Fig 1: Biochemical consequences of the G164D and F194S mutations of FBP1 in association with decreased protein expression and aggregation in ER due to protein misfolding.In vitro functional analysis of the G164D and F194S FBP1 mutants in FBP1-KO HepG2 cells. FBP1 protein expression was examined. a Protein expression analysis demonstrated that Myc-tagged FBP1 was decreased in G164D, F194S, and cotransfected constructs compared with that in the WT construct. RT‒qPCR analysis detected sufficient quantities of mRNA among the constructs (n = 2). b Immunofluorescence analysis showed that G164D and F194S FBP1 mutants resulted in the aggregation of FBP1 in the cytoplasm. The scale bars indicate 50 µm. c Immunofluorescence analysis of a liver biopsy specimen from the patient. FBP1 aggregated in the cytoplasm. The scale bar indicates 20 µm. d Venn diagram of the proteins identified in the FBP1 interactome based on MS analysis. e The main function of FBP1 interactome proteins based on Ingenuity Pathway Analysis (IPA). f Clusters of the enriched biological processes in the FBP1 interactome. Data from the STRING interaction database were visualized using Cytoscape. g Intracellular localization of the endoplasmic reticulum (green) and Myc-tagged FBP1 (red). The G164D and F194S FBP1 mutants colocalized, but not completely, in the endoplasmic reticulum. The scale bars indicate 10 µm. h Immunoblot analyses of FBP1 and ATF6. ATF6 is an endoplasmic reticulum, stress-regulated, transmembrane transcription factor. It was enriched in the endoplasmic reticulum fractions. FBP1 protein was detected in the whole cell lysate and endoplasmic reticulum fractions. Protein expression of the G164D and F194S FBP1 mutants was relatively high in the endoplasmic reticulum compared to the whole cell lysate. i HepG2 cells were treated with the mannosidase inhibitor kifunensine (200 µM) for 48 h, which increased the protein expression of the G164D and F194S FBP1 mutants. j Immunoprecipitation with anti-Myc and immunoblot analysis of FBP1, HSP70, HSP90, HSP60, and TCP1. k G164D and F194S FBP1 mutants demonstrated greater interactions with HSP70, HSP90, HSP60 and TCP1 compared with wild-type (WT) FBP1. The data are presented as the mean ± SD. *P < 0.05; **P < 0.01 versus WT (one-way ANOVA test followed by Dunnett’s multiple comparison test). WT: n = 3–6, G164D: n = 3–6, F164S: n = 3–6. Vertically stacked strips of bands in in (a, h, i, and j) were evaluated in the same experimental conditions respectively while they were not in fact all derived from the same gel.