| Oxidative stress refers to when the body subjected to a variety of stimuli,a serious imbalance between the generation of free radicals and antioxidant defenses,which is the main cause of cell damage,and greatly affects diverse biological phenomena,including cell death,aging and various disorders accompanied by the occurrence and development of many diseases(including cardiovascular disease,autoirnune disease,neurodegenerative diseases,cancer and aging,etc.).However,the molecular mechanisms associated with oxidative stress inside cells remains to be thorough.Reactive oxygen species(ROS)accumulation of oxidative stress is the main cause of cell damage.ROS is also known as reactive oxygen species,which is generated in cell metabolism or oxidative stress.Many marmalian cell types also produce HZOZ in response to a variety of extracellular stimuli,with the H2O2 so produced serving as a signaling molecule that regulates various biological processes.ROS includes H2O2,hydroxyl radicals,in which H2O2 can act as an intracellular second messenger and plays an important physiological function.When H2O2 acts as a second messenger,that oxidizes critical eysteine residues of effectors in response to activation of cell surface receptors,and its concentration is strictly controlled,Despite its oxidative toxicity,while when a large number of ROS gathered,which can cause DNA breakage,protein cross-linking and lipid peroxidation leading to the process of cell or tissue injury,which will cause pathological response.So antioxidant enzymes within cells maintain intracellular redox homeostasis to protect cells and tissues from oxidative damage.Intracellular antioxidant enzymes:There are different types of mammalian peroxidases playing a eommon antioxidant effect to the reduction of hydrogen peroxide to water.There are three classes of peroxidases:catalase,glutathione peroxidases(GPxs)and peroxiredoxins(Prxs).Catalase is a heme-containing H2O2 dismutase,exclusively localized in the peroxisome,a subcellular organelle devoted to oxidizing organic substances,and catalase is believed either to remove H2O2 that passively diffuses into the organelie or to prevent H2O2 leakage.Five GPx isoforms are distinguishable by their tissue and subcellular distribution and substrate specificity.Among them,GPxl is the only cytosolic enzyme that reduces H2O2 by utilizing the electrons donated by NADPH via the glutathione-glutathione reductase system.GPxl is a selenoprotein that is produced at low levels and is dispensable in unstressed animal cells,its concentration is smaller than that of Prxlor Prx2 in most tissues.Peroxiredoxins is one of antioxidant enzyme,which is widely expressed by catalytic reduction of H2O2 and lipid hydroperoxide,to avoid further damage in cells subjected to oxidative stress process.prdxs(Prdx1-6)are a superfamily of small nonseleno peroxidases(22-27 kDa)currently known to comprise six mammalian isoforms,including atypical 2-Cys Prxs(PrxV),typical 2-Cys Prxs(Prx?-?),1-Cys Prxs three categories(PrxVI),Tipical 2-Cys Prxs in content is more,with unique catalytic properties,and which has been most studied.While 2-Cys Prxs peroxide and peroxidation slow reversal,H2O2 can protect it from damage and have enough time to complete the signaling cascade,and then was cleared by the restonng active 2-Cys Prxs when it is completed by the messenger function.Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H2O2 generated in response to receptor stimulation.2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H2O2 reduction reaction in the presence of thioredoxin,thioredoxin reductase and NADPH.Prx enzymes are efficient in eliminating low concentrations of H2O2 because of their low Kmvalues for this substrate.The catalytic efficiency of RoS of Prxs is about 105·M-1·S-1,of the relative catalase approximately 106·M-1·S-1 and of glutathione peroxidase approximately 108·M-1·S-1,which is lowest catalytic efficiency as H2O2 scavenging enzyme compared with the latter two.However,due to the 2-Cys Prxs having high affinity for H2O2(km<20 ?mol)and large number of widely exist in the cell(Prx?-? in the most cutured cells accounted for 1-10ug in per mg total soluble pratein).Therefore Prxs in the body plays as an important ROS scavenging.Later also found that Prxl in human can act as chaperone function.The chaperone function of 2-Cys Prxs can effectively prevent oxygen free radical-induced cytoplasmic protein aggregation to assist cell repair.After successful repaired peroxide 2-Cys Prxs restored by Srx reduction are involved in receptor signaling regulation.In sum,2-Cys Prxs has great potential in research and development of diseases treatment assotiated with active oxygen(such as cancer and cardiovascular diseases).Prxl is a typical 2-Cys Prxs,because there are two redox-active cysteine residue in its structure.In the catalytic reaction process,the peroxidatic cysteine(Cys51 in Prxl)reduces H2O2 to H2O and becomes oxidized to sulfenic acid.The resolving cysteine(Cysl73 in Prdxl)of another subunit reacts with the sulfenic acid to form an intramolecular disulfide,which can be reduced by thioredoxin(Trx).Trx is then reduced by NADPH-dependent Trx reductase.Oxidation state took advantage of the reduction of electronic NADPH Trx provide recovery in the thioredoxin reductase into a reduced state.Efficient catalytic reduction of peroxide,oxidative stress plays an important role in the process.In the catalytic process,Cys51-SOH peroxide H2O2 may be occasionally formed Cys51-SO2H,which will cause Prxl peroxide inactivation,inactivation of such peroxide,which may be twisted by sulfiredoxin,prokaryotes do not express sulfiredoxin.Therefore,Prxl peroxidation is blocked in prokaryotes,eukaryotes rely on the peroxidation,which can cause the accumulation of H2O2 under specific conditions so that as to needed concentration for signals transduction.Prx1 main plays role in intracellular antioxidant effect and regulation of hydrogen peroxide(H2O2)-mediated signal transduction,and is also involved in cell cycle progression and play the role of molecular chaperones.In particular,Prxl appears to provide selective,specific and localized control of receptor-mediated signal transduction.It also transient reversible oxidation by adjusting the concentration of H2O2 inactivation,H2O2,which plays as a messenger needs the eoncentration rises rapidly to reach the threshold value and i5 not widespread destruction by Prxl.While reported:Prxl associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune reeeptors in vitro.The loealized inactivation of Prxl allows for the transient accumulation of H2O2 around membranes,where signaling components are concentrated,while preventing the toxie accumulation of H2O2 elsewhere.This space is restrictive Prxl on inactivation to generate favorable intracellular H2O2 concentration gradient,played a major role,so that the membrane surrounding the signal protein was gathering and transmitting signals,which will not be generated in the whole cell high levels of toxic concentrations of H2O2.We found that Prx1 in vivo with multiple important functions,which is not ouly closely related to a variety of metabolic activity in tissue and cells,but also closely related to the occurrence and development of many diseases.According to the literature,Prxl was inactived by tyrosine phosphorylation or peroxidation,however,while the specific mechanisms of regulating the activity of Prx1 is still lacking,so in this study we aimed to study about the activity state of Prx1 in vivo and the regulatory mechanisms on it so as to enrich the understanding of Prx1 and guide research,diagnosis and treatment of many diseases.Organism's life activities is closely related to dynamic changes of proteins,with the in-depth study of proteomics research,we come to realize that in many eases the absolute amount of certain proteins do not change significantly,a large number of proteins play a biological effect through either direct interaction with other protein molecules or regulation downstream moleeules through a series of post-translational modification or being regulated.Our previous experient results:by doing 2-D Fluorescence Difference Gel Electrophoresis(2D-DIGE)and mass spectrometry found that the phosphorylation level of Prxl was significantly elevated in PRAK+/+cells while in PRAK-/-cells had no obvious changes.In view of PRAK is located in the downstream of the MAPK signaling pathway.After the in vitro kinase reaction,Prxl phosphorylation levels were detected utilizing western blot technique and found that there is no significant phosphorylation of PRAK on Prxl,serine,threonine and tyrosine residues.And then detecting the phosphorylation levels of Prxl by 2D-DIGE technology,we didn,t found obvious different protein spots,suggesting that there may be no direct phosphorylation regulation relationship between PRAK and Prxl.And then respectively treated with 200 ?mol/L NaASO2 to PRAK+/+,PRAK-/-cell for 45 min,lysed to obtain protein,Western Blot detection performed and found in both kinds of cells Prxl threonine sites did not occur significant phosphorylation;while Prxl serine sites existed a certain level of phosphorylation in PRAK-/-cell,but lower than in PRAK+/+ cells,suggesting the existence of PRAK has a role in promoting Prxl serine phosphorylation.However,it was unclear the influence is related to oxidative stress conditions;and Prxl tyrosine sites did not occur significant phosphorylation in PRAK-/-cells,while in PRAK+/+ cells detected obvious tyrosine phosphorylation,and Prx1 tyrosine phosphorylation has been enhanced by the oxidation stimulation.Suggesting the existence of PRAK also affects the level of tyrosine phosphorylation of Prxl,and closely associated with oxidative stimuli.PRAK is one of serine/threonine kinase phosphorylation,so we speculate that PRAK regulates the nevel of Prxl tyrosine phosphorylation by the impact of one middle