Studies On The Cytoplasm Phosphorylation Proteomics Of Liver Cells From Mice With Endotoxic Shock

Sepsis is a syndrome of systemic inflammatorome induced by infection,of which the severe complication,sepsis shock,is the major cause of death when the patients suffered from wound,burn,and post operation.Statistic indicates that half of the sepsis is caused by Gram-negative bacteria,and that lipopolysaccharide(LPS),which release from the cytoderm of Gram-negative bacteria,is considered to be the major molecule which is responsible for the endotoxin sepsis.LPS can stimulates endothelium,macrophage and neutrophil,the 3 main targets,producing a large number of cytokine,which leads to inflammatory cascade reaction out of control,and leads to endotoxin shock,tissue damage and multiple organ dysfunction syndrome at last.In past,more attention focused on the 3 main tagets of LPS,but less on other cells or tissues that were waiting for exploiting.Liver is another important organ involved in sepsis.It's well known that besides playing a key role in metabolism,liver carries important immunologic function.It can produce and release a large number of acute phase proteins in response to inflammatory stress,playing an important role in prompt defense.However,more mechanism about liver acting in sepsis and the changes of itself during development of the disease is under exploiting,which attracts us.Endotoxin induced signal transduction pathway is the important component in the process of endotoxin pathopoiesis.The key research of cellular signal transduction is to feel environmental stimuli,transduct signals to regulate the metabolism of physiological responses and gene expression of the molecular pathway.Phosphorylation modification itself with the simple,flexible,reversible characteristics,as well as phosphate donor ATP is easy to be got, has been selected to become one of the most common means of regulation in eukaryotic cells. Protein phosphorylation,which plays an irreplaceable role in signal transduction,is a well-known truth.Protein phosphorylation and dephosphorylation almost regulates all life activities, such as cell proliferation,development,differentiation,signal transduction,apoptosis,neural activity,muscle contraction,the process of tumor and so on.Now,lots of human diseases have reported to be caused by unusual phosphorylation,and some unnormal phosphorylation is caused by certain disease.So,what happened to the cytoplasm in early endotoxic shock? What about their specific molecular mechanism? We know very little about it.Thus,it is important to study cytoplasmic phosphoproteins during endotoxic shock by large-scale proteomics analysis, which is useful to further understand the mechanism of the occurrence and development of endotoxic shock.Most previous studies are focus on several moleculars to analyze and find out few moleculars which properly have great significance to the occurrence and development of diseases,so as to further study its function.This is a traditional research strategy.Its merit is that the research questions can be focused and studied deeply,and the question can be answered thoroughly on a point.But if put this question in a system level,its function has become blurred trickle.It is caused by the limitations of research tools.Only accumulate enough results of such a "point",the question can be clear,but this process usually takes a long time.The proteome is highly dynamic,even if the same cell is in different physiological or pathological environment,its protein expression are different,and such different proteins are often a sign of certain diseases happened.Through comparing the protein expression level between normal and disease cell(organization),differential expression proteins are identified, which can be used to screen and find out potential drug target from the scale on the overall level,as well as for early diagnosis,intervention and treatment protein markers of disease. However,The quantitative analysis of changes of proteins often limit our research perspective. Because a lot of important activity of the protein is regulated by post-translational modifcation, Sometimes protein level does not show significant changes while the level of post-translational modification has been significant change.Therefore,it is very useful to analysis the cytoplasmic phosphoproteome before and after endotoxin stimulation by using quantitative techniques,which can help us to understand molecular mechanism of cytoplasm variation by endotoxin,and to find out more specific and sensitive protein markers which are closely related to the development of liver damage during endotoxic shock.Fluorescence differential gel electrophoresis(DIGE) is a method which labels protein samples with different fluorescent dyes before 2-D electrophoresis,and then 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.Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye multiple labeling method and DeCyder difference 2-D analysis software.DeCyder software takes the advantage of the spots co-detecting algorithm,which can automatically detected fluorescence images,eliminate background,quantifiy,normalize and match spots in gel,thus,systematic errors caused by different operators can be eliminated.In order to understand the molecule mechanism of cytoplasm variation of endotoxic shock, we design a differential proteomics experiments by duplicating BALB/c endotoxic shock model as 0 rain(normal control) and 1 h after peritoneal injection of LPS.We fractionated highly purified two groups BALB/c mice liver cytoplasm by differential centrifugation combination with density-gradient centrifugation.Then cytoplasm phosphoprotein were extracted by the phosphate metal affinity chromatography resin(PMAC).The result of WesternBlot showed that the enrichment of cytoplasmic phosphoprotein was efficient.Finally, the differential expression of protein profile of two groups has been established by using DIGE technology.Through analysis by use of DeCyder difference 2-D analysis software,37 differential protein spots were found.The numbers of up-regulated proteins is 23;while the numbers of down-regulated proteins is 14.At last,a total of 28 proteins have been identified among these differential protein spots by using matrix-assisted laser desorption ionization mass spectrometry(MALDI-TOF/TOF).19 of them are confirmed to be phosphorylated.We predicted the subcellular localization of the differential proteins and made a result that, among 28 proteins,2 proteins locate in cytoskeleton;20 proteins are in both cytoplasm and other subcellular position;7 proteins are in other subcellular organelle but not in cytoplasm. After subcellular localization analysis,the protein functional analysis has been done.Through analyzing protein domain and motif database,the identified proteins roughly included chaperone,oxidoreductase,anti-apoptosis protein,cell cycle protein,cytoskeletal protein. However,we also predicted protein interactions using the "String" protein interaction databases.We found that 8 of identified proteins are interacted with each other and they constitute an interaction network.Based on the researches above,we have some conclusion:1.We found that differential centrifugation and dens centrifugation is an effective approach to isolate cytoplasm.2.Cytoplasm phosphoproteins were enriched through the phosphate metal affinity chromatography resin(PMAC).Subsequently,the phosphoproteins of normal control and endotoxic shock(LPS 1h) BALB/c liver were separated by DIGE.37 differential protein spots with statistical significance between normal control and LPS 1 h were detected.3.37 differential protein spots were analyzed by mass spectrometry.28 proteins were identified after deleting keratin and duplication proteins.19 of them are had confirmed to be phosphorylated.4.We predicted the subcellular localization of the differential proteins and made a result that,among 28 proteins,2 proteins locate in cytoskeleton;20 proteins locate in both cytoplasm and other subcellular position;7 proteins are in other subcellular organelle,but not in cytoplasm.5.Through analyzing protein domain and motif database,the identified proteins roughly included chaperone,oxidoreductase,anti-apoptosis protein,cell cycle protein,cytoskeletal protein.8 of identified proteins are interacted with each other and they constitute an interaction network.