ZHAO Jing,CAO Yu,ZHOU Chuanren, et al. Design and activity evaluation of FGF19 mutant based on FGFR asymmetric dimerization model[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(8): 614-622.
Abstract：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 (FGF19F159A) and wild-type FGF19 (FGF19WT) 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 FGF19WT and FGF19F159A; 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 (PFRS2),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 FGF19WT and FGF19F159A 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,FGF19F159A was successfully constructed, and protein with high purity was obtained through expression and purification; PLA experiments showed that FGF19F159A significantly reduced the degree of receptor dimerization compared with the FGF19WT group (P<0.01); MTT experiments showed that the proliferative activity of FGF19F159A was significantly reduced compared to the FGF19WT group (P<0.05); Western blot experiments showed that FGF19F159A significantly decreased the expression level of P-FRS2 and P-ERK, compared with the FGF19WT group (P<0.05); Immunohistochemical analysis showed that FGF19F159A significantly declined the expression level of proliferation markers such as Ki67 and PCNA in the liver (P<0.01), compared with the FGF19WT group; FGF19F159A prominently suppressed the relative mRNA levels of AFP, CCNA2 and EGFR in the liver, compared with the FGF19WT 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 FGF19F159A group compared
to the FGF19WT group (P>0.05); There was basically no difference between FGF19F159A and FGF19WT in the ability to lower blood glucose and improve glucose tolerance (P>0.05). Conclusion: The FGF19F159A 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.
