptic density protein 95 (PSD95)* mixed lineage kinase 3 (MLK3) signaling module induced by cerebral ischemia-reperfusion. The results also show that GluR6 downstream MLK3* mitogen activated protein kinase kinase 4/7* JNK signaling module and nuclear or non-nuclear apoptosis pathways are involved in the above signaling route. However, dithiothreitol (DTT) antagonizes the neuroprotection of SNP. Treatment with DTT alone, as a negative control, prevents S-nitrosylation of proteins, which indicates the existence of endogenously produced S-nitrosylation. These data suggest that GluR6 is S-nitrosylated by endogenous NO in cerebral ischemia-reperfusion, which is possibly correlated with NMDAR* PSD95* nNOS signaling module, and further activates GluR6* PSD95* MLK3 signaling module and JNK signaling pathway. In contrast, exogenous NO donor antagonizes the above action of endogenous NO generated from nNOS. Thus, our results provide the coupling of nNOS with GluR6 by S-nitrosylation during the early stages of ischemia-reperfusion, which can be a new approach for stroke therapy.
S-亚硝基化作为一种蛋白质翻译后修饰，近年来在中风研究中受到越来越多的关注。S-亚硝基化通过共价连接机制调节蛋白质功能，该机制控制一氧化氮（NO）从半胱氨酸硫醇的添加或去除。NO的产生是通过证明在脑神经元中，NO主要在再灌注早期由神经元型一氧化氮合酶（nNOS）产生而确定的。在过去的研究中，我们证明全脑缺血再灌注促进谷氨酸受体6（GluR6）介导的c-Jun氨基末端激酶（JNK）信号通路的激活。本研究的主要目的是在大鼠四血管闭塞（4-VO）缺血模型再灌注早期，确定nNOS衍生的NO是否通过主要由生物素开关法进行的S-亚硝基化影响GluR6介导的JNK信号途径。在这里，我们表明给予nNOS抑制剂7-硝基吲唑或N-甲基-D-天冬氨酸受体（NMDAR）拮抗剂氯胺酮，可减少脑缺血再灌注诱导的GluR6增加的S-亚硝基化。相反，诱导型一氧化氮合酶抑制剂2-氨基-5,6-二氢-6-甲基-4H-1,3-噻嗪不影响GluR6的S-亚硝基化。此外，用外源性NO供体硝普钠（SNP）处理可增加nNOS的S-亚硝基化和磷酸化，导致脑缺血再灌注诱导的GluR6增加的S-亚硝基化以及GluR6突触后致密蛋白95（PSD95）混合谱系激酶3（MLK3）信号模块的组装减弱。结果还表明，GluR6下游的MLK3丝裂原活化蛋白激酶激酶4/7JNK信号模块以及核或非核凋亡途径参与上述信号途径。然而，二硫苏糖醇（DTT）拮抗SNP的神经保护作用。单独用DTT作为阴性对照处理可防止蛋白质的S-亚硝基化，这表明存在内源性产生的S-亚硝基化。这些数据表明，在脑缺血再灌注中，GluR6被内源性NO进行S-亚硝基化，这可能与NMDARPSD95nNOS信号模块相关，并进一步激活GluR6PSD95MLK3信号模块和JNK信号通路。相反，外源性NO供体拮抗nNOS产生的内源性NO的上述作用。因此，我们的结果提供了缺血再灌注早期通过S-亚硝基化使nNOS与GluR6偶联的机制，这可能是中风治疗的一种新方法。
