Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7 macrophages
| Autor: | Gerald N. Rechberger, Elfriede Zenzmaier, Manfred Kollroser, Albin Hermetter, Bojana Stojcic, Ute Stemmer, Claudia Ramprecht |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
Poxno-PC
1-palmitoyl-2-(9-oxo-nonanoyl)-sn-glycero-3-phosphocholine aSMase acid sphingomyelinase Plasma protein binding medicine.disease_cause VDAC voltage dependent anion channel LDL low density lipoprotein PC phosphatidylcholine Mice chemistry.chemical_compound Tandem Mass Spectrometry Protein targeting PGPC 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine Electrophoresis Gel Two-Dimensional Paze-PC 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine oxPL oxidized phospholipids DMEM Dulbecco's modified Eagle's medium POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Molecular Structure Chemistry Phospholipid Ethers Serum Albumin Bovine mmLDL minimally modified LDL Aldehydophospholipid PS phosphatidylserine Lipoproteins LDL Biochemistry Covalent bond Phosphatidylcholines Electrophoresis Polyacrylamide Gel lipids (amino acids peptides and proteins) BY BODIPY™ HAEC human aortic endothelial cells Oxidation-Reduction Protein Binding Boron Compounds Oxidized LDL PE phosphatidylethanolamine TCA trichloroacetic acid Article Cell Line Schiff base ROS reactive oxygen species Phosphatidylcholine medicine PAPC 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Animals Humans POVPC 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine oxLDL oxidized LDL Phospholipid exchange TLR toll-like receptor Molecular Biology Fluorescent Dyes PAF platelet activating factor TLC thin layer chromatography Macrophages Albumin Membrane Proteins Proteins Lipid metabolism Cell Biology Atherosclerosis Functional proteomic analysis IL interleukin Microscopy Fluorescence Models Chemical Membrane protein Cell culture BSA bovine serum albumin Cattle MAPK mitogen-activated protein kinase |
| Zdroj: | Biochimica et Biophysica Acta |
| ISSN: | 1388-1981 |
| DOI: | 10.1016/j.bbalip.2012.01.014 |
| Popis: | The truncated phospholipids 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are oxidation products of 1-palmitoyl-2-arachidonoyl phosphatidylcholine. Depending on concentration and the extent of modification, these compounds induce growth and death, differentiation and inflammation of vascular cells thus playing a role in the development of atherosclerosis. Here we describe the import of fluorescent POVPC and PGPC analogs into cultured RAW 264.7 macrophages and the identification of their primary protein targets. We found that the fluorescent oxidized phospholipids were rapidly taken up by the cells. The cellular target sites depended on the chemical reactivity of these compounds but not on the donor (aqueous lipid suspension, albumin or LDL). The great differences in cellular uptake of PGPC and POVPC are a direct consequence of the subtle structural differences between both molecules. The former compound (carboxyl lipid) can only physically interact with the molecules in its immediate vicinity. In contrast, the aldehydo-lipid covalently reacts with free amino groups of proteins by forming covalent Schiff bases, and thus becomes trapped in the cell surface. Despite covalent binding, POVPC is exchangeable between (lipo)proteins and cells, since imines are subject to proton-catalyzed base exchange. Protein targeting by POVPC is a selective process since only a limited subfraction of the total proteome was labeled by the fluorescent aldehydo-phospholipid. Chemically stabilized lipid–protein conjugates were identified by MS/MS. The respective proteins are involved in apoptosis, stress response, lipid metabolism and transport. The identified target proteins may be considered primary signaling platforms of the oxidized phospholipid. Highlights ► Identification of primary protein targets of truncated phospholipids. ► Covalent modification of proteins by aldehydophospholipids. ► Exchange of fluorescent aldehydophospholipids between cells, lipoproteins and albumin. ► Imaging of fluorescent oxidized phospholipids in live cells. |
| Databáze: | OpenAIRE |
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