Research On The Tyrosine Nitration In P53Induced By Peroxynitrite In And Its Activity

p53protein is a kind of tumor suppressor protein that encoded by gene TP53inhuman body. It is very important to physiological process such as cell cycle regulating,DNA repairing, cell differentiation, apoptosis and genetic stability. p53protein canInduce cells to stop dividing and active DNA repair proteins, at the same time, it canInitiate programmed cell death. So it is also called“the guardian of genome”. The lossof function and the damage of structure will cause the loss of its regulatory role to cellcycle, and then lead to unlimited cell division, at last the formation of tumor.Peroxynitrite ion （ONOO^-） is a kind of cytotoxic substance in organisms thatformed by the rapidly bonding of nitric oxide free radical （·NO） and superoxideanion(O₂^·-). Under physiological conditions, ONOO^-can be protonated to beperoxynitrite （ONOOH） to across the cell membrane. The strong oxidation capacityand nitrification ability of it can lead to the damage of the biomacromoleculesincluding protein, lipid and DNA. The nitration of protein tyrosine is a kind of quiteimportant protein post-translational modification process; this process will causeprotein denaturation and a series of cytotoxic effects, then induce diseases. So Thenitration of protein tyrosine has important physiology and pathology effects.In this study, p53protein was regarded as target molecule, ONOO^-was regardedas revulsant. Analytical instruments such as microenvironment electrophoresisapparatus trophoresis, ultraviolet and visible spectrophotometer, high-pressure liquidchromatography （HPLC） were used, and study methods such as Western Blot,molecular dynamics simulation were applied here. Combined with microenvironmentof organisms, the change of protein structure and the damage after protein translationon the level of modification caused by ONOO^-were studied.Firstly, GST-p53fusion protein was prepared through recombinant plasmidprokaryotic expression system of E.coli, and the separation and purification of thefusion protein was did by glutathione gel chromatography column. To prepare humanrecombination p53protein has important application value. Purified GST-p53fusionprotein has laid the foundation of later experiments.Secondly, HPLC-ESI-ITMS was applied to study the reaction process andselectivity of the nitration of p53protein tyrosine mediated by ONOO^-. Through thephysical simulation of nitrate concentration and reaction system, liquidchromatography-mass spectrometry system and the optimization of conditions, Theprotein residues identification fraction of coverage reached85%. Six tyrosine of ninewere identified, and prior nitration sites were also identified as Tyr107, Tyr163andTyr327. Then the damage that made by ONOO^-to the protein activity of p53has testedand verified by Western Blot. And the result showed that when the concentration of ONOO^-was on10mM level, p53protein would lose its activity.At last, molecular dynamics simulations were done to the nitration products ofp53protein base on the experiments before. The effect that made by the production of3-nitrotyrosine to the secondary structure of p53protein was clarified. Inference andexplanations about the selectivity of tyrosine nitration in p53protein were given.Effected by electronegativity and hydrophobic amino acid residues nearby, Tyr107can be easily nitrified. The conjugation of п-and space hydrophobic formed byTyr163and Arg249, the exposing of Tyr327residues could all make them be the sitesthat can be easily nitrified. Nitrified Tyr107, Tyr163and Tyr327has made greatimpact on the secondary structure of p53protein.p53protein was prepared and purified in the experiments, the prior nitrationsites of p53protein tyrosine induced by ONOO^-and the effect on protein activitymade by ONOO^-were clarified. Combined with Molecular dynamics simulations,reasonable explanation was given to initially clarify the damage made by ONOO^-andthe mechanism that ONOO^-can cause cancer. Based on this study, theoretical supportswere provided to prevent cancer and develop drugs.