| Reactive oxygen species(ROS)are products of normal cellular metabolism product.ROS is well recognized for playing a dual as both deleterious and beneficial species,since they can be either harmful or beneficial to living systems.The appropriate concentration of ROS is beneficial for cellular immune response,as for example it plays a crucial role in defence against infectious agents and in the function of a number of cellular signaling systems.While the harmful effect of free radical occuring at high concentrations and causing potential biological damage is termed oxidative stress.Oxidative stress is attribute to the excess production of ROS or its metabolic disorders and excess of eliminating capacity of endogenous antioxidant system,thus the oxidation and antioxidant systems become inbalance,and then the excess of ROS participate in the process of biological macromolecules oxidation,eventually leading to the cellular lipid peroxidation and damage of lysosome,mitochondrial.It is an adaptive reaction which finally lead to cell and tissue damage.Because the body is constantly exposed to a variety of sources of free radicals,in order to maintain the stability of the intracellular redox state,the living organisms have a series of anti-oxidation mechanisms,for example:preventative mechanisms,repair mechanisms,physical defences and antioxidant defences.And in the process of antioxidant defense,a number of studies reported that the serine/threonine kinases of the MAPK family can be regulated by oxidants.There are four known MAPK families:extracellular-regulated(ERKs),c-jun-NH2-terminal kinase(JNKs),p38 MAPK and the big MAPK-1(BMK-1).p38 regulated/activated protein kinase(PRAK)was originally described as a protein kinase activated downstream of the p38 mitogen-activated protein kinase(p38 MAPK).PRAK is a member of MAPK-activated protein kinase(MAPKAP kinase/MK)family.The molecular weight of PRAK is about 54 kDa.A PRAK gene was found in most vertebrates,with predominant expression in the heart and skeletal muscle.When over-expressed,PRAK localizes to the nucleus of quiescent cells,but upon cellular stress,PRAK translocates to the cytoplasm.While the subcellular localization of endogenous PRAK was somewhat controversial with reports that the endogenous protein was either in nuclear or cytoplamic.PRAK is mainly regulated by p38 MAPK signaling pathway involved in cell stress response process.Early data has shown that with the stimulation of cellular stress or proinflammatory cytokines,PRAK was phosphorylated on Thr182 by p38 MAPK,then activated PRAK phosphorylates small heat shock protein HSP27,resulting in cytoskeletal reorganization and responses to cellular stress.p38 MAPK-mediated phosphorylation of PRAK can lead to redistribution of a significant fraction of PRAK from the nucleus to the cytosol.But it remains unclear what is the function of PRAK translocation.Despite the fact that PRAK can clearly be activated by p38 MAPK in over-expressed cells,doubts began to surface as to whether or not PRAK is a target of the p38 MAPK pathway in vivo.Recent data indicated that no significant activation of endogenous PRAK could be detected in response to either cellular stress or inflammatory cytokines,despite the concomitant activation of p38 MAPK,and endogenous PRAK was not able to phosphorylate HSP27 in vitro and in vivo.Furthermore,an analysis of protein-protein interactions between endogenous PRAK and p38 MAPK failed to find any evidence for complex formation.Therefore,it is necessary to reevaluate the relationships between p38 MAPK and PRAK,and the substrates of PRAK.In summary,PRAK may exert comprehensive and complex biological functions.But so far our knowledge about PRAK is still very limited,and there are still a lot of controversies about PRAK.In addition,whether there are some unknown regulation mechanisms and functions of PRAK remains to be studied.Traditional study of protein function was mostly focus at a few proteins on the basis of the existing work and theoretically deduced proteins which may have important implications on the development of the disease,and then followed by in-depth and detailed functional study.But this kind of "hypothesis driven"research strategy often exist shortcomings,such as unilateralism,subjectivity and low efficiency.As the human genome project(HGP)completed ahead,proteomics theory and the technical method has become the theme of post genome era.As a large-scale,high throughput,high sensitivity method,proteomics can be effectively,objectively analyzed of the overall intracellular proteins.Through comparing the protein expression level between differences samples,differential expression proteins were