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

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FGF19^(WT)组相比,FGF19^(F159A)组肝脏中Cyp7A1的蛋白表达水平,胆汁酸CA、DCA、UDCA、CDCA的含量差异无统计学意义(P>0.05);与FGF19^(WT)组相比,FGF19^(F159A)组的降糖能力和改善糖耐量的能力差异无统计学意义(P>0.05)。结论:基于FGFR非对称二聚化模型设计的FGF19改构体FGF19^(F159A)能够在极大减弱细胞增殖活性的同时保留其代谢活性,有望成为治疗胆汁淤积症的候选药物。

Objective: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 showed that FGF19^(F159A)significantly reduced the degree of receptor dimerization compared with the FGF19^(WT)group(P<0.01);MTT experiments showed that the proliferative activity of FGF19^(F159A)was significantly reduced compared to the FGF19^(WT)group(P<0.05);Western blot experiments showed that FGF19^(F159A)significantly decreased the expression level of P-FRS2 and P-ERK,compared with the FGF19^(WT)group(P<0.05);Immunohistochemical analysis showed that FGF19^(F159A)significantly declined the expression level of proliferation markers such as Ki67 and PCNA in the liver(P<0.01),compared with the FGF19^(WT)group;FGF19^(F159A)prominently suppressed the relative mRNA levels of AFP,CCNA2 and EGFR in the liver,compared with the FGF19^(WT)group(P<0.01);There was no significant difference in the protein expression level of Cyp7A1 and the content of CA,DCA,UDCA and CDCA in the liver of the FGF19^(F159A)group compared to the FGF19^(WT)group(P>0.05);There was basically no difference between FGF19^(F159A)and FGF19^(WT)in the ability to lower blood glucose and improve glucose tolerance(P>0.05).Conclusion:The FGF19^(F159A)designed based on the asymmetric dimerization model of FGFR significantly reduces proliferative activity while retaining its metabolic activity,which is expected to become a potential drug for cholestasis.