Design

Our research aims to investigate the binding interactions of vitamin B6 and B12 with the PLP-1 protein using molecular docking techniques (Agu et al., 2023). We divided the project into three modules: the protein structure modeling module, the small molecule preparation module, and the docking analysis module. We utilized AlphaFold3 to model the target protein PLP-1 and employed Maestro computational software to optimize the structures of the protein and vitamins B6 and B12. The predicted protein structure and small molecules were optimized to ensure stability and minimize energy, significantly increasing the likelihood and accuracy of successful docking. Subsequently, we employed a semi-flexible docking method to analyze the binding interactions. We expect to identify the optimal binding modes of vitamin B6 and B12 with PLP-1, contributing to our understanding of their anti-inflammatory effects.

BUILD

1.Pheochromacytoma (PC12) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin(Kiernan et al., 2016). The cells were maintained in a humidified incubator at 37℃ with 5% CO2. The growth medium was replaced daily, and the cells were passaged every two days to ensure optimal growth conditions. To establish a cell injury model, the normal culture medium was replaced with a sugar-free medium (2 mL per well in a 6-well plate) and placed in a sealed anaerobic glass container for 2 hours. This anaerobic treatment simulates conditions that induce cellular stress and damage. Following this incubation, the cells were switched back to the normal culture medium to assess recovery. Concurrently, vitamin B6, vitamin B12, and their combinations were added in varying ratios: 1:1 (group 1), 1:10 (group 2), 10:1 (group 3), and 20:1 (group 4). The vitamins were allowed to act for a duration of 24 hours.

2. For the overexpression of PLP1, a membrane protein, we can select either a mammalian or insect cell expression system, depending on the desired post-translational modifications and protein yield (Ikuo et al., 1998). The plasmid designed for this purpose incorporates essential elements for effective expression. The design includes a strong promoter to drive high levels of transcription, a suitable multiple cloning site for the insertion of the PLP1 gene, and a selection marker to facilitate the identification of successfully transformed cells. The PLP1 gene is linked to the expression vector, ensuring that the protein is produced in sufficient quantities for subsequent analysis. The canonical sequence of PLP1, identified as P60201-1, serves as a reference for the design, ensuring that all positional information corresponds to this isoform. This sequence is also provided in downloadable formats for further research. The successful construction of this plasmid will enable the study of PLP1's functional roles and its potential implications in cellular processes.

3.Introduce the overexpression vector into expression-deficient cells for the purpose of interaction validation or to apply stress treatment to the overexpressing cells.

TEST

1. Through Western blot analysis, we successfully validated the interaction between vitamins B6 and B12 with the PLP-1 protein. The results indicated that the combinations of vitamins B6 and B12 at ratios of 10:1 and 20:1 exhibited the most potent anti-inflammatory effects, characterized by the up-regulation of PLP-1 and PGC-1α proteins, alongside the down-regulation of TNF-α protein.

2. Conduct morphological, protein, and molecular level assessments on cells successfully transfected with the plasmid to confirm the successful construction of the vector and the functionality of the protein pathway.

Learn

mins B6 and B12 and the PLP-1 protein. This pathway exhibits a nourishing effect on neurons and vestibular functional areas (Baltrusch, 2021), manifested as the alleviation of neuronal edema symptoms, thereby mitigating damage to key functional regions, including vision, hearing, and taste. 2. We found that combinations of vitamins B6 and B12 at ratios of 10:1 and 20:1 significantly upregulated the expression of PLP-1 and PGC-1α proteins while downregulating TNF-α protein. Based on these characteristics, we formulated a soothing nasal suction complex containing PLP-1 protein and vitamins.

Reference