谷胱甘肽化修饰对Hsp70家族分子伴侣功能的影响

Glutathionylation of cysteine residues plays an important role in fine-tuning the regulation of proteins involved in diverse physiological functions in animal and plant cells. Hsp70 chaperones, which are essential for cell survival, take part in a wide range of cellular activities, so have earned the name "molecular supertool" . Glutathionylation of Hsp70 proteins has been detected in several different types of cells under conditions of oxidative stress. However, there is still little information available about the effects of glutathionylation on the structure and function of Hsp70 proteins, or the resulting physiological consequences. This study will focus on in vitro investigation of the conditions required for glutathionylation of Hsp70 proteins to occur, the mechanism of the reaction, and the mechanism of the emzymatically-catalysed degluthionylation reaction. In particular, we will investigate the effect of glutathionylation on the structure of Hsp70 proteins, and how this affects their ATPase and chaperone activities. In addition, we will investigate what factors are able to trigger glutathionylation of Hsp70 proteins in the cell and the consequences of this. This work will provide an integrated picture of the biochemical events before and after Hsp70 glutathionylation. The results will shed new light on the mechanisms by which the different functions of Hsp70 proteins are regulated, and will further our understanding of glutathionylation as a regulatory mechanism.

蛋白质巯基的谷胱甘肽化修饰参与动植物多种生理活动的精细调节。Hsp70家族分子伴侣（Hsp70s）作为生命必需的分子伴侣，在细胞活动的多领域都有影响，被誉为"分子超级工具"。Hsp70s的谷胱甘肽化修饰已在多种细胞的氧化应激条件下被检测到，然而谷胱甘肽化修饰对Hsp70s的结构和功能的影响及其细胞效应仍然鲜有研究数据。本项目将致力于体外研究Hsp70s发生谷胱甘肽化修饰的基本条件、反应机制及去谷胱甘肽化的酶促催化反应，重点研究谷胱甘肽化修饰对Hsp70s结构的扰动及对其ATPase活性和分子伴侣功能的影响，进一步研究细胞内Hsp70s发生谷胱甘肽化修饰的促发因素和后续生理效应，阐述Hsp70s发生谷胱甘肽化修饰前后的系列生化事件。本项目成果将为Hsp70s功能的调节机制提供新思路，并为谷胱甘肽化修饰研究提供基础数据。

蛋白质巯基的谷胱甘肽化修饰参与动植物多种生理活动的精细调节。Hsp70 家族分子伴侣（Hsp70s）作为生命必需的分子伴侣，在细胞活动的多领域都有影响。Hsp70s 的谷胱甘肽化修饰已在多种细胞的氧化应激条件下被检测到，而其生物学意义和相关机制仍不清楚。本项目致力于体外研究Hsp70s 发生谷胱甘肽化修饰的基本条件、反应机制及去谷胱甘肽化的酶促催化反应，重点研究谷胱甘肽化修饰对Hsp70s 结构的扰动及对其ATPase 活性和分子伴侣功能的影响，进一步研究细胞内Hsp70s 发生谷胱甘肽化修饰相关的生理意义。通过本项目研究，我们发现大肠杆菌Hsp70（DnaK），酵母Hsp70（Ssa1），人源应激型Hsp70（hHspA1A）和人源保守型Hsp70（hHsc70, hHspA8）的核苷酸结合结构域（NBD）上的Cys能在核苷酸耗竭状态，底物结合结构域（SBD）上的Cys不受核苷酸和底物结合限制，由自由基或氧化剂引发，发生谷胱甘肽化修饰。我们发现DnaK唯一的Cys发生谷胱甘肽化修饰可以导致NBD发生部分去折叠，NBD和SBD之间的结构域通讯发生改变，从而引起ATPase活性下降，底物结合能力下降，和辅分子伴侣的相互作用减弱，重折叠酶活性降低；由于氧化应激条件引发的DnaK的谷胱甘肽化修饰导致DnaK和热休克因子Sigma32之间的相互作用减弱，有利于热休克反应的促发，我们推测DnaK的谷胱甘肽化修饰可能作为氧化应激和热休克反应之间的信号传递机制。我们发现hHsp70A1A的SBD上的两个Cys发生谷胱甘肽化修饰后，导致其盖子结构域的三螺旋结构发生去折叠，变成无规卷曲结构，从而作为自身底物结合腔的底物，通过Leu542为中心的疏水区结合于底物结合腔，导致ATPase活性升高，底物结合能力下降，发生谷胱甘肽化修饰的hHsp70A1A也和相应的热休克因子Hsf1之间的相互作用减弱，促进热休克反应的促发，进一步提示Hsp70的谷胱甘肽化修饰在氧化应激信号传递中的保守意义。项目成果为Hsp70s 功能的调节机制提供新思路，并为谷胱甘肽化修饰研究提供基础数据，为基于巯基修饰的Hsp70的共价抑制剂设计提供了良好的基础。