title: LDL protein nitration: implication for LDL protein unfolding.
abstract: Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.
authors: Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA.
authors: Ryan T
authors: RT
authors: Hamilton
authors: Liana
authors: L
authors: Asatryan
authors: Jon T
authors: JT
authors: Nilsen
authors: Jose M
authors: JM
authors: Isas
authors: Timothy K
authors: TK
authors: Gallaher
authors: Tatsuya
authors: T
authors: Sawamura
authors: Tzung K
authors: TK
authors: Hsiai
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D053299:Apolipoprotein B-100 / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008054:Lipid Peroxides / Q000032:analysis; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008956:Models, Chemical; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D053299:Apolipoprotein B-100; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; D011506:Proteins; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D003545:Cysteine