Regulation Of Prx1Phosphorylation By Prak Under Oxidative Sterss

Oxidative stress points to the process that when the body suffered from various harmful stimulus, the production of free radical and antioxidant defense became imbalance, leading to a cluster of reactive oxygen species in the body or within cells and cause cell toxicity, leading to the process of tissue damage. Oxidative stress is the most important reason of cell damage,leading to the occurrence and development of many diseases, Including cardiovascular disease, autoimmune diseases, neurodegenerative diseases, cancer,aging and so on. In the appropriate concentration,ROS is critical to the cellular immune response,such as in the mitosis response process, in the function of a number of cellular signaling systems and in the defence against infectious agents.But in some pathological conditions, the production of large Numbers of free radicals, and at the same time the antioxidant defense ability fell, the oxidation ability is far more than oxidation resistance,which initiates oxidative stress,leading to the peroxidation of biological membrane lipid, the degeneration of cellular proteins and enzymes, the damage of DNA, apoptosis or tissue damage, which eventually led to the happening of the disease.When ROS levels exceed the antioxidant capacity of cells, the body will start oxidative response through a series of antioxidant mechanisms to maintain the dynamic balance of oxidation-reduction, such as the production of large Numbers of antioxidant proteins and repair proteins. This redox dynamic equilibrium have a great relationship with the intracellular antioxidant enzymes.Peroxidase is a is widely expressed antioxidant enzymes in biology, through catalyzing H2O2and lipid hydrogen peroxide reduction, protect cells from further damage in the oxidative stress process.According to the number and location of the cysteine participated in catalytic reaction, prxs can be divided into three major subclasses:typical2-cysteine (2-Cys) Prxs (Prxl-4), atypical2-Cys Prdx (Prdx5) and1-Cys Prdx (Prdx6). Prxl is a typical2-Cys Prxs, because there are two redox-active cysteine residues in its structure.In the catalytic reaction process, the peroxidatic cysteine (Cys-SH) residues attack the oxide substrate and restore itself oxidized to cysteine sulfonic acid (Cys-SOH), and thenPrxl react with the resolving cysteine(Cys-SH) in another Prxl molecule and formate intermolecular disulfide bonds. At this time,the reduction system consisted of thioredoxin (Thioredoxin, Trx), thioredoxin reductase (Thioredoxin reductase, TrxR) and NADPH can re-reduction of Prx1, and efficiently catalyze peroxide reduction,which plays an important role in the oxidative stress.A large number of studies have confirmed that Prx1not only exists as a dimer, playing the role of peroxidase, can also forms a decame, acting as a molecular chaperone.This structural change gives the diversity of Prxl function,and participates in the regulation of a variety of cellular signal transduction pathways associated with oxidative.Prxl has a close relationship with a variety of signaling pathways,such as H2O2-mediated signaling pathway, apoptosis signal-regulated kinase1(ASK1)-mediated signaling pathways, PTEN/AKT-mediated signaling pathways, c-Jun N-terminal kinase (JNK)-mediated signaling pathway, androgen receptor (AR)-mediated signaling pathway, p38/JNK stress activated protein kinase (SAPK)-mediated signaling pathways. Therefore, the study of Prxl function not only helps us to fully understand its function,but also has a great significance for the diagnosis and treatment of a variety of related diseases.We know that under stress conditions, a protein exerting its biological activity, is inseparable with the interaction between the protein and protein and protein modification.In other words, the functional activity of a protein has a direct relationship with the signaling pathway. In the process of oxidative stress, how does the oxidation signal transfer to Prx1?Studies found that the mitogen-activated protein kinase signal transduction pathway plays an important role in maintaining normal physiological function of cells, such as growth, development, differentiation, and apoptosis. A series of studies have shown that in eukaryotic cells, this pathway also plays a key role in the cellular stress response. Studies have shown that a low dose of ROS produced in moderate stress can introduce the production of antioxidant enzymes, through activating of MAPK signaling kinase,thereby enhancing the scavenging ability of oxygen free radicals, preventing cells from further injury.p38MAPK can be activated by a variety of stimulation, such as ROS, UV radiation, heat shock, cytokines, etc.. Activated p38MAPK can regulate the transcription of genes involved in cellular response through phosphorylation of different substrate, for example, on the one hand by activating transcription factor2,myocyte enhancer factor2C and C/EBP homologous protein, on the other hand can also activate intracellular protein kinases such as p38regulated/activated protein kinase, MAPK-activated protein kinase2/3, MAPK-interacting kinase1/2etc. to participate in many important biological processes, including cell signal transduction, cytoskeleton remodeling, stress and growth factor-induced gene expression.In our laboratory,we have much studies on p38MAPK downstream protein