s Protein Phosphatase Mg 2+ /Mn 2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg 2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg 2+ in the active site. Specifically, three Mg 2+ were located in the active site of the PPM1K N94K instead of two Mg 2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.
Background Atherosclerosis (AS) is a chronic inflammatory vascular disease. Macrophage reprogramming plays a crucial role in the progression of AS. Ophiopogonin D (OP-D), a bioactive constituent of Ophiopogon japonicus , has exhibited potent anti-atherosclerotic property. However, whether OP-D exerts its atheroprotective effect via reprogramming macrophage remains unclear. This study aimed to investigate the effect of OP-D on AS and elucidate its underlying mechanism. Methods The atheroprotective effect of OP-D on atherosclerotic mice was evaluated by histological and molecular assays. The crucial role of macrophage in OP-D's atheroprotective effect was detected by flow cytometry and verified through macrophage depletion/infusion. The direct effect of OP-D on macrophages were assessed in vitro by RT-qPCR, flow cytometry, and immunofluorescence. RNA sequencing and amino acid-targeted metabolomics analysis were employed to identify the associated amino acid metabolic pathways. In addition, PPM1K deficient was established to validate the role of branched-chain amino acids (BCAAs) in macrophage reprogramming and OP-D's therapeutic effect. Results Our results demonstrated that OP-D attenuated the progression of AS and directly prevented the M1-like polarization of macrophages. Meanwhile, the therapeutic benefits of OP-D were abolished after macrophages depletion, suggesting macrophage was a crucial mediator of OP-D's therapeutic effect. Further mechanistic studies revealed that dysregulated BCAAs metabolism-mediated macrophage polarization underlay the anti-atherosclerotic effect of OP-D. Conclusion This study elucidated that OP-D reprogrammed the polarization of macrophages through modulating BCAAs catabolism to delay atherogenesis. These findings will deepen the understanding of OP-D's atheroprotective mechanisms and provide a potential therapeutic target for AS.
危险品化学品经营许可证（带存储）