海洋碱性蛋白酶MP及其抑制剂lupI相互作用机制研究

Inhibitor lupI located downstream of marine alkaline protease MP, the inhibitor could inhibit the protease MP activity in vitro, but in the intracellular its existence and function as well as the enzyme and inhibitor interaction mechanism is not very clear. It is postulated that this class of inhibitors inhibit protease activity in the intracellular, thereby prevent the protease degradation of cell proteins. Proteinase MP, belongs to Serralysins, and its inhibitor lupI as the research object, study on the interaction of enzyme and inhibitor. First by homologous recombination technology of lupI deletion mutation we detect the strain whether normal secretes the proteinase MP , and then mutate the lupI in vitro and crystal structure of the compound for determination the interaction of MP and inhibitor; finally, combined with the crystal structure and biochemical data elucidate the function of lupI in the secretion process of MP and the role of enzyme and inhibitor interaction mechanism, thus laid the foundation for the interaction mechanism of Serralysins and its inhibitor.

抑制剂lupI基因位于海洋碱性蛋白酶MP基因下游，该抑制剂在体外可完全抑制蛋白酶MP的活性，但在胞内的存在方式及功能以及酶与抑制剂的相互作用机制目前还不是很清楚，有假说认为这类抑制剂在胞内抑制蛋白酶的活性，从而阻止蛋白酶对细胞蛋白的降解。本课题以属于沙雷氏蛋白酶家族的蛋白酶MP及其抑制剂lupI为研究对象，研究其胞内相互作用及酶与抑制剂相互作用机制。首先通过同源重组技术缺失突变lupI基因，进而检测该菌株是否能正常分泌蛋白酶MP，然后体外突变表达lupI基因，测定其与MP是如何相互作用的，对其复合物进行结晶和晶体结构解析；最后，结合晶体结构和生化数据阐明lupI在MP分泌过程中的作用以及酶与抑制剂的相互作用机制，进而为沙雷氏蛋白酶及其抑制剂的相互作用机制奠定基础。

本项目以属于沙雷氏蛋白酶家族的蛋白酶MP及其专一性抑制剂LupI为研究对象，通过构建自杀质粒pLP12-LupI来敲除抑制剂LupI基因，敲除后对菌株80的生长和蛋白酶MP的分泌均不产生影响，表明lupI并不是细胞生长所必需的基因，它在蛋白酶MP分泌过程中并没有起到保护性作用。通过体外表达抑制剂LupI的关键位点的不同突变体，对其抑制活性进行了测定，同时对酶与抑制剂形成的复合物进行了晶体生长及结构解析，阐述了抑制剂LupI中N端Trunk结构、第一位氨基酸、不规则卷曲c4等结构与蛋白酶MP的相互作用机制。上述研究结果有力的推动了沙雷氏蛋白酶家族的基础研究工作，为其未来进入生物医药领域的实际应用奠定了重要的理论基础。项目执行期内已发表文章7篇，其中SCI文章4篇，申请中国发明专利1项。