Fig. 3.

Butyryl-CoA binds to CPT1A to antagonize malonyl-CoA mediated repression. (A) Functional comparison between butyrate (But) and butyryl-CoA (BCoA) for the regulation of CPT1A activity. (Left) Mitochondria or (Right) cell lysates from iTreg cells cultured as described in Fig. 1A for 24 h were incubated with CPT1 detection buffer containing EDTA, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and palmitoyl-CoA in the presence of malnoyl-CoA (MCoA) (80 μM), But (125 μM), or BCoA (125 μM) for 5 min prior to CPT1A activity analysis. (B) Etomoxir (Eto) blocked BCoA-mediated up-regulation in CPT1A activity. In the presence of BCoA (125 μM) or/and Eto (5 μM), naive CD4⁺ T cells were cultured under iTreg-polarization condition as described in Fig. 1A for 24 h prior to the analysis of CPT1A activity. (C) Effect of BCoA on MCoA-mediated CPT1A inhibition. Recombinant CPT1A protein was incubated with MCoA (80 μM) and indicated concentrations of BCoA at 30 °C for 5 min. CPT1A activity was tested using CPT1 Activity Assay Kit. (D) Effect of cerulenin (Cer) on iTreg differentiation. In the presence of Cer (4 μM) or/and BCoA (125 μM), naive CD4⁺ T cells were cultured under iTreg-polarization condition as described in Fig. 1A for 72 h. iTreg cells positive for CD4, CD25, and Foxp3 were analyzed by flow cytometry (Left) and quantified accordingly (Right). (E–G) Assay for BCoA- and MCoA-binding to CPT1A protein. Recombinant (E and F) CPT1A^WT or (G) CPT1A^R243A were loaded onto the Ni-NTA biosensors in the biolayer interferometry analysis and incubated with BCoA or MCoA to detect their association. Data represent mean ± SD (n = 3) of three independent experiments, with significance determined by one-way ANOVA test. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant.