Studies On The Interaction Between PRDX1and PRAK

Protein is the performer of all the biological life activities. However, in body function the protein could not play role independently, they usually go though many kinds of modifications and form protein complex again with other protein to play roles in the body. When protein complex is in particular time and space, it preforms its own unique function. The progress of protein interaction exist in all life activities, only if the protein interaction smoothly, our normal cell activities can be ensure. Now, based on the chemical, physical and biological, bioinformatics technology cross each other and fusion, make the research of protein interaction present development trend of high throughput, high sensitivity, fast, real-time qualitative and quantitative detection, Reveals the interaction between the proteins and establish the mutual relationship between network diagrams have become a hot spot in the study of proteomics researches. Therefore, the study of protein interactions has the important meaning for us to understand the function of proteins and expounds the regularity of the activities in the life.Our laboratory use the difference gel electrophoresis (DIGE) technology and mass spectrum analysis technology to identify the changes in the phosphorylated proteins using mouse embryo fibroblasts cell PRAK+/+and PRAK-/-stimulated by arsenic acid sodium (NaAsO2)45min, and got32protein points in statistically significant difference, which include Peroxiredoxin1(PRDX1). After the arsenic acid sodium stimulated, phosphorylation level of PRDX1is significantly reduced in PRAK-/-cell.Through the analysis between PRDX1and PRAK, we found that there are some common in both. For example, the proteins are position in the nucleus; both are involved in signal transduction process; and both play a very important role in inflammation stimulation process. Accordingly, we assume that after stimulating by As, p38MAPK signal pathway in cells is activated, and further activate PRAK protein, activated PRAK and PRDX1get interact in some way and make the phosphorylation level of PRDX1protein changes, then the function of PRDX1changes and final effect the physiological function of antioxidant of PRDX1. On the above basis, we establish this study, that is using the related experimental methods to verify the interaction between PRDX1and PRAK, and demonstration the forms they involved in inflammation signal transduction. The results will provide the basis for in-depth understanding the function of PRDX1and PRAK.In order to verify whether PRDX1and PRAK have interaction, we first use Pull Down technology and Co-IP technology to check the relationship between the two proteins in vitro and in vivo, then we choose immunofluorescence technology to observe the position and express conditions of the two proteins. Through the above research, we get the following conclusion:1. With the experiments in vitro, the results show that PRDX1and PRAK do not have specificity interaction when use GST Beads and His Beads.2. With the experiments of Co-immunoprecipitation (Co-IP), the results show that we cannot separate the light chain and weight chain of the antibodies from PRDX1and PRAK protein, suggest we should choose other appropriate methods to test.3. With the experiments of Immunofluorescence experiments, the results show that exogenous PRDX1protein exists in nucleus and cytoplasm and exogenous PRAK protein is mainly distributed in the nucleus.4. With the experiments of immunofluorescence experiments, the results show that PRDX1and PRAK have colocalization in nucleus and cytoplasm.5. With the experiments of immunofluorescence experiments, the results show that, overexpress PRDX1protein can activate PRAK protein shift out nuclear to cytoplasm, this suggest that PRAK protein is activated by PRDX1.6. All experiments show that, PRDX1protein and PRAK protein has the possibility of indirectly interaction.The results of this topic research rich the information of protein interaction and provide a new idea and method for protein interaction research; it also provide a new direction and new therapeutic targets for protein related to diseases.