p38regulated/activated protein kinase(PRAK).We found that the apoptosis rate of PRAK-/-cells was significantly higher than PRAK+/+cells when the cells were stimulated by exogenous hydrogen peroxide. This shows that PRAK protein may play an important role in cells when subjected to oxidative stress, but the specific mechanism is not yet clear.At the same time, studies have shown that when the environmental stress or pro-inflammatory response occurs,PRAK is activated to play a role in cells. Stimulating PRAK+/+and PRAK-/-cells with strong oxidants arsenite sodium, enrichment of phosphorylated proteins, doing2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry found that in PRAK+/+cells, the phosphorylation level of Prxl was significantly elevated,but in PRAK-/-cells had no obvious changes.Studies found that the peroxidase activity of Prxl has a close relationship with its phosphorylation. In view of PRAK is located in the downstream of the MAPK signaling pathway, we assume that PRAK may be involved in cellular oxidative stress response through regulating the phosphoryIation level and peroxidase activity of Prxl. Therefore,this study was designed to reveal the influence of PRAK on the phosphorylation levels of Prxl in the process of cellular oxidative stress.The enzyme activity of Prxl can be measured by Trx reduction system,which is consisted of thioredoxin, thioredoxin reductase and NADPH.The life activities of organisms is closely related to proteins’dynamic changes. In many cases, the absolute amount of certain proteins is not changed significantly,but they fulfill their biological functions through a variety of post-translational modification.Protein phosphorylation is one of a very important post-translational modification which most frequently occurs in serine, threonine and tyrosine sites,causing changes in the nature of protein, changing the position of the protein, enhancing the targeted degradation of proteins, or influencing the affinity capacity between the protein and protein, protein and nucleic acid. These changes will eventually lead to cascade amplifier in many biological processes, participating in a variety of biological processes.The current research on protein phosphorylation is most based on electrophoresis technology, detected by Western blot using anti-phosphorylated antibody to detecting the results of Western blot. This method has the advantages of high resolution and specificity. However, for unknown proteins, we don’t know the specific phosphorylation sites, it is difficult to use the method because the antibodies are not specific. The emergence of Fluorescent two di-mensional difference gel electrophoresis(2D-DIGE) technology provides a powerful tool for proteomics.Protein phosphorylation might cause the isoelectric point changes, in the two-dimensional gel,the points will migrate, presenting a series of homologous proteins.Digging the phosphorylated migration protein points,using a mass spectrometry combined with tandem mass spectrometry,then we can determine the phosphorylation sites.Based on the researches above, we have some conclusion:1. Successfully constructed the recombinant plasmid of pET14b-Prxl for prokaryotic expression and purification of His-Prx1protein.2. After the in vitro kinase reaction, we detected the phosphorylation levels of Prxl utilizing western blot. No significant phosphorylation of PRAK on Prxl serine, threonine or tyrosine residues was found. And then detecting the phosphorylation levels of Prxl by2D-DIGE technology, we didn’t found obvious different protein spots, suggesting that there may be no direct phosphorylation regulation relationship between PRAK and Prxl.3. Cultured PRAK-/-, PRAK+/+cells, treated with NaAsO2for45min and lysised cells to obtain proteins,we found that no significant phosphorylation of PRAK on Prx1was tested; There was a certain level of phosphorylation on Prxl serine sites in PRAK-/-cells, but the level was lower than that in PRAK+/+cells, Suggesting that the presence of PRAK promots the phosphorylation of Prxl on serine sites, but whether this effection was impacted by oxidative stress was not clear. No significant phosphorylation of Prxl on tyrosine sites in PRAK-/-cells was found, but there was significant phosphorylation in PRAK+/+cells, and the level of phosphorylation was enhanced after oxide stimulation. Suggesting that the presence of PRAK also affected the phosphorylation level of Prxl tyrosine residues,which was associated with oxidation.4. Extracted of yeast cell genome, and successfully constructed pET14b-yTrxl and pET14b-yTrxR recombinant plasmid and prokaryotic expression and purification of His-yTrxl and His-yTrxR protein.5. Optimized the reaction conditions of the yTrx activity detection system, and detected the peroxidase activity of Prxl to proving that we successfully established the activity detection system.6. Using yTrx activity detection system, we found that the peroxidase activity of Prx1in PRAK+/+cells was stronger than that in PRAK-/-cells, and when the cells was stimulated by NaAsO2, the activity of Prxl was decreased. Suggesting that PRAK may regulate the peroxidase activity of Prxl through regulating the phosphorylation levels of Prx1, which resulting the instantaneous accumulation of H2O2and other second messenger molecules, launching its downstream cell signaling pathways, and thus involved in cellular oxidative stress response.