SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI).
SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI).
复制标题
DOI：
10.1186/1742-2094-9-237
复制
发表时间：
2012-10-12
影响因子：
9.3
通讯作者：
Kong J
中科院分区：
文献类型：
--
作者：
Chen X;Guan T;Li C;Shang H;Cui L;Li XM;Kong J
泛素化蛋白聚集体参与脑缺血/再灌注损伤。这些泛素化蛋白聚集体的存在是异常的，似乎与疾病有关。然而，目前尚不清楚是什么导致聚集体形成，以及聚集体是否代表对聚集体介导的神经变性的反应。为了研究亚硝化应激诱导的蛋白聚集在脑缺血/再灌注损伤中的作用，我们使用原代星形胶质细胞培养物作为细胞模型，系统地检测了它们在氧葡萄糖剥夺和再灌注后的iNOS表达和随后的NO产生。蛋白质二硫键异构酶（PDI）和铜锌超氧化物歧化酶（SOD 1）的表达也进行了检查，并通过免疫沉淀法测定PDI和SOD 1之间的生化相互作用。此外，培养的星形胶质细胞缺氧缺糖和再灌注处理后的S-亚硝基化PDI的水平进行了测量，使用生物素开关测定。通过免疫印迹和免疫荧光染色检测泛素化蛋白聚集体的形成。我们的数据表明，在氧糖剥夺和再灌注处理后，iNOS表达上调导致过量的NO产生。在培养的星形胶质细胞中，缺氧缺糖再灌注后，PDI和SOD 1的表达也上调，并且发现这两种蛋白质相互结合。此外，由于缺血/再灌注损伤增加的亚硝化应激与NO诱导的PDI的S-亚硝基化高度相关，并且PDI的S-亚硝基化与泛素化蛋白聚集体的形成相关; S-亚硝基化PDI的水平与聚集体的形成平行增加。当iNOS特异性抑制剂1400 W抑制NO生成时，PDI的S-亚硝基化反应被显著阻断。此外，1400 W还抑制了缺氧缺糖再灌注后培养的星形胶质细胞中泛素化蛋白聚集体的形成。有趣的是，这些聚集体与SOD 1共定位，发现SOD 1与PDI共免疫沉淀。NO介导的PDI的S-亚硝基化可能参与了脑缺血/再灌注损伤中SOD 1连接的泛素化蛋白聚集体的形成。
Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI) and copper-zinc superoxide dismutase (SOD1) were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the formation of ubiquitinated-protein aggregates in cultured astrocytes following oxygen glucose deprivation and reperfusion was also suppressed by 1400W. Interestingly, these aggregates were colocalized with SOD1, which was found to co-immunoprecipitate with PDI. NO-mediated S-nitrosylation of PDI may be involved in the formation of the SOD1-linked ubiquitinated-protein aggregates in cerebral ischemia/reperfusion injury.
登录
查看更多内容
DOI：
10.1073/pnas.0631696100
发表时间：
2003-05-27
影响因子：
11.1
作者：
Lee, J;Chuang, TH;Cottam, HB
通讯作者：
Cottam, HB
影响因子：
2.9
作者：
He, J;Kang, HJ;Chen, C
通讯作者：
Chen, C
影响因子：
11.4
作者：
Lee, Seong-Ok;Cho, Kwangmin;Ahn, Kwangseog
通讯作者：
Ahn, Kwangseog
DOI：
10.1073/pnas.89.22.10945
发表时间：
1992-11-15
影响因子：
11.1
作者：
GALEA, E;FEINSTEIN, DL;REIS, DJ
通讯作者：
REIS, DJ
影响因子：
4.8
作者：
Ko, HS;Uehara, T;Nomura, Y
通讯作者：
Nomura, Y