| Background: Myeloid differentiation protein-2(MD-2), is one of binding receptorsbetween Toll like receptor (TLRs) and lipopolysaccharide (LPS), considered as animportant regulatory molecule of innate immune recognition, to play an important role inthe process of LPS signal conduction. Recent studies suggest that MD-2is required for LPSactivation of TLR4receptor molecule transmembrane signaling pathways, inhibition of thepathway activation is an effective strategy for prevention and treatment of LPS inducedsepsis. The distance function of secretory or soluble MD-2(sMD-2) has been found, whichshows that MD-2interaction sites on the cell membrane is not only one, looking for newMD-2interaction sites or proteins have an important theoretical significance and potentialapplication value for the prevention of excessive inflammatory reaction induced byLPS/endotoxin.Our project team in the previous NSFC project (No.30700289), based on the study ofthe relationship between structure and function of MD-2, designed and built a newsimulation peptide originating from MD-2with two unique binding domain, called MD-2mimetic peptide(MDMP), the amino acid sequence is: CHGHDDDYSFCFSFEGILFPKGH-YR, including MD-2and TLR4binding domain (Cys95-Cys105) and MD-2and LPSbinding domain (Phe119-Lys132), and proved that MDMP can effectively inhibit excessiveinflammatory response induced by LPS in vitro and in vivo.This paper makes further study on the characteristics of molecular structure of MDMPon the basis of previous studies, through the9-Fmoc solid-phase synthesis method(Fmoc)for the synthesis of MDMP and its derivatives: including FITC-MDMP for cellularlocalization of and6XHis-MDMP for Pull Down the MDMP interaction protein/sites inRAW264.7cells; to explore the membrane localization features, and get interacting proteinon the membrane of RAW264.7cells by His-Pull Down, separate with Tricine-SDS-PAGE electrophoresis and identify protein by ESI-Q-TOF, discuss the possible mechanism ofin-depth study of MDMP antagonist excessive inflammatory response.In addition, in order to further fishing interacting protein of MDMP in the model ofhuman monocyte-derived macrophage, phorbol myristate acetate (PMA) was used to inducedifferentiation of THP-1into human mature macrophages as a cell model, to confirm theMDMP interaction proteins/sites on human cells are the same. In view of there is nouniform standard that the method the current of through PMA to induce THP-1todifferentiate into mature macrophages, for the establishment of stable and reliable tosimulate THP-1derived macrophage inflammatory model of endotoxin, we made someoptimization of the induction process, detection and analysis of influence of MD-2expression, localization and TNF-α, IL-6secretion process and the PMA in the induction ofTPH-1become mature human macrophages, and lay the foundation for the effect andmechanism of MDMP on human cells in the next stage.The main experimental results:1. Using EasyModeller software with MD-2as template, to access MDMP intuitivebased on the spatial structure theory of homology modeling and, and energy optimization,simulation structure can better show MD-2mimetic peptide (MDMP) level with secondaryor tertiary structure; the clear positioning of FITC-MDMP was observed by confocal laserscanning microscopy (Confocal laser scanning microscope, CLSM) in the membrane ofRAW264.7cells.2. Through the improvement and optimization of technical conditions of His-PullDown experiments to get the MDMP interaction proteins in RAW264.7cell membrane, toseparate interaction proteins with Tricine-SDS-PAGE gel electrophoresis, Coomassiebrilliant blue staining showed different band; further to identify different protein bands byESI-Q-TOF mass spectrometry,to analysis mass spectrometry data through the proteinwebsite database http://www.uniprot.org/, after preliminary screening of large interactioneffect of protein,the possible interaction proteins of MDMP include CH60(60kDa heatshock protein), S10A4(Protein S100-A4),RAB10(Ras-related protein Rab-10), IRAK2(Interleukin-1receptor-associated kinase-like2), ILRL1(Interleukin-1receptor-like1),MSRE (Macrophage scavenger receptor types I and II), and so on.3. PMA gradient dose (1~100ng/ml) can induce THP-1cells differentiation into mature macrophage successfully, mRNA and protein expression of MD-2detected byRT-qPCR and Western blot in mature THP-1cell macrophage were significantly higher thanbefore induction (P<0.01), TNF-α, IL-6secretion levels, in cell culture supernatant afterinduction with PMA gradient dose, detected by ELISA were significantly increased(P<0.01), sMD-2only before and after induction with significant statistical difference(P<0.01); laser scanning confocal microscope was used to observe the distribution of MD-2,which shows that the expression of MD-2protein ncrease significantly after induction ofPMA relative to before, no significant differences between the dose groups.Conclusion：1.Constructed unique MDMP’s secondary or tertiary structure spatial conformationwith Homology modeling system theory, which provides a theoretical basis for thesubsequent interaction sites; confocal laser scanning microscopy show exactly positioningcharacteristics of MDMP in RAW264.7cell membrane of mouse macrophage cells.2.Improved and Optimized His-Pull Down experimental conditions combined withTricine-SDS-PAGE electrophoretic was used to separation different bands and identified byESI-Q-TOF mass spectrometrycation, interacting protein of MDMP fished successfullyfrom the cell membrane of RAW264.7, which shows that the site of action of excessiveinflammatory MDMP antagonist induced by endotoxin is not single, there may be multiplesites of action and a variety of modes of action.3. Low dose of (1ng/ml) PMA is able to establish human THP-1macrophages modeleffectively, no obvious effects of structural expression on macrophage membrane receptorMD-2, and can not increase the basis of inflammatory factor TNF-α, IL-6secretion,conducive to study of MDMP anti-endotoxin effect and its mechanism... |
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