Identification Of Differential Phosphoproteome From Liver Nuclei Of Mice With The Early Stage Of Endotoxic Shock

Sepsis is a kind of systemic inflammatory response syndrome induced by infection,and the sepsis shock,a severe complication of sepsis,is one of the major causes of death for patients suffered from trauma,burn,and post operation.Statistics indicate that half of sepsis in clinic is caused by Gram-negative bacteria infection,and that lipopolysaccharide(LPS),or called endotoxin,which is one of composing components of Gram-negative bacteria cell wall,is considered to be the major molecule responsible for sepsis caused by Gram-negative bacteria.LPS can widely act on human multiple organs and tissues,among which endotheliocyte,macrophagus and neutrophil are the main effector cells.Stimulated by LPS,they produce a large number of cytokines,namely "cytokine storm",and then lead to excess inflammatory cascade,and eventually result in endotoxin shock,tissue damage and multiple organ dysfunction.Liver is annoter important organ involved in sepsis.It’s well known that besides playing a key role in metabolism,Liver carries important immunologic function.It can produces and releases 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 exploit,which attracts us.The genetic material localized mainly in the nucleus.Genetic information is stored here,and in accordance with the life of cells,transcription activity is regulated, so that cells adapt to the environment and their own life-cycle changes in cells.For this reason the nucleus is called the control center of the cell life activity.Nuclear protein group include mainly the nuclear matrix protein,nuclear membrane protein, nucleolar protein,the proteins which involve in the regulation of protein transcription process,the histones and non-histones participating in the formation of the chromosomes.Together,they participated in the function cases occurring in the nucleus,the implementation of nucleus function.In the life process or disease process, The composition and post-translational modification status of these proteins would undergo corresponding changes,such as when the cells are stimulated by some signals from the extracellular to the intracellular,intracellular proteins have been modified or the structure corresponding the changes occurs,so that these proteins are activated or in-activated,thereby making immediate responses.There is also a part of the shuttle proteins from the nucleus to participate in the expression of certain genes to stimulate the outside world to make long-lasting response.As for the post-translational modification of proteins,the phosphorylation(dephosphorylation) is recognized the most,responsible for on / off control duty in biochemical function of many proteins and the key adjustment mechanism to much of life phenomenon, including regulation of cell proliferation,growth,differentiation,signal transduction, apoptosis,neural activity,muscle contraction and tumor processes et al.It is well known that many human diseases are due to abnormal phosphorylation modification arising,and some phosphorylation modifications are the consequences caused by a disease.Therefore,through understanding nuclear phosphoproteome changes happening in the endotoxic shock,this can help us know the liver response mechanism in endotoxic shock.However,a problem we encounter is to choose two strategies of research:one is traditional based on single molecule research scale,and another is innovational, intraducing the principle of "omics".The traditional one is senior in explaining the detail of a single problem,but junior in answering systematic one,while using "omics" method is on the contrast side.In this research,we want to have a systematic view of the problem and form a framework for ongoing studying.Using this strategy may be more effective and easy to graph important aspects underlying the problems. So we start our study with the strategy of proteomics.Proteomics characterize large-scale,high-throughput and high-sensitivity,the total proteins of a cell can be analyzed effectively by it.But the proteome of any cell, tissue or organism is a complex mixture of proteins that span a wide range of size, relative abundance,acidity/basicity,and hydrophaobicity,so subcellular proteomics emerge.Subcellar proteomics research can not only provide information about subcellar location of certain protein and imply its function,but also tell us the whole-protein components of the specific subcellular fraction(organelle or other multi-protein complex) and then help understand their structures and biological functions.And recently developed proteomics study new methods,such as: fluorescence difference gel electrophoresis(difference gel electrophoresis,DIGE) technology because of the succession of the two-dimensional gel electrophoresis (two-dimensional electrophoresis,2-DE) high-resolution and at the same time with high reproducibility,high sensitivity,high-throughput and high dynamic range and other advantages,the more we set up a good research platform.Based on the above analysis,the BALB / c mouse liver was chosen for the study and we prepared the animal model of endotoxic shock with peritoneal injection of LPS in order to gain the liver tissue of the under the disease.Then we aquied LPS stimulus check 1 hour(hour,h) group and normal control group of mice. Subsequently we collected mice liver tissues after anesthesia,fractionated highly purified two groups BALB/c mice nucleus through differential centrifugation and sucrose dense centrifugation,and lysed the nuleus with Qiagen lysis buffer to gain the whole nuclear protein.Thus we enriched the nuclear phosphoproteins through phosphoprotein enrichment column.Finally we got the expression patterns of liver nuclear differential phosphoproteome of endotoxic shock mice by using DIGE technology.Through analysis by the use of DeCyder difference 2-D analysis software, 31 differential protein spots were found.There was upregulation of the 21 points and downregulation ten points.Of these differences in protein spots,a total of 29 kinds of proteins were identified by using MALDI-TOF/TOF mass spectra.This study,we obtained the following conclusions:1.Successfully extracted and purified the nuclei from mouse liver tissues,and respectively identified with fluorescent microscope,transmission electron microscopy and mass spectrometry.2.Set up the nuclear phosphoproteome map of differences between the normal group and endotoxic shock early BALB / c mouse liver cell through DIGE technology.3.Found 31 protein spots with different expression by differential software analysis, then identified 29 different proteins using mass spectrometry identification.4.We predicted the subcellular localization of the differential proteins and made a result that,among 29 proteins,there is only 1 protein completely localized in nucleus; 21 proteins in both nucleus and cytoplasm;1 proteins in nucleus but also in other sub-cellular structure;6 proteins not in nucleus and other sub-cellular structure.5.Through bioinformatics analysis,the identified proteins are involved in splicing RNA processing,nucleic acid binding protein,gene expression regulation,protein transport,transcription regulation and cell signal transduction.This experiment,we explored a technology program with combining purification technology and 2D-DIGE gel electrophoresis to study of the nuclear phosphoprotein expression profile differences in the mouse liver.Conclusionally,this not only provide a new technical method on revealing corresponding changes in the nucleus for early endotoxic shock,but also new opportunities for an in-depth study of the pathogenesis of sepsis and its prevention and control strategies.