氧化应激中calpain对GSK-3β的片段化在其活性调节中的作用及机制

发布时间：2018-01-08 10:04

本文关键词：氧化应激中calpain对GSK-3β的片段化在其活性调节中的作用及机制　出处：《华中科技大学》2012年硕士论文　论文类型：学位论文

更多相关文章： GSK-3β 过氧化氢 HEK293/Tau细胞 calpain

【摘要】：研究背景：糖原合成酶激酶-3β（GSK-3β）是一种多功能的丝苏氨酸蛋白激酶，其功能失调可导致多种疾病例如精神疾病、代谢系统疾病以及神经系统退行性疾病的发生发展，而在此过程中，氧化应激也发挥重要作用。多条细胞信号通路参与调节GSK-3β的活性，酪氨酸216位点的磷酸化可以增加GSK-3β的活性，而其丝氨酸9位点的磷酸化则降低GSK-3β的活性，另外，胰岛素途径、wnt信号通路以及与GSK-3结合蛋白之间相互作用等都可调控GSK-3β的活性。我们先前的实验结果已报道过在氧化应激的情况下，GSK-3β活性增高，但是氧化应激是如何参与调节GSK-3β激酶活性的机制不明。已有研究报道，calpain对GSK-3βN端的片段化可导致GSK-3β的抑制结构域丧失，并将GSK-3β切割为两个具有激酶活性的片段，即calpain的激活可截断GSK-3β并使其活性升高。目的：探讨在HEK293/Tau441细胞系中，过氧化氢（H2O2）如何影响GSK-3β的活性及其潜在机制，进一步研究氧化应激中calpain对GSK-3β的片段化在其活性调节中的作用及机制。方法：细胞培养，药物处理后，用CCK-8试剂盒测定细胞活力，同时测定SOD和MDA含量，评估氧化应激模型是否成功建立，检测GSK-3β活性，并运用westernblot技术检测GSK-3β不同位点磷酸化程度以及可能参与磷酸化的相关激酶活性形式的表达；用I×71共聚焦显微镜检测药物处理后不同时间点荧光探针Fura-2-AM标记的HEK293/Tau441细胞内钙离子浓度变化，并运用蛋白免疫印迹分析GSK-3β的片段化程度；随后又用上述实验中所使用的过氧化氢浓度以及calpain的特异性抑制剂Ⅳ同时处理HEK293/Tau441细胞，探究calpain介导的GSK-3β特异性切割。结果：（1）在过氧化氢处理的HEK293/Tau441细胞中，GSK-3β的活性与空白对照组相比增加了两倍，然而GSK-3β的丝氨酸9位点的磷酸化程度也明显升高，即非激活形式的GSK-3β水平升高，同时GSK-3β的酪氨酸216位点的磷酸化程度未出现明显改变；（2）用过氧化氢处理HEK293/Tau441细胞1分钟后，细胞内钙离子浓度开始上升，3分钟细胞内钙离子浓度达到高峰，而后免疫印迹分析结果显示GSK-3β被切割为两个片段；（3）用过氧化氢和calpain特异性抑制剂Ⅳ同时处理HEK293/Tau441细胞，发现GSK-3β的片段化效应明显受到抑制。结论：过氧化氢通过增加细胞钙离子内流激活calpain，引起GSK-3β的片段化，进而对抗由过氧化氢磷酸化丝氨酸9位点诱导的GSK-3β酶活性的下降，，从而上调GSK-3β的活性。
[Abstract]:Background: glycogen synthase kinase -3 beta (GSK-3 beta) is a multifunctional serine / threonine protein kinase, its dysfunction can lead to various diseases such as mental illness, the occurrence and development of metabolic diseases and neurodegenerative diseases, and in this process, oxidative stress also plays an important role in several cellular signaling pathways. Participate in the regulation of GSK-3 beta activity, tyrosine 216 phosphorylation can increase GSK-3 beta activity, while its serine 9 phosphorylation decreased GSK-3 beta activity, in addition, the insulin pathway, Wnt pathway and GSK-3 binding protein interaction can regulate GSK-3 beta activity. Our previous experiments the results have been reported in situations of oxidative stress, increased GSK-3 activity, but the oxidative stress is how to participate in the mechanisms regulating GSK-3 beta kinase activity is unknown. It has been reported that the calpain of GSK Fragmentation of -3 beta N ends can lead to loss of domain inhibition of GSK-3 beta and cut GSK-3 beta into two fragments with kinase activity, that is, activation of calpain can truncate GSK-3 beta and increase its activity.
Objective: To investigate the effect of hydrogen peroxide (H2O2) on the activity and potential mechanism of GSK-3 beta in HEK293/Tau441 cell line, and further study the role and mechanism of calpain in the regulation of GSK-3 beta activity in oxidative stress.
Methods: cell culture, drug treatment, cell viability was determined by CCK-8 kit, the simultaneous determination of SOD and MDA content, the evaluation of oxidative stress models, detection of GSK-3 beta activity, and using Westernblot technology to detect GSK-3 beta sites phosphorylation and expression of related kinase activity forms may be involved in the phosphorylation of the use; I * 71 confocal microscopy to detect drug treatment at different time points after Fura-2-AM labeled HEK293/Tau441 changes in intracellular calcium ion concentration, the degree of fragmentation and use the Western blot analysis of GSK-3 beta; then by using the experiment of hydrogen peroxide concentration and specific inhibitor of calpain IV and HEK293/Tau441 cells, explore calpain GSK-3 mediated by beta specific cutting.
Results: (1) in H2O2 treated HEK293/Tau441 cells, GSK-3 beta activity increased two times compared with the control group, but GSK-3 beta serine 9 phosphorylation level was significantly increased, namely increased expression and activation of GSK-3 beta level, while GSK-3 beta tyrosine 216 phosphorylation there is no obvious change degree;
(2) after 1 minutes of treatment with hydrogen peroxide, the intracellular calcium concentration began to increase, and the intracellular calcium concentration reached its peak in 3 minutes. Western blotting analysis showed that GSK-3 HEK293/Tau441 was cut into two fragments.
(3) HEK293/Tau441 cells were treated with hydrogen peroxide and calpain specific inhibitor IV, and the fragmentation effect of GSK-3 beta was obviously inhibited.
Conclusion: hydrogen peroxide increases GSK-3 beta fragmentation by increasing intracellular calcium influx and activating calpain, thereby antagonized the decrease of GSK-3 beta activity induced by hydrogen peroxide phosphorylation serine 9 site, thereby increasing the activity of GSK-3 beta.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

【共引文献】