identified,which can be used to solve various biological problems.At present there are many novel differential proteomics analysis methods,among which the difference gel electrophoresis(difference gel electrophoresis,DIGE)technology becomes one of the most popular one,because it's not only have the high-resolution feature,inherited from two-dimensional gel electrophoresis(2-DE),but also have high reproducibility,high sensitivity,high throughput and high dynamic range.DIGE is a method which can labele protein samples with different fluorescent dyes before 2-D electrophoresis,and can separate up to three different protein samples at the same time in one two-dimensional gel.The application of the internal standard could further increase the credibility of the experiment,and ensure the results could reflect the real biological differences,while avoid influence of systematic errors on experimental results.Based on the previous research,the subject was mostly focus on the proteins associated with PRAK under early stage of oxidative stress.Therefore we chooses mice fiber cells with the type of PRAK+/+and PRAK-/-as the research object,using the arsenic acid sodium as oxidatie stress molecules,using a technology platform based on difference gel electrophoresis,MALDI TOF/TOF mass spectrometry and bioinformatics analysis to find the proteins associated with PRAK under early stage of oxidative stress.It can provide the data for further deepening the understanding of PRAK and ultimately offer the opportunity for revealing related function proteins associated with PRAK in the oxidative stress process.According to the analysis of differential protein spots combining different maps of DIGE and Decyder software,we set these which were up-regulated or down-regulated 1.5 times as differential protein spots.Comparing the stimulation group with the control group of PRAt+/+,6 differential protein spots were detected,The number of up-regulated is 1 and on the contrary are 5.Comparing the stimulation group with the control group of PRAK-/-,46 differential protein spots were found,The number of up-regulated are 17 and on the contrary are 29.Comparing with the control group of PRAK-/-to PRAK+/+,26 differential protein spots were found,The number of up-regulated are 21 and on the contrary are 5.Some differential protein spots between a few groups all change,we finally analyse 60 differential protein spots.Protein spots of interest were excised by automatic Spot picker,and then the picked spots were carried out with in-gel digestion,followed by using ABI 4800 MALDI TOF/TOF mass spectrometry.Retrieving SWISSPROT database through the mascot software,geting rid of the same protein within appraisal result and 8 unidentified protein spots,38 differential proteins were identified.We carried out the bioinformatics analysis of the function of all 38 identified differential proteins.Firstly the identified differential proteins were processed by subcellular localization analysis with subcellular localization software WoLF PSORT.The results indicated that 32 proteins predicted localized in the cytoplasm;26 proteins predicted localized in nucleus;25 proteins predicted localized in cytoplasm or nucleus;13 proteins predicted localized in mitochondria;7 proteins predicted localized in extracellular;2 proteins predicted localized in cytoplasm or mitochondria;1 protein predicted localized in cytoplasm or membrane;1 localized in skelemin and 1 localized in endoplasmic reticulum.These results speculate that most differential proteins shuttle among cytoplasm and nucleus.This may attribute to functional regulation of cellular oxidation system and antioxidation system under early stage of oxidative stress.After subcellular localization analysis,the protein functional analysis had been done.Through analyzing protein domain and motif database,the identified proteins were roughly categorized into six groups based on functions,which include nucleotide-binding proteins,cytoskeleton proteins,molecular chaperone,membrane transport proteins,functional regulating proteins,metabolic enzymes proteins.The protein function is usually carried out by the interaction between proteins.To study the relationship among proteins,we use the "String" protein interaction databases.The result showed that there are 21 differential proteins exising at protein interaction network simultaneously,which indicated that these differential proteins have functional relation and may associated with cellular antioxidation response.These proteins were analyzed by bioinformatics software and the literature investigation,we choose "peroxiredoxin 4"(Prdx4)as the interesting protein-Since Prdx4 was involved in