基于FGFR非对称二聚化模型的FGF19改构体的设计及活性评估  

Design and activity evaluation of FGF19 mutant based on FGFR asymmetric dimerization model

作　　者：赵静[1] 操宇 周传仁 吴佳敏 ZHAO Jing;CAO Yu;ZHOU Chuanren;WU Jiamin(Department of Pharmacy,Zhejiang Hospital,Hangzhou 310012,China;School of Pharmacy,Wenzhou Medical University,Wenzhou 325035,China)

机构地区：[1]浙江医院药剂科,浙江杭州310012 [2]温州医科大学药学院,浙江温州325035

出　　处：《温州医科大学学报》2024年第8期614-622,共9页Journal of Wenzhou Medical University

基　　金：浙江省自然科学基金项目(Q19H300011)。

摘　　要：目的:探究以成纤维细胞生长因子受体(FGFR)非对称二聚化模型为结构基础设计成纤维细胞生长因子19(FGF19)的非促肿瘤型改构体,并对其增殖活性和代谢活性进行评估,使其成为治疗胆汁淤积症的候选药物之一。方法:利用pymol软件分析蛋白晶体结构并设计FGF19改构体,将构建好的FGF19改构体(FGF19^(F159A))与FGF19野生型(FGF19^(WT))的质粒转化到大肠杆菌感受态细胞中,经过诱导表达、变性、复性获得正确构象的蛋白,经过镍柱亲和层析与分子排阻色谱方法纯化得到目的蛋白;通过蛋白邻位连接技术(PLA)对比FGF19^(WT)和FGF19^(F159A)诱导受体二聚化的程度;MTT实验检测细胞增殖活性;Western blot实验检测磷酸化成纤维细胞生长因子受体底物2(P-FRS2)、磷酸化细胞外调节蛋白激酶(P-ERK)、胆固醇7α-羟化酶(Cyp7A1)的蛋白表达水平;免疫组化法检测动物肝脏中促增殖指标Ki67和增殖细胞核抗原(PCNA)的蛋白表达水平;RT-qPCR法检测动物肝脏中肿瘤指标甲胎蛋白(AFP)、细胞周期蛋白A2(CCNA2)、表皮生长因子受体(EGFR)的相对mRNA水平;质谱法测定肝脏中胆酸(CA)、脱氧胆酸(DCA)、熊去氧胆酸(UDCA)、鹅去氧胆酸(CDCA)的含量;以db/db小鼠为模型,连续给药1个月,监测药物的慢性降糖效果,糖耐量实验检测其对糖耐量的改善能力。结果:以蛋白结构为基础成功构建了FGF19^(F159A),经过表达纯化得到了高纯度的目的蛋白;PLA结果显示,与FGF19^(WT)组相比,FGF19^(F159A)诱导受体二聚化的能力明显减弱(P<0.01);MTT实验显示,与FGF19^(WT)组相比,FGF19^(F159A)的促增殖活性明显减弱(P<0.05);Western blot实验表明,与FGF19^(WT)组相比,FGF19^(F159A)明显下调P-FRS2和P-ERK的蛋白表达水平(P<0.05);免疫组化结果显示,与FGF19^(WT)组相比,FGF19^(F159A)组Ki67和PCNA的蛋白表达水平显著降低(P<0.01);与FGF19^(WT)组相比,FGF19^(F159A)组AFP、CCNA2、EGFR的mRNA水平明显降低(P<0.01);与FGObjective:To explore the non-tumorigenic mutant of fibroblast growth factor 19(FGF19)based on the asymmetric dimerization model of fibroblast growth factor receptor(FGFR)and to evaluate its proliferative and metabolic activities with a view to make it a potential drug for the treatment of cholestasis.Methods:Pymol software was used to analyze the protein crystal structure and design the FGF19 mutant;the modified FGF19 mutant(FGF19^(F159A))and wild-type FGF19(FGF19^(WT))plasmids were transformed into E.coli competent cells,and then the protein with the correct conformation was obtained through induction expression,denaturation,and renaturation.The target protein was purified by nickel column affinity chromatography and molecular exclusion chromatography;Proximity Ligation Assay technique was used to compare the receptor dimerization abilities induced by FGF19^(WT)and FGF19^(F159A);Cell proliferation activity was measured by MTT method;Western blot assay was used to detect protein expression levels of phospho-fibroblast growth factor receptor substrate 2(P-FRS2),phospho-extracellular regulated protein kinase(P-ERK),and cholesterol 7alpha-hydroxylase(Cyp7A1);Immunohistochemistry was used to detect the expression levels of proliferation indicators,Ki67 and proliferating cell nuclear antigen(PCNA);RT-qPCR was implemented to determine the relative mRNA levels of tumor indicators,such as alpha-fetoprotein(AFP),cyclin A2(CCNA2)and epidermal growth factor receptor(EGFR);Mass spectrometry was used to determine the content of cholic acid(CA),deoxycholic acid(DCA),ursodeoxycholic acid(UDCA),chenodeoxycholic acid(CDCA)in the liver;after db/db mice were injected with FGF19^(WT)and FGF19^(F159A)for one month,their blood glucose level was monitored and compared and the ability to improve glucose tolerance was tested through glucose tolerance experiments.Results:Based on the structural design,FGF19^(F159A)was successfully constructed,and protein with high purity was obtained through expression and purification;PLA experiments sho

关 键 词：FGF19^(F159A) FGF19^(WT) 非对称二聚化模型 胆汁酸 肿瘤活性 

分 类 号：R975[医药卫生—药品]