Methodology Application Of Mouse Liver Phosphoteome And HBx Involved In The Development Of HCC Through Regulating PDK1

Protein phosphorylation is an important post-translational modification that controls a wide range of protein functions including intracellular signal transduction、apoptosis、muscle contraction、metabolism and other biological processes, but due to the low stoichiometry of protein phosphorylation in cells and limitations ofexperimental techniques, many of phosphorylation events has not yet been found. Numerous developments of mass spectrometry techniques have been make, combined with enrichment procedures makes it possible to identify a large scale of phosphoprotein, combined with bioinformatics analysis, comprehensive information of phosphorylated protein is achieved, provide a powerful platform for the study of molecular mechanisms of cell biology activities、mechanisms of disease and various of cellular process. Phosphoproteomics is the subject of identification and separation of phosphoprotein through a high-through put mass spectrometry and data analysis, to detect disease-associated diagnostic markers and therapeutic target, the dynamic change of protein phosphorylation site has very important significance.Protein phosphorylation plays a major role in a wide range of important cellular functions, many diseases are known to have aberrant phosphotylation. phosphorylation-based network is formed by protein kinase、phosphate-binding protein、protein phosphatase. Protein kinase is an important regulator, Many diseasesincluding cancer are known to have aberrant activation of kinase signalling pathways that impart significant changes on the dynamic regulation of protein phosphorylation. Detailed information on the nature of the targetsubstrates to determine site-specific changes in phosphorylation,variation in protein abundance, and the impact on interacting partnersand their associated functions are required to understand thecomplex regulation of protein phosphorylation in human diseases.HBV is double-strand circular DNA virus, there are four known genes encoded by the genome, called C, X, P, and S. The core protein is coded for by gene C(HBc Ag), the DNA polymerase is encoded by gene P, gene S is the gene that codes for the surface antigen(HBs Ag), gene X is the gene which coded for the HBx protein. HBx protein,a 154-amino acid polypeptide with a molecular weight of 17 k Da, is highly conserved among all mammalian hepadnaviruses.Hepatocellular carcinoma is the most common malignant tumors, the second largest cancer mortality, serious threat to people’s health. In our country 80% of hepatocellular carcinoma associated with HBV infection, HBx conservative presence in the viral DNA, and some studies suggest that HBx RNA and protein expression in human HCC tumorous cells in the absence of HBV replication. The function of the protein HBx is associated with the development of liver cancer. HBx protein is a multifunctional regulator that modulatestranscriptional activation、epigenetic、apoptosis、DNA repair,promote cell proliferation、metastasis and invasion, play a important role in the development and progression of HCC, so HBx is considered to be an importantregulatory proteins in HCC.In the first chaptor, we constructsa method of mouse liver phosphoproteomics, phosphorylated kinases were analyzed to provide valuable information for integrating mouse kinase phosphorylation regulatory networks. Our study constructed a method to identify phosphoproteome from mouse liver.First of all, liver protein was digested with trypsin, then the resulting peptides were subjected to a two-step phosphopeptide enrichment and separation procedure consisting of Ti O2 chromaphy enrichment combined with high p H HPLCseparation. Samples were injected onto a Nano LC-Ultra-2Dplus system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument. Then data analysis were performed to provide information of new identified phosphorylation sites of kinase. Using our efficiencyand high-throughoutplatform, we report the identification of5386 phosophorylation sites and 4553 phosopho-peptide from 1533 proteins ofmouse liver.Identified 126 new phosphorylation site from 116 kinase, which provide valuable information for the phosphorylation networks in mouse liver.During the last decade quantitative phosphoproteomicshave evolved from a highly specialized area to a powerful and versatile platform for analyzing proteinphosphorylation at a system-wide scale and has become the intuitive strategy for comprehensive characterizationof signaling networks.SILAC-based differential proteomics in phosphorylation is widely used in the analysis of cancer and the adjacent tissues, potential biomarkers and drug targets for cancer are obtained by data analysis.In the second chapter, HBx transgenic mouse liver and normal mouse liver wereused for large-scale high-throughput proteomic analysis of differences in phosphorylation, differential proteomics data are obtained, and further data digging and analysis are performed.The use of HBx gene insertion of 24 months and 24 months of wild type mice liver cancer model of phosphorylated proteins group differences between mouse model, we found that the function of PDK1 and WNK1 two kinase.In HBx group identification to the data stream PDK1 and WNK1 phosphorylation level increases obviously, so we focus on the relationship between the two kinases and HBx, and build the HBx overexpressing cell linesto further the instantaneous expression and stable expression cell lines to HBx for regulation of PDK1 found in HBx over expression of cell line, p-PDK1 obvious increase, at the same time, the phosphorylation of p-WNK1 raised obviously.According to the existing research, we know that PDK1 involved in regulating cell proliferation, when we use PI3 K and PDK1 kinase inhibitor processing cell, cell proliferation can be effectively suppressed, in HBx stable expressing cells, however, PI3 K and PDK1 will weaken the effect of the inhibitor, and p-PDK1 and p-WNK1 there will be a lower dose dependence, so can conclude that HBx can effectively restrain PI3 K and PDK1 inhibitorinhibit the proliferation, and HBx is likely to be through PDK1/WNK1 this pathway to give play to the role of inhibition of proliferation.In HBV related HCC clinical samples, we have our hypothesis for further verification, found in HBV positive HCC, compared to the tissue adjacent to carcinoma p-PDK1 increases significantly.