redox regulation process,and it could also activate NF-?B by phosphorylating NF-?B inhibitor protein alpha(I-?B?)in the cytoplasm,and the Prdx4 gene was also showed to have dependent thioredoxin(TRX)peroxidase activity and functions about cell proliferation and differentiation.What's the relationship between differential expression of Prdx4 and PRAK?Whether Prdx4 gene was regulated by PRAK?Whether Prdx4 is a downstream substrate of PRAK?What's the biological function of Prdx4 in the oxidative stress?They are valuable to study.Western blot analysis was performed to verify the expression of Prdx4 in PRAK+/+and PRAK-/-cells,and two rows of relatively clear bands were found,combined with molecular weight of the binding proteins,after referring to literatures,it was suggested that these two protein bands are oxidate or peroxide dimers of Prdx4.The molecular weight of the above band is about 60 kDa,and presumably is the oxidized homo dimers of Prdx4,the molecular weight of the low one is about 50 kDa,presumably is the oxidate heterologous dimer of Prdx4-Prdxl.The expression of Homo dimers and hetero dimers of Prdx4 in PRAK+/+ cells was at a very low level,but in cells of PRAK+/+treating group,increased levels of homo dimer of Prdx4 was indicated;however,in the PRAK-/-control group and PRAK-/-treat group,the level of homo dimer and hetero dimer of Prdx4 is high,but there is no significant difference between before oxidative stress or after.Based on the experimental results,we speculated that the ROS in PRAK+/+cells was in a relatively low level,when acute oxidative stress happened,the expression of Prdx4 increased rapidly to generate the homo dimer of Prdx4;in PRAK-/-cells,the PRAK gene had been knockout,the cells appeared in a compensatory increase of Prdx4,the dimer of Prdx4 also increased,which made the ROS stay at a low level,in the early stage of oxidative stress,the impact to the level of ROS is very small,therefore,the dimer levels of Prdx4 do not change significantly.By real time PCR experiments we found that the mRNA levels of Prdx4 in PRAK+/+control group was low,but the level in PRAK+/+ treat group raised quickly,and nearly 1.4 times about PRAK+/+control group;while the mRNA levels of Prdx4 in PRAK-/-control group and PRAK-/-treat group were both relatively high,and with no significant difference between the two groups,expression was nearly 2 times about PRAK+/+ control group.According to the experimental results,we speculated that the PRAK+/+ control group was in the relative balance state of redox,so the expression of Prdx4 was at a low level,but once oxidative stress occurs,the transcription of Prdx4 gene would start quickly;while the expression of Prdx4 in PRAK-/-control group and PRAK-/-treat group were relatively high,which indicated that after the gene knockout of PRAK,Prdx4 played a compensatory role of antioxidant,to balance the level of intracellular ROS.To test whether Prdx4 can activate NF-?B by phosphorylation of I?B?,the results of Western blot showed that,the phosphorylation level of I?B? was consistent with the expression levels of the homo dimer of Prdx4.The experimental results suggested that,in PRAK+/+cells group,the PRAK signal can activate NF-?B through p38-MAPK signal pathway,so the phosphorylation level of I?B? in PRAK+/+control group was low,and the phosphorylation level of I?B? in oxidative stress would increase to play the role of antioxidant;and because the protein of PRAK is deficient in PRAK-/-cells group,the homodimer of Prdx4 may take part in the activation of NF-?B,to start the transcription of antioxidant protein in cells.Therefore,the homo dimer of Prdx4 may be involved in the phosphorylation of I?B?,which plays an important role in the reaction oxidative stress.By immunofluorescence experiments,we can observe the localization and transfer of Prdx4 more clearly.The results show that Prdx4 mainly distributed in the cytoplasm in the PRAK+/+cell lines,and the expression and localization phenomenon of Prdx4 was increasing along with the stimulation of sodium arsenite(Oh,lh,2h,4h).In addition,the nucleus shape gradually shrink in the PRAK+/+cells.On the contrary,the expression of Prdx4 in the PRAK-/-cell lines was the same trend at Oh,1h and 2h and peaked at 4h,the nuclear localization phenomena and shape also have the same trend.Therefore,we can presume that the expression of Prdx4 is lower in PRAK+/+control cells,and a sudden increase of ROS levels can quickly start signal transduction,leading to a gradual increase of Prdx4 under acute oxidative stress.In addition,the nuclear localization phenomenon also gradually increased,which was probably because of the chaperone role of Prdx4 multimers