unknown protein.Since Prxl activity status been reported are closely related to its phosphorylation levels,we consider the establishment of a sound Prxl activity detection system at first,through monitoring Prxl active trends under different oxidative stimulate concentrations,to sdudy its change of activity and phosphorylation levels and protein complex in Prxl and identify statistically significant different protein complexes with different active state and then to Improve of the knowledge of the regulating mechanisms about the activity of Prxl.Therefore,this study was designed to find the change of Prxl itself polymerized state and related protein complex making use of co-immunoprecipitation,BN-PAGE combined with mass spectrometry experimental techniques in NIH3T3 cells in the process of oxidative stress.And to detect the activity trends of the Prxl in the NIH3T3 cells after stimulation by the gradient NaASO2,utilizing Trx activity detection system,which is composed with yeast thioredoxin(Trx)proteins,yeast thioredoxin reductase(TrxR)and coenzyme ?(NADPH),for further studying the specific regulatory mechanisms on Prxl activity in the process of cellular oxidative stress.Through this research,we draw the following conclusions.1.By optimizing the reaction conditions of yeast Trx activity detection system even low concentration of prokaryotic purified protein His-Prdx1 can be successfully detected activity.2.Cultured two groups of NIH3T3 cells,One group was given p38 pathway inhibitor for lh,the other group was given the same volume medium,and lh later the cells were given 200 ?mol/L NaASO2 treatment for 45 min,after stimulation the cells were lysed to obtain the endogenous Prxl protein by IP technology,and to detect its activity trend through optimized yeast Trx activity detection system.We find that the activity trends of the Prxl in two groups were almost identical.Suggesting p38 pathway inhibitor does not directly inhibit the activity of Prx1.3.NIH3T3 cells were treated respectively 0,200,300,400,500,600 ?mol/L NaASO2 for 45 min,after stimulation the cells were lysed to obtain the endogenous Prx1 protein by IP technology,and to detect its activity trend through optimized yeast Trx activity detection system.We find that with the stimulation concentration of NaASO2 increaseing,the activity of Prxl detected firstly increased then reducted or even lower than non-stimulated group,in 400 ?mol/L NaASO2 stimulation group Prx1 is detected the most active of all groups,while in 600 ?mol/L group the activity of Prxl is almost below no stimulation group.Suggesting Prxl-Cys52-SH peroxide is not be peroxided after 400 ?mol/L NaASO2 stimulation,still in the presence of highly active state of the dimer,with increasing concentrations of NaASO2,Prxl redox activity is gradually suppressed,which maybe related to gradually lose of its redox activity after being oxidized,and may also be associated with inactive polymers develepted among high activity among dimmers.Specific mechanism is not clear,which needs further study.4.NIH3T3 cells were cultured for transfecting plasmid PCDNA3-HA-PRX1,with 400 ?mol/L NaASO2 stimulation for 45min to detected whether expressed HA-Prx1 has the same response to oxidative stimuli as endogenous Prxl.This section is still in progress.5.Treated NIH3T3 cells respectively 0,200,400,600 ?mol/L NaASO2 for 45 min,after stimulation the cells were lysed to obtain the endogenous Prxl protein by IP technology,detected by Western-blot and found successful obtain the endogenous Prx1 protein.and detected by SDS-PAGE with the same samples found that there are significant bands existed in 200 and 400 ?mol/L stimulate groups in the molecular weight of about 40 kDa and 70 kDa,while not detected in the other groups in the same molecular weight,and also in the molecular weight of about 55 kDa band was exsited in 600 ?mol/L group.Differences bands were digging for further identifing by mass spectrometry to identify protein spots.6.NIH3T3 cells were treated respectively 0,200,400,600 ?mol/L NaASO2 for 45 min after stimulation the cells were lysed to obtain the endogenous total protein,to detected by BN-PAGE.We find that there are significant different complex between stimulatedand non-stimulated groups.Result 5 and result 6 both suggest after NaASO2 stimulation it appears to there exist different interactional complexes with Prx1.7.Treated respectively 0,200,400,600 ?mol/LNaASO2 in NIH3T3 cells for 45 min,after stimulation the cells were lysed to obtain the total protein,to detect the change of 194-tyrosine phosphorylation nevel of Prxl with gradient NaASO2 stimulation.We find that the result is there is non-bands of phosphorylation detected in cantrol 200 and 400 ?mol/L groups,while significant bands detected in 600?mol/L group.It was suggested that Prxl maybe inactived when stimulated with 600?mol/L NaASO2,because its tyrosine was phosphorylated. |
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