Zhang Huan, Fei Rui, Xue Baigong, Yu Shanshan, Zhang Zuoming, Zhong Sheng, Gao Yuanqi, Zhou Xiaoli
Department of Cell Biology, College of Basic Medical Sciences, Jilin University, Changchun 130021, China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
Int J Mol Sci. 2017 Jan 7;18(1):113. doi: 10.3390/ijms18010113.
Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile , was overexpressed in for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.
丝氨酸蛋白酶抑制剂（丝氨酸蛋白酶抑制剂超家族）是丝氨酸蛋白酶的天然抑制剂，构成了一个庞大的蛋白质家族，其成员分布于真核生物和原核生物中。然而，迄今为止，只有极少数原核丝氨酸蛋白酶抑制剂，特别是来自嗜极端微生物的，得到了表征。在本研究中，一种来自嗜热菌的假定丝氨酸蛋白酶抑制剂Pnserpin在大肠杆菌中过量表达，用于纯化和表征。它在20至100°C的温度范围内以浓度依赖的方式不可逆地抑制胰凝乳蛋白酶、胰蛋白酶、弹性蛋白酶和枯草杆菌蛋白酶样蛋白酶。随着温度升高，Pnserpin对蛋白酶的抑制化学计量比（SI）降低，表明Pnserpin的抑制活性随温度升高而增加。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）显示，Pnserpin通过形成抗SDS的共价复合物来抑制蛋白酶。同源建模和分子动力学模拟预测，Pnserpin可以形成稳定的常见丝氨酸蛋白酶抑制剂折叠。本研究结果将有助于理解嗜热丝氨酸蛋白酶抑制剂的结构和功能特性，并将拓宽目前关于嗜极端微生物丝氨酸蛋白酶抑制剂的知识。
