Fig. 3: AA005 targets the mitochondrial trifunctional enzyme α subunit (HADHA).
a Preadipocyte 3T3-L1 cells were induced to undergo adipogenesis and concomitantly treated with different concentrations of AA005 on day 8. The cells were stained with Oil-Red-O. Scale bar: 10 μm. Three different biological replicates were tested. b Workflow illustrating the use of biotin-tagged AA005. c On day 8 after induction of adipogenesis concomitant treatment with biotin or biotin-AA005, 3T3-L1 cells were stained with Oil-Red-O. Scale bar, 10 μm. Three different biological replicates were tested. d Immunofluorescence imaging showing the localization of biotin-AA005 and HADHA in cells. Mitotracker was used to label mitochondria, and DAPI was used to label nuclei. Three different biological replicates were tested. Scale bar, 10 μm. e Schematic representation of the discovery of interacting proteins of AA005 using chemical proteomics. f Mitochondrial fractions of 3T3-L1 cells were incubated with biotin or biotin-AA005 in the presence or absence of a 5- or 10-fold excess of unlabeled AA005. The proteins were then pulled down with streptavidin-agarose, and western blotting was performed to detect HADHA. Three different biological replicates were tested. g Structural overview of the predicted HADHA–AA005 complex model. The docking result was performed by Swiss-docking method. The protein is shown in cartoon, and AA005 is shown in a stick representation. Residues around the predicted binding pocket were highlighted with a red rectangle. On the right is the zoomed-in view of the predicted HADHA–AA005 interface. Potential key interface residues (V174, L175, L202, T203, R205, T294, Q293, E500, K505, M506, P581, V582, E590, G708, G709, and L708) in HADHA were colored in green and labeled. h Microscale thermophoresis (MST) binding assay determined the Kd value (WT, Kd = 1.1 μΜ; Mut-V174, Kd = 9.6 μΜ; L175, Kd = 20 μΜ; L202, Kd = 6.1 μΜ) for the binding of AA005 toward wild and mutant HADHA. Three different biological replicates were tested. i Immunoprecipitation analysis of the physical interaction between wild and mutant HADHA and biotin-AA005. Three different biological replicates were tested. j On day 8 after induction of adipogenesis, 3T3-L1 cells with normal HADHA expression (3T3-L1-NC) or cells with Hadha knockdown (3T3-L1-siHadha) were stained with Oil-Red-O. Scale bar: 10 μm. Three different biological replicates were tested. Data are mean ± SEM. Source data are provided as a Source Data file.
