Journal: Experimental & Molecular Medicine
Article Title: Targeting COL6A3-C5 with nigericin suppresses endotrophin formation and enhances insulin sensitivity in obesity
doi: 10.1038/s12276-026-01661-y
Figure Lengend Snippet: a Validation of the interaction between MMP9 and C5 domain of COL6A3 with various concentrations of NGC. HEK293T cells were transiently transfected with MMP9 and COL6A3-C5, and incubated with various concentrations of NGC. b Validation of COL6A3-C5 interaction with MMP9 with or without NGC by using SPR analysis. The recombinant COL6A3-C5 proteins were immobilized on the surface of the CM5 chip and then flowed over the MMP9 with NGC ranging from 0.001 to 1 µM. SPR binding curves for NGC and MMP9, binding to the captured COL6A3-C5 and fit to a 1:1 binding model. The half-maximal inhibitory concentration (IC 50 ) value of NGC was approximately 0.08 µM. c , d Quantitative SPR measurements of immobilized either COL6A3-C5 ( c ) or MMP9 ( d ) interacting with NGC. e Molecular docking model illustrates the NGC and COL6A3-C5 binding interface. NGC adopts a stable binding pose within the COL6A3-C5 pocket (CDocker energy −8.5 kcal/mol, interaction energy −20.45 kcal/mol). The docking analysis identified R3122, D3123, F3124 and W3141 of COL6A3-C5 as key residues directly interacting with NGC. R3122 forms a carbon–hydrogen bond (2.98 Å) with the oxygen atom of the C-11 methoxy group of NGC, while D3123 establishes a conventional hydrogen bond (2.16 Å) with the hydroxyl group adjacent to the C-3 carbonyl. F3124 engages in both a carbon–hydrogen bond with the C-3 carbonyl oxygen (2.75 Å) and a π–alkyl interaction with the C-4 methyl group (4.40 Å). Similarly, W3141 forms a π–alkyl interaction (4.87 Å) with the C-4 methyl group. f NGC disrupts the structural stability of the MMP9–C5 complex. MD of the MMP9–C5 complex, performed with or without NGC, showed a stable conformation in the absence of NGC (RMSD 2.27 Å), whereas the presence of NGC markedly disrupted the binding interface (RMSD 47.99 Å). g Insulin responsiveness. The 3T3-L1 adipocytes were treated with CoCl 2 (150 mM) for 48 h, followed by NGC for 16 h, before stimulation with insulin (25 nM) for 15 min. The levels of insulin-stimulated AKT phosphorylation were determined using immunoblotting. GAPDH was used as a loading control. h , i Insulin responsiveness of NGC in genetic depletion or supplementation of COL6A3/ETP. h , i A3KO ( h ) and ETP-reconstituted A3KO ( i ) adipocytes were treated with CoCl₂ (150 μM) for 48 h, followed by NGC for 16 h, and then stimulated with insulin (25 nM) for 15 min. Insulin-stimulated AKT phosphorylation was assessed by immunoblotting, with GAPDH serving as a loading control. The recombinant mouseETP (100ng/ml) was added to A3KO adipocytes to restore ETP levels. j , k The effect of NGC on inflammasome activation was assessed. The 3T3-L1 adipocytes ( j ) and peritoneal macrophages ( k ) were primed with LPS (1 µg/ml) or TNFα (50 ng/ml) for 6 h, followed by treatment with NGC. After 16 h, IL-1β concentrations in the culture supernatants were measured using enzyme-linked immunosorbent assay.
Article Snippet: Molecular docking between the C5 domain of COL6A3 and the FN II domain of MMP9 was performed using the CDOCKER and ZDOCK modules implemented in BIOVIA Discovery Studio 4.0 (Accelrys).
Techniques: Biomarker Discovery, Transfection, Incubation, Recombinant, Binding Assay, Concentration Assay, Phospho-proteomics, Western Blot, Control, Activation Assay, Enzyme-linked Immunosorbent Assay
