巯基氧化剂诱导的氧化胁迫对骨骼肌型钙释放通道门控和蛋白交联的影响

发布时间：2018-08-12 07:35

【摘要】： 当人体处于极端环境和病理生理状态下,体内的氧化还原平衡被打破,氧化水平升高,可以形成胞内局部的氧化胁迫环境。研究表明一些心肌和骨骼肌疾病都与肌质网膜的离子通道的氧化损伤有关。而肌质网肌质网(sarcoplasmicreticulum,SR)中的钙释放通道(ryanodine receptor,RyR)富含巯基,对氧化还原环境异常敏感,是氧化胁迫的作用目标。前期工作表明,在氧化胁迫条件下RyR被严重抑制,蛋白内的自由巯基数量剧减。钙释放通道RyR的功能状态与其它膜蛋白和RyR之间的相互作用密切相关,因此,我们选择蛋白间的相互作用作为主要研究内容,初步探讨氧化胁迫对RyR的影响如何和造成这种影响的机制。本研究以氧化胁迫浓度下的Na_2SeO_3和1,4NQ模拟氧化胁迫环境,以兔骨骼肌肌质网(SR)囊泡和钙释放通道(RyR1)为研究对象,利用【3H】-ryanodine结合实验,SDS-PAGE和Western Blotting,光子相干光谱法(PCS)和DPH荧光偏振法,考察氧化胁迫条件下氧化型通道调控剂对RyR1通道活性,SR蛋白分布,RyR1颗粒粒径分布和SR膜流动性的影响。实验表明,氧化胁迫浓度的Na_2SeO_3和1,4NQ的预处理导致了RyR1通道活性和SR膜的流动性降低;并导致了SR膜蛋白交联,形成大分子交联复合物电泳条带并被DTT逆转;RyR、钙泵、myosin和钙结合蛋白等蛋白参与了复合物的形成;进而发现,Na_2SeO_3和1,4NQ处理使得RyR蛋白之间形成了大粒径的聚合物,导致RyR的平均粒径增大,而DTT也可以部分逆转这种粒径的增大。上述结果显示,氧化胁迫浓度的氧化剂对RyR1通道的抑制作用,可能是由于氧化了负责关闭通道的职能巯基群,并由此产生了蛋白间错误交联而形成蛋白异常堆积,因此可能改变了钙释放通道的功能结构;SR膜流动性的下降,也可能诱导了蛋白和脂质分子、以及脂质分子间的交联,由此也可能导致蛋白变构的灵活性下降。另外所导致的RyR之间相互连接的增强也可能影响RyR之间的正常的协同性。
[Abstract]:When the human body is in extreme environment and pathophysiological state, the redox balance is broken and the oxidation level is increased, which can form the local oxidative stress environment in the cell. Studies have shown that some myocardial and skeletal muscle diseases are associated with oxidative damage of ion channels in the sarcoplasmic omentum. The ryanodine receptor RyR in sarcoplasmic reticulum SR, which is rich in sulfhydryl, is sensitive to redox environment and is the target of oxidative stress. Previous work showed that RyR was severely inhibited under oxidative stress, and the number of free sulfhydryl groups in the protein was greatly reduced. The functional state of calcium release channel (RyR) is closely related to the interaction between other membrane proteins and RyR. To explore the effect of oxidative stress on RyR and its mechanism. In this study, (SR) vesicles and calcium release channel (RyR1) of rabbit skeletal muscle reticulum were used to simulate oxidative stress environment under oxidative stress concentration of Na_2SeO_3 and 1nq, and calcium release channel (RyR1) of rabbit skeletal muscle sarcoplasmic reticulum was studied. Using [3H] -ryanodine binding experiment, SDS-PAGE and Western blotting. photonic coherence spectroscopy (PCS) and DPH fluorescence polarization method were used to investigate the effect of oxidation channel regulator on the particle size distribution and SR membrane fluidity of RyR1 channel active SR protein. The results showed that the pretreatment of Na_2SeO_3 and 1NQ under oxidative stress concentration resulted in the decrease of RyR1 channel activity and the fluidity of SR membrane, and the cross-linking of SR membrane protein. The formation of macromolecular cross-linked complex electrophoresis bands was reversed by DTT, and proteins such as calcium pump myosin and calcium-binding protein were involved in the formation of the complex, and it was found that the treatment of Na _ 2SeO _ 3 and 1N _ (4) NQ resulted in the formation of large particle size polymers between RyR proteins. The average particle size of RyR increases, and DTT can partially reverse the increase. These results suggest that the inhibition of oxidant concentration on RyR1 channel may be due to the oxidation of the functional sulfhydryl group responsible for closing the channel and the formation of abnormal protein accumulation as a result of cross-linking between proteins. Therefore, it may change the functional structure of calcium release channel and decrease the fluidity of SR membrane. It may also induce protein and lipid molecules and the cross-linking between lipid molecules, which may lead to the decrease of flexibility of protein structure. The resulting enhancement of RyR interconnections may also affect the normal synergy between RyR.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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