and transfering of the nuclear transcription factor into the nucleus.The cells started the process of apoptosis and the nuclei gradually shrinking with prolonged exposure.In PRAK-/-cells,since the lack of PRAK protein,the expression of Prdx4 was under high levels and the expression of ROS was under low level,therefore,the ROS regulation of cellular signal transduction can not be activated rapidly under acute oxidative stress,resulting in the relatively stable expression of the Prdx4 from 1 to 3 hours and sharply increase expression until 4h.The nuclear localization phenomenon also explains the time of transcriptional regulation of the cell nucleus is relatively late.The low level of ROS in PRAK-/-cells may play a protective role,and lead to the slow reaction to the external environment.In order to observe intracellular ROS levels at different time points of PRAK+/+and PRAK-/-cells stimulated with NaAs02,we have detected the abundance of intracellular ROS.A phenomenon has been observed from the data:ROS level of PRAK+/+cells is double of PRAK-/-cells.With the extension of stimulation time,PRAK+/+cell ROS levels rise rapidly to 1.5 times of the normal level,then stabilize at this level.The ROS level of PRAK-/-cells is relatively low and stable in 1-4h,but rapidly rises at time points of 5h and 6h.Finally the levels of ROS reached about 2 times the normal level.Presumably,in PRAK+/+cells,Prdx4 expression ratio is low.When subjected to oxidative stress,ROS can initiate a rapid increase in cellular antioxidant system,resulting in a stable level of ROS;for PRAK-/-cell lines,due to the high amount of Prdx4 compensatory expression,ROS stays at a low level.When PRAK-/-cells are under oxidative stress,high expression levels of Prdx4 can play as antioxidant.ROS increased relatively slowly until Prdx4 stop compensated,then have a sharp increase.We use real-time quantitative polymerase chain reaction(real-time PCR)to observe the changes of expression of diffienetial proteins on the level of the mRNA.The experimental results showed the mRNA level of Actg1 remains the same under arsenic acid sodium stimulation comparing with the unstimulated group of PRAK+/+cells or PRAK-/-cells.However,compraring PRAK-/-cells with PRAK+/+cells,Actgl have increased significantly on the level of mRNA.And the mRNA level of Anxa4 have significant differences between PRAK-/-control and PRAK+/+ control cells,but have no obvious change among other groups of cells.So we can conclude that PRAK gene may be involved in the regulation of Actgl and Anxa4 gene expression.MTT was used to observe the cell survival at different time points of PRAK+/+and PRAK-/-cells stimulated with NaAso2.PRAK+/+cells survival rate was significantly higher than PRAK-/-cells in oxidative stress response.Presumably,the knockout PRAK gene may impacts the function about anti-oxidative stress.The compensatory highly expressed of Prdx4 in cells not only play a protective function,but also inhibit the signal transduction pathways of anti-oxidation,resulting PRAK-/-cells intolerance to oxidative stress.To look for PRAK related protein complexes,we have established a BN-PAGE system.The differential protein complexes associated with PRAK protein was detected and cut,then SDS-PAGE and mass spectrometry were performed.We found 18 components from the differential protein complexes related to PRAK by mass spectrometry.After the research,three conclusions were drawn as follows:1.Differential maps of proteins of PRAK+/+and PRAK-/-cells were separated by DIGE.60 differential protein spots with statistical significance were detected,and 38 proteins have been identified with mass spectrometry.The differential proteins may be associated with PRAK on the early stage of oxidative stress.2.In oxidative stress reaction for PRAK gene knockout cells,Prdx4 gene may play a compensatoric role at transcriptional level.Additional,Prdx4 has a antioxidant function,and their homodimers may activate NF-?B by regulating the phosphorylation level of I?B?,starting transcriptional regulation of antioxidant genes,in order to maintain the balance of intracellular ROS.3.We established BN-SDS PAGE coupling with MS technology platform which was used for separation and identification of protein complexes,and we found 1 differential protein complex and analyzed the 18 compoments with mass spectrometer.This study not only provided the experiment data for re-understanding of PRAK activation mechanism,the downstream substrate and the biological function of PRAK,but also laid the foundation for exploring the molecular mechanism of PRAK at the early stage of oxidative stress response. |
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