Journal: Advanced Science

Article Title: HMGCR‐Driven Cholesterol Metabolism Promotes Osteoarthritis Progression by Accelerating Synovial Fibroblast Senescence

doi: 10.1002/advs.76498

Figure Lengend Snippet: Phosphorylation of AKT1 at Ser473 enhances its binding to Lys140 of Insig1. (A) The interaction between endogenous AKT1 and Insig1 in NIH‐3T3 cells was demonstrated by co‐immunoprecipitation. (B) The molecular docking mode of the interaction between AKT1 and Insig1. (C) RMSD (up) and RMSF (down) analysis showing the binding of AKT1 and Insig1. Red, black, and blue represent AKT1, Insig1, and the complex, respectively. (D) Molecular mechanics‐Poisson‐Boltzmann surface area (MM‐PBSA) analysis of AKT1–Insig1 and pAKT1 (Ser473+Thr308)–Insig1 complexes. Calculation of energy terms (kcal per mol) from the 100 ns trajectory. Van der Waals Energy (ΔvdW) indicated nonpolar interactions between molecules. Electrostatic Energy (ΔEEL) indicated electrostatic interaction energy between charges. Polar solvation energy (ΔEPB) represents the solvation energy of molecules in polar solvents, calculated by employing the Poisson–Boltzmann equation. Nonpolar Solvation Energy (ΔESA) was typically calculated based on the solvent‐accessible surface area multiplied by an empirical coefficient. Δ G bind (Δ E tot ) represented the overall binding affinity between interacting molecules. (E) FEL of the two complexes, constructed using RMSD and R g as variables, displaying the energy distribution in different conformational states between 0 and 100 ns (left). H‐bonds and hydrophobic interactions by LigPlus illustrating interactions between AKT1 and Insig1 in two different states, which are displayed in green dotted lines and red or pink spikes, respectively. The main panel shows hydrogen bonds and hydrophobic interactions surrounding AKT1 Ser473 and pSer473 with Insig1 (right). (F,G) Co‐IP analysis to assess AKT1–Insig1 interaction in the dTNF‐InSen NIH‐3T3 cells after transfection of Insig1 K140A vector under conditions of AKT1 WT overexpression or AKT1 S473A vector under the condition of Insig1 WT overexpression. (H,I) The expression of γ‐H2AX ( n = 60 per group), Il‐6 ( n = 3 per group), and Mmp13 ( n = 3 per group) in the dTNF‐InSen FLSs after the transfection of AKT1 WT or AKT1 S473A vector under the condition of Insig1 WT overexpression, compared to the controls. All statistical tests were two‐sided. Fisher's exact test (H). Student's t ‐test (I). dTNF‐InSen, induction of senescence by double treatment of TNF‐α; RMSD, protein backbone root mean square deviation; RMSF, root mean square fluctuation; FEL, free energy landscape; R g , radius of gyration.

Article Snippet: The primary antibodies were as follows: HMGCR (Huabo, ET1702‐41), p16 (Abcam, ab189034), p53 (CST, 2524S), p21 (CST, 37543T), AKT1 (CST, 2938S), p‐AKT ser473 (ABclonal, AP0637), Insig1 (Santa, sc‐390504), phosphoserine/threonine (p‐Ser/Thr; BD Biosciences, 612549), SCAP (Proteintech, 12266‐1‐AP), IL‐6 (Proteintech, 26404‐1‐AP), MMP13 (Abcam, ab39012), Myc (CST, 2276), Flag (CST, 14793), β‐actin (Proteintech, 66009‐1‐AP).

Techniques: Phospho-proteomics, Binding Assay, Immunoprecipitation, Solvent, Construct, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Over Expression, Expressing