Fig 1: Murine GID is dispensable for the regulation of gluconeogenic enzymes FBP1 and PCK1.A, B. Western blot analysis of control- and GID siRNA-treated primary mouse hepatocytes and HEK-293 cells shifting from gluconeogenesis (GNG) to glycolysis (GLY) over the indicated period of time. C, D. qRT-PCR of PCK1 and FBP1 transcripts in HEK-293 and primary hepatocytes in control and GID-depleted cells during a metabolic shift from GNG to GLY. Data is shown as mean fold change relative to control ± SD, n = 3.
Fig 2: Compromised GID activity results in an intermittent cellular growth defect.(A) Cell proliferation of RPE control and MAEA-KO cells was quantified with MTT assays between days 6 and 9 after lentiviral transfection. Data are shown as mean of triplicates and % change in signal relative to control gRNA-treated cells ± SD, n = 3, ***p=0.0006. (B) In vitro autoubiquitination assay of native GID particles isolated via the stably expressed HSS-tagged RING protein Rmnd5a from HEK-293 control or MAEA-KO cells in the presence of Ube2H. Deletion of MAEA results in complete loss of the catalytic activity of the GID complex as shown by immunoblotting using an antibody directed against Ubiquitin. (C) Cell proliferation of RPE MAEA-KO and WDR26-KO cells was measured by MTT assays between days 6–9 (naïve) and 15–17 (adapted) after lentiviral transfection. Data are shown as mean of triplicates and % change in signal relative to control gRNA-treated cells ± SD, n ==2, **p=0.0016, ***p=0.0007. (D) Cell extracts prepared from naïve and adapted RPE MAEA-KO and WDR26-KO cells were analyzed for cell cycle and growth pathway markers by immunoblotting. Note that the adapted GID-KO cells overcome the proliferation defect as judged by the presence of phosphorylated Histone H3, Rb (S780 and S807/811) and restored levels of the cell cycle markers Cyclin A and Cyclin D1. (E) Untreated (naïve) control and MAEA-KO RPE cells express comparable FBP1 protein levels in contrast to the autoregulated and stabilized GID subunit YPEL5. (F and G) The murine GID complex is dispensable for degradation of the gluconeogenic enzymes FBP1 and PCK1 in HEK-293 and primary murine hepatocytes. Cells were starved after control and GID siRNA treatment before switching them back to glycolytic conditions. Samples for immunoblot analysis were taken at the indicated time points and probed for FBP1 and PCK1 protein levels (n = 2). The metabolic switch in these cells was controlled by phosphorylation of Akt (Ser473) and the efficiency of GID complex depletion was probed using an antibody directed against the subunit RanBP9 of the complex.
Fig 3: Categorization of FBP1 missense mutations based on genotype-functional phenotype associations.a Schematic distribution of all previously reported FBP1 missense mutations. Type 1 mutations, which are those without a change in amino acid hydrophobicity, are direct substitutions of key residues within an enzymatically active site. Type 2 mutations are those with a change in hydrophobicity, except for G294V, and Type 3 mutations are those that are not located at pivotal residues in functional motifs. b–d Structure of the FBP1 dimer based on the protein data bank (DOI: 10.2210/pdb1FBP/pdb). Locations of the mutations are shown. b Type 1 mutations include D119N (red), P120L (green), N213K (yellow), and E281K (orange). These mutations are directly located at pivotal residues in functional sites of substrate or metal binding sites. c Type 2 mutations include R158W (salmon), G164D/S (smudge), A177D (light blue), F194S (lemon), G260R (light pink), P284R (teal), and G294E/V (yellow orange). These mutations are likely to be located outside of the important amino acid residues in the functional motif and appear to cluster around the substrate binding pocket. d Type 3 mutations include G207R (white) and V325A (white), and these mutations are structurally distant from the sites associated with the enzyme activity motif and substrate binding pocket. e Binding ability of HSP70 and HSP90 to FBP1 mutant proteins. Type 2 mutants showed increased HSP70 and HSP90 interactions compared to WT FBP1 and Type 1 and Type 3 mutants. f Scatterplot of the signal intensity ratios of HSP70 (upper) or HSP90 (lower) binding ability (mutant/WT) and the number of cells with FBP1 aggregates (%). Both HSP70 and HSP90 binding ability were significantly correlated with FBP1 aggregates.
Fig 4: Characterization of the enzyme activity and protein expression of all previously reported FBP1 missense mutations.a Schematic distribution of all previously reported FBP1 missense mutations. Substrate binding site, metal binding site, and AMP binding sites are shown according to the NCBI and UniProt databases. b FBPase enzyme activity of all FBP1 missense mutants. All these mutants, except for G207R and V325A (NS: not significant), exhibited a loss in enzymatic activity (**P < 0.01 versus WT) (one-way ANOVA test followed by Dunnett’s multiple comparison test). The data are presented as the mean ± SD; n = 6–12. c, d Protein expression of all FBP1 missense mutants in FBP1-KO HepG2 cells. All the mutants that did not change their hydrophobicity, except for G294V, exhibited sufficient protein expression compared with that of WT FBP1, whereas all the mutants that changed their hydrophobicity, except for G207R, exhibited decreased protein expression. The data are presented as the mean ± SD. **P < 0.01 versus WT (one-way ANOVA test followed by Dunnett’s multiple comparison test) (n = 3–4). Vertically stacked strips of bands in a figure were evaluated in the same experimental conditions respectively while they were not in fact all derived from the same gel.
Fig 5: FBP1 mutations identified in an adult patient with severe hypoglycemic acidosis and its clinico-endocrinological profiles.Panels a and b demonstrate the results of the oral fructose tolerance test. Oral fructose loading resulted in hypoglycemia accompanied by increased lactate levels. In addition, the 3-hydroxybutyrate levels mildly increased, whereas the free fatty acid (FFA) levels exhibited a robust increase. The patient and a healthy volunteer are shown in red and black, respectively. Panels c–e show the results of the genetic analyses of the FBP1 gene. c Whole-exome sequencing results of the patient and her family. IGV browser visualization of the whole-exome sequencing results revealed a novel compound heterozygous missense mutation in the FBP1 gene: c.491G > A p.G164D and c.581T > C p.F194S. The patient inherited the G164D and F194S mutations from her father and mother, respectively. d Compound heterozygous missense mutations in the FBP1 gene, G164D and F194S, were identified in the patient and validated using Sanger sequence analysis. e Pedigree and genotypes of the family. Solid symbols indicate the G164D allele, and half-solid symbols indicate the F194S allele. Squares denote males, and circles denote females. The arrow indicates the patient. f The FBPase enzyme activity was markedly decreased for the G164D, F194S, and cotransfected constructs compared to that for the WT construct. The data are shown as the mean ± SD. *P < 0.05; **P < 0.01 versus WT (one-way ANOVA test followed by Dunnett’s multiple comparison test). Mock: n = 4, WT: n = 9, G164D: n = 5, F164S: n = 5, G164D + F194S: n = 3. g Structure of the FBP1 dimer based on the protein data bank (DOI: 10.2210/pdb1FBP/pdb) is shown. Cyan sphere: substrate binding site; magenta sphere: metal binding site; blue sphere: AMP binding site. G164D and F194S are shown as a smudge and in lemon color, respectively.
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