Application of saturation dye 2D-DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages.
Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.
Dupont
Annabelle
A
INSERM, U744, Lille, France, Institut Pasteur de Lille, Lille, France.
Chwastyniak
Maggy
M
Beseme
Olivia
O
Guihot
Anne-Laure
AL
Drobecq
Hervé
H
Amouyel
Philippe
P
Pinet
Florence
F
D000199:Actins / Q000096:biosynthesis; D051347:CapZ Actin Capping Protein / Q000096:biosynthesis; D002232:Carbocyanines; D002374:Catalase / Q000096:biosynthesis; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes; D018260:Gelsolin / Q000096:biosynthesis; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000378:metabolism* / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D008840:Microfilament Proteins / Q000096:biosynthesis; D009687:Nuclear Proteins / Q000096:biosynthesis; D020543:Proteome / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000096:biosynthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000199:Actins; C495842:CAPZA1 protein, human; D051347:CapZ Actin Capping Protein; D002232:Carbocyanines; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D018260:Gelsolin; D006360:Heat-Shock Proteins; D008077:Lipoproteins, LDL; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D020543:Proteome; C089187:cyanine dye 3; C085321:cyanine dye 5; C099252:oxidized low density lipoprotein; C066575:CAPG protein, human; D002374:Catalase; D011770:Pyruvate Kinase