Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

Background: With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK₁) or menadione (VK₃) via the expression ofHomo sapiensUBIAD1 (HsUBIAD1). The methylotrophic yeast,Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered inP. pastoris.Results: Firstly, we constructed a novel synthetic pathway inP. pastorisfor the production of menaquinone-4 (MK-4) via heterologous expression ofHsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (P_GAP) appeared to be mostsuitable for the expression ofHsUBIAD1 for various reasons. By optimizing the expression conditions ofHsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We foundHsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK₁and VK₃as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression ofSaGGPPS andPpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK₃was the isopentenyl acceptor.Conclusions: In this study, we constructed a novel synthetic pathway inP. pastorisfor the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression ofHsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

Glucagon-like peptide-1 (GLP-1) is easily degraded by dipeptidyl peptidase-4 (DPP-4) in the human body, limiting its therapeutic effect on type II diabetes. Therefore, improving GLP-1 receptor agonist (GLP-1RA) stability is a major obstacle for drug development. We analyzed human GLP-1, DPP-4, and GLP-1 receptor structures and designed three GLP-1RAs, which were introduced into fusion protein fragments and changed in the overall conformation. This modification effectively prevented GLP-1RAs from entering the DPP-4 active center without affecting GLP-1RAs’ ability to bind to GLP-1R, the new GLP-1RA hypoglycemic effect lasting for >24 h. Through molecular modeling, molecular dynamics calculation, and simulation, possible tertiary structure models of GLP-1RAs were obtained; molecular docking with DPP-4 and GLP-1R showed access to the fusion protein. The overall conformational change of GLP-1RAs prevented DPP-4 binding, without affecting GLP-1RAs’ affinity to GLP-1R. This study provides important drug design ideas for GLP-1RA development and a new example for application of structural biology-based protein design in drug development.

Nutritional food supplements and pharmaceutical products produced with vitamin K₂ as raw materials a very promising market in the global scope. The main production method of vitamin K₂ is microbial fermentation, but approximately 50% of vitamin K₂ synthesized by the main production strain Bacillus subtilis natto exists in extracellular form, which is not easy to separate and extract. In order to solve this problem, in this study, we synthesized a novel cellulose flocculant, MCC-g-LMA, by grafting reaction using microcrystalline cellulose (MCC) and lauryl methacrylate (LMA) as monomers, and ammonium persulfate as an initiator to flocculate VK₂ from the fermentation supernatant. The flocculant was characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM), and the grafting reaction was successful. When the flocculant dosage was 48.0 mg/L and pH was 5.0, the flocculation rate of the MCC-g-LMA on the fermentation supernatant reached 85.3%, and the enrichment rate of VK₂ reached 90.0%. Furthermore, we explored the flocculation mechanism of VK₂ by the MCC-g-LMA and speculated that the flocculation mechanism mainly included adsorption bridging, hydrophobic association and net trapping and sweep effect. In this study, the extraction method for trace high-value biological products in the fermentation supernatant was improved, which provided a method and theoretical basis for the efficient separation and purification of VK₂ and other terpenoids.