Fig. 7: The structure–function relationships in CtBP2 reveals critical coupling of metabolite-sensing with transcriptional regulation.
a Either wild-type CtBP2 (WT), CtBP2 mutated in its Rossmann fold (mut) or a control gene (GUS) was expressed in the liver of diet-induced obese (HFD) mice by adenoviral transduction and the expression of metabolic genes was analyzed (n = 7, biologically independent animals, 6-h fasted, p values for GUS vs WT/WT vs mut are follows: 8.6 × 10−4/5.4 × 10−4 (G6pc), 1.1 × 10−3/0.048 (Scd1), 4.0 × 10−3/3.3 × 10−3 (Fasn), and 2.9 × 10−3/8.2 × 10−4 (Srebf1c), respectively). b Heatmap showing gene expression levels examined by RNA-seq. c–e Gene ontology analysis of RNA-seq data. Biological process (c), cellular component (d), molecular function (e). f Three-dimensional principal component analysis. Pink: GUS, blue: wild-type CtBP2, green: mutant CtBP2. g The CtBP2/FoxO1 complex formation was analyzed using purified recombinant proteins in vitro. Increasing concentrations of oleoyl-CoA (0, 15, 50 μM) with indicated concentrations of NADH (0 or 10 μM) were included. The p values are as follows: 9.0 × 10−3 for control vs NADH+/oleoyl-CoA 0 μM, 0.021 for NADH+/oleoyl-CoA 0 μM vs NADH+/oleoyl-CoA 15 μM, 0.035 for NADH+/oleoyl-CoA 0 μM vs NADH+/oleoyl-CoA 50 μM. h Computer-assisted structural analysis of CtBP2 with NADH and palmitoyl-CoA. For comparison, images of NADH and palmitoyl-CoA were overlaid. i The effect of acetyl-CoA on CtBP2/FoxO1 interaction using recombinant proteins in the same setting as in g. The p values are as follows: 0.026 for control vs NADH+/acetyl-CoA 0 μM, 0.81 for NADH+/acetyl-CoA 0 μM vs NADH+/acetyl-CoA 15 μM, 0.048 for NADH+/acetyl-CoA 0 μM vs NADH+/acetyl-CoA 50 μM. Quantification of the recombinant protein interactions represented in Fig. 7g, i is shown to the right of each blot. Experiments were repeated three times for each. j Strucural modeling of CtBP2 complexed with palmitoyl-CoA by the first-principles calculations FMO. Part of the acyl-chain moiety is depicted in green. Data are expressed as the mean ± SEM. *, **, and NS denote p < 0.05, p < 0.01, and non-statistical significance evaluated by unpaired two-tailed Student’s t-test, respectively. Source Data are provided as a Source Data file.
