小鼠精子热休克蛋白90的棕榈酰化

发布时间：2018-11-09 21:07

【摘要】：蛋白质棕榈酰化是指饱和十六碳的棕榈酸盐通过硫酯键或者酰胺键连接在蛋白质肽链的半胱氨酸上,属翻译后修饰,可影响蛋白质的相互作用、稳定性及定位等功能。热休克蛋白90(heat shock protein 90,Hsp90)是一种重要的分子伴侣,已证明其参与精子获能等生理过程。然而,哺乳动物精子中是否存在蛋白质棕榈酰化,Hsp90在精子不同生理状态下是否发生棕榈酰化,目前尚不清楚。本研究首先采用酰基-生物素置换法检测小鼠附睾尾部成熟精子总蛋白质棕榈酰化情况,然后通过CSS-Palm 4.0软件预测Hsp90的棕榈酰化位点,再进一步结合免疫沉淀方法检测附睾头部、附睾尾部精子的Hsp90棕榈酰化情况。结果显示,小鼠附睾尾部精子多种蛋白质存在棕榈酰化,其中分子量大小约50、65、72、85和130 k Da的蛋白质发生棕榈酰化最为显著;软件预测显示Hsp90两个亚型共有5个棕榈酰化位点;免疫沉淀结果也显示小鼠精子存在棕榈酰化的Hsp90,且其棕榈酰化水平与小鼠精子的生理状态有关:附睾尾部棕榈酰化水平比附睾头部高,而获能后的棕榈酰化水平比获能前明显升高。以上结果表明,哺乳动物精子中存在蛋白棕榈酰化,且Hsp90棕榈酰化可能参与精子生理状态的调节。
[Abstract]:Protein palmitoacylation means that the hexadecyl palmitate is connected to the cysteine of the protein peptide by thioester bond or amide bond. It is a post-translational modification which can affect the interaction, stability and localization of protein. Heat shock protein 90 (Hsp90) is an important molecular chaperone, which has been proved to be involved in physiological processes such as sperm capacitation. However, it is not clear whether there is protein palmitylation in mammalian sperm and whether Hsp90 occurs in different physiological state of sperm. In this study, the total protein palmitylation of mouse epididymal tail mature spermatozoa was detected by acyl-biotin replacement method, and then the CSS-Palm 4.0 software was used to predict the palmitoacylation site of Hsp90. The Hsp90 palmitylation of epididymal head and tail sperm was detected by immunoprecipitation. The results showed that there was palmitylation of many proteins in mouse epididymal tail sperm, among which the proteins with molecular weight of 5065kmol / 72O85 and 130k Da were the most significant, and the software prediction showed that the two subtypes of Hsp90 shared five palmitoacylation sites. The results of immunoprecipitation also showed that there was palmitoacylated Hsp90, in mouse spermatozoa and the level of palmitoacylation was related to the physiological state of mouse spermatozoa: the level of palmitoacylation in tail of epididymis was higher than that in head of epididymis. The level of palmitoacylation after capacitation was significantly higher than that before capacitation. These results suggest that there is protein palmitylation in mammalian spermatozoa and that Hsp90 palmitylation may be involved in the regulation of the physiological state of spermatozoa.
【作者单位】：温州医科大学检验医学院生命科学学院;浙江省医学科学院生殖医学研究中心;
【基金】：supported by the National Natural Science Foundation of China(No.81571426,81170554) the Natural Science Foundation of Zhejiang Province,China(No.LY14H040012) Public Project for Technological Research and Social Development,the Science and Technology Plan of Zhejiang Province,China(No.2013C31066)
【分类号】：R321.1

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