| Angiogenesis is the sprouting of endothelial cells to form vascular system from already existing vessels,and it occurs frequently in the adult during would healing not only in the embryo as vasculogenesis.Liver regeneration is an angiogenesis-associated phenomenon,and received continuous attentions.Angiogenesis is a complicated highly regulated process dependent on the interplay and balance between several angiogenic and anti-angiogenic factors favouring or inhibiting angiogenesis respectively.Several angiogenic factors initiated the angiogenesis after partial hepatectomy,such as HIF1 a and IL6.The angiogenic signaling enters cell and triggers a serial biochemical reaction through interacted with membrane receptors,and the researches done in this field still have their limitations.Lipid raft is the major functional compartment by clustering of receptors and components of receptor-activated signaling cascades,showed the high effective of the signaling.The angiogenesis related receptor which is recruited by lipid raft may contribute the discovering of the angiogenic signal network.There are overlaps between receptors identified in lipid raft and angiogenesis,such as Receptor tyrosine kinases(RTKs),Protein phosphatases,GPI-anchored proteins,G protein and G-protein-coupled receptors(GPCRs).Others like u PAR,MMP-9,flotillin-1 and caveolin-1located in lipid rafts are all reported to be involved in angiogenesis.This paper conducts the research on angiogenesis related membrane proteins during liver regeneration from three aspects.In the first part,the phenomenon that angiogenesis proteins were recruited in lipid raft was demonstrated by the quantitative study.In the second part,the phosphoproteomics study was conducted in lipid raft,which partically illustrates the rapid actions in signal transduction on membrane microdomain.In the third part,the level of tyrosine nitration was detected in liver sinusoidal endothelial cells plasma membrane to find out the association between protein nitration and the termination of angiogenesis.by isobaric tags for telative and absolute quantitation and reverse transcription-polymerase chain reaction.Firstly,the classic method developed by our laboratory for isolating and purifying the plasma membrane was used here to quantitate the angiogenesis related lipid raft proteins.The obtained proteins were quantified by isobaric tags for telative and absolute quantitation,a total of734 proteins were identified,among which the difference expression ratio of 258 proteins is greater than 1.2.These proteins are mainly involved in signal transduction,vesicle transport,and cell adhesion.In order to detect the differences between the level of transcription and translation,we selected several angiogenesis related proteins through bioinformatics method,and verified their quantitative data with quantitative RTPCR.11 genes increased their expression at the transcriptional level,while 10 genes down regulated their expressions.The expression of protease activated receptor 4(PAR4)was significantly up-regulated in i TRAQ but down-regulated in RTPCR,and the activation model of PAR4 matches the pattern of protein located in lipid raft.So we further verified its expression on whole cell lysate,plasma membrane and lipid rate by western blotting.The immunoblotting results showed that PAR4 were highly expressed in plasma membrane and lipid raft at 48 h and 72 h after partial hepatectomy,which suggested that PAR4 is recruited by lipid raft for angiogenesis.The clustering of proteins in lipid raft decreased the spatial steric hindrance,which is beneficial to signal communication.Multisite phosphorylation can produce ultrasensitivity in signaling networks,involving in cross-talk between cells.The researches in phosphorylation of lipid raft proteins could provide the mentality and the method for the following researches on angiogenesis.Here,the combination of SDS-PAGE and pro-Q staining were used to identify the phosphorylated proteins in lipid raft and the potential phosphorylated peptides were enriched by Ti O2 before MS/MS identification.327 and 271 phosphorylated peptides correspond to 106 and 105 phosphorylated proteins were identified at 0 h and 72 h after partial hepatectomy.A total of 166 phosphoproteins were identified and 45 were both indentified at 0h and 72 h after PH,among which 17 proteins showed the different phosphorylation sites.These proteins participate in multiple processes according to gene ontology analysis,including metabolic process(69),response to stimulus(43),localization(31),and developmental process(22).These data are consequence with the function orientation of lipid raft,and indicated that lipid raft may participate in intercellular communication.After deeply excavation of the quantitative data,we found that the apoptosis molecular PKC were up-regulated at PH72,and previous research demonstrated that the peak of LSEC proliferation were at PH96. Thus we speculated that the accumulation of reactive oxygen species(ROS)and reactive nitrogen species(RNS)produced by the proliferation of the liver parenchymal cells could initiate the tyrosine nitration,and inhibit the prolife of LSEC.The nitroproteins in LSEC plasma membrane were identified in this study,Anti-nitrotyrosine immunoreaction showed that the high level of protein tyrosine nitration in LSEC PM after PH,indicating that tyrosine nitration may contribute the termination signal of liver regeneration.A total of 639 proteins(nitrated and non-nitrated)in nitrotyrosine immunopositive 2D gel spots were identified,and 16 nitroproteins and their 17 nitrotyrosine sites were identified with MS/MS analysis.These nitroproteins participate in multiple processes such as the apoptosis and cardiovascular diseases related proteins,including Phosphatidylinositol 4-phosphate 3-kinase and Hexokinase-1,and protein related cell cycle including Solute carrier family 25 member 34(Slc25a34),hexokinase,inositol hexakisphosphate(Ins P6),structural maintenance of chromosomes(SMC).This study could provide new insights into tyrosine nitration and its potential role in the termination signal of liver regeneration. |
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