Study On The Protein Expression And Molecular Mechanism Of G72 Regulation Oxidative Stress

G72 is a primate-specific gene encoding a protein with 153 amino acids. G72 can interact with D amino acid oxidase (DAO) which is a flavo-enzyme to metabolite D-amino acids, especially brain D-serine, for regulating synaptic neurotransmission. The synaptic D-serine activates N-methyl-D-aspartate (NMDA) receptors in neurotransmissions. In neurological or psychiatric diseases, the alteration of D-serine concentration can make NMDA receptors hyper-or hypo-function. This study aims to provide evidences to describe the reduction of synapstic D-serine may result from the DAO activity inhibited by G72. The plasmids, pMAL-C2G-DAO, pET23a-DAO, pGEX-6p-l-G72 and pET23a-G72 were constructed. After protein expression and purification, we found that MBP tag would reduce the DAO activity, and the purified GST-G72 would be cleaved automatically. Therefore, DAO-6H and the refolded G72-6H were used in this study. On the other hand, three point mutants of G72 at the 30th amino acid residue from Arg to positively charged Lys, to negatively charged Glu and to uncharged Ala were generated. To analyze G72 functions, G72-6H variants were mixed with DAO, and the DAO enzymatic activity was measured. With the increasing concentrations of G72-6H, G72R30A-6H and G72R30k-6H, the DAO activity were inhibited. However G72R30d-6H did not inhibit DAO activity even at the molar ratio (G72:DAO) up to 16. Here we provide evidences that the 30th residue of G72, Arginine, may play a crucial role in DAO-G72 interaction, and this result may explain part of the studies which indicated higher proportion of G72R30k (rs2391191) found in patients with mental disorders.Further application of gene chip for screening, looking for G72 increased oxidative stress gene expression changes. A large number of positive results were obtained, which indicated that G72 had a broad and important role in the oxidative stress. G72 transfected cells and the control group showed a significant difference between the expression of anti oxidative stress gene expression. The upregulation of the antioxidant gene expression may be an adaptive compensation of G72 transfected cells to the elevated ROS level. This study found that G72 protein will be combined with the normal wild-type SOD1 protein in glioma U87 cell line over expression of G72 protein will induce the production of ROS, and further confirmed that G72 is involved in the regulation of oxidative stress. Glioma U87 cell line overexpression G72 protein induced HADHB (mitochondrial membrane protein), p21, p53, STAT1, AMPKr-1, Cytochrom C, p38 alpha and MAPK expression levels are rising, suggesting that over expression G72 protein may through autophagy, cell cycle arrest, and cell apoptosis of three kinds of path cell damage; G72 gene into the ALS model mice (SOD1 G93A) cerebral motor cortex, found that mice decreased exercise capacity and shorten life, prompt G72 protein may will destroy the nerve cells in the mice caused a decline in exercise capacity. This study with the sod mimetic Tempol treatment G72 gene transfected cells, in order to improve the status of oxidative stress in cells, application of the antioxidant can effectively reduce the ROS level of G72 gene transfected cells. The results show that oxidative stress is the key effect of G72 gene transfected cells, G72 induced by ROS, promote the occurrence of oxidative stress. Some studies have showed that the G72 vector was transfected into animals, the increase in ROS caused by excessive G72 induced oxidative stress, indicating that G72 is likely to be an important factor in the oxidative stress. Therefore, we evaluated the level of oxidative stress in G72 transfected cells. The first is to detect the ROS level of G72 transfected cells, and ROS was found to be significantly increased in G72 transfected cells, suggesting that the oxidative stress status. We also analyzed the activity of anti oxidative enzyme SOD from the point of body clearance of ROS, and decreased the viability of G72 transfected SOD cells. The results showed that G72 transfected cells were in oxidative stress state, which suffered from oxidative stress injury, and the antioxidant capacity was affected by some degree of damage.To sum up, the over expression of G72 will form complexes with SOD1 protein in cytoplasm, resulting in SOD1 accumulation in the mitochondria, the SOD1 accumulation resulting in decreased protein activity and cannot effectively remove superoxide, oxidative stress increased, eventually leading to neuronal injury or death.