基于幽门螺旋杆菌毒力因子HtrA三维结构的拮抗肽设计及其在预防胃癌发生发展中的作用研究

Helicobacter pylori infection is a major pathogenic factor of gastric cancer as one of the most common malignant tumor severely threatening human health. Recently, a new virulence factor HtrA specifically shearing E-cadherin was discovered in Helicobacter pylori. Cleaved E-cadherin has been proven to be an important biomarker of gastric cancer. The mechanism of such shearing, however, remains to be determined. In this project, we aim to firstly uncover the correlation between HtrA and gastric mucosal injury by analyzing clinical data related to Helicobacter pylori and expression of E-cadherin on gastric mucosa in patients; and to determine the 3D structure of HtrA by means of X-ray crystallography. On the basis of structural analysis of the HtrA protein, key amino acids in the active center will be identified and the molecular mechanisms of HtrA cutting E-cadherin could be revealed. Targeting against the active site of HtrA, antagonist peptides will be designed in this project, and modified later before the verification at cell level. Our research would provide a new approach for potential treatment of Helicobacter pylori infection inducing gastric mucosal injury and prevention of Helicobacter pylori inducing gastric cancer.

胃癌是威胁人类健康最常见的恶性肿瘤之一，而幽门螺杆菌感染是胃癌发生的重要致病因素之一。最近发现一种幽门螺杆菌新型毒力因子HtrA，能特异性剪切E钙粘蛋白（E-cadherin）的胞外段，而E钙粘蛋白已被证实是胃癌发生和转移的重要标志物。然而这种酶切的分子机制并不清楚。我们拟通过分析临床上幽门螺杆菌定植密度和E-cadherin在病人胃黏膜上的表达情况，阐释HtrA与胃黏膜损伤的相关性，然后通过原核表达系统表达纯化HtrA蛋白并结晶，利用X射线衍射获得HtrA蛋白的衍射数据，通过解析蛋白三维结构和确认活性中心，揭示HtrA剪切E-cadherin的分子机制。并针对活性位点的三维结构，设计并改造拮抗肽，然后在细胞水平验证该拮抗肽的有效性，从而为治疗幽门螺旋杆菌感染引起的胃黏膜损伤和预防幽门螺旋杆菌诱导的胃癌发生发展提供一种新的途径。

胃癌是威胁人类健康最常见的恶性肿瘤之一，而幽门螺杆菌感染是胃癌发生的重要致病因素之一。最近发现一种幽门螺杆菌新型毒力因子HtrA，能特异性剪切E钙粘蛋白(E-cadherin )的胞外段，而E钙粘蛋白已被证实是胃癌发生和转移的重要标志物。然而如何阻止这种剪切的发生对幽门螺旋杆菌感染引起的胃癌具有重要意义。本项目在确认幽门螺旋杆菌感染、胃黏膜病理损伤与E-cadherin表达之间的相关性后，通过原核表达系统得到HtrA蛋白200mg，纯度达到95%以上。经多轮结晶筛选，获得了HtrA蛋白的晶体结构，分辨率达到2.1A。利用突变体和蛋白同源比对确定其酶活性位点，经过多位点突变筛选，发现Ser221和Ser205是该蛋白酶活性中心的重要氨基酸。我们利用HtrA蛋白活性位点结构和E-cadherin蛋白序列的同源建模筛选出拮抗肽（TGTLLLILSDVNDNAPIPEPR），初步实验显示该拮抗肽具有抑制HtrA蛋白对E-cadherin蛋白剪切。该拮抗肽的发现为治疗幽门螺旋杆菌感染引起的胃黏膜损伤和预防幽门螺旋杆菌诱导的胃癌发生发展提供一种新的途径。