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LDL protein nitration: implication for LDL protein unfolding |
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| Authors: | Hamilton Ryan T Asatryan Liana Nilsen Jon T Isas Jose M Gallaher Timothy K Sawamura Tatsuya Hsiai Tzung K |
| Institution: | a Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA b Department of Biomedical Engineering and Division of Cardiovascular Medicine, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA c Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA d Department of Pharmaceutical Sciences and Division of Cell Biology, Department of Bioscience, National Cardiovascular Center Research Institute, Osaka University, Japan |
| 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 α-helical structures and β2 sheet as well as cysteine oxidation to cysteic acid in β1 sheet. Circular dichroism analyses showed that the α-helical content of LDL− was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL− showed greater content of β-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 α-helical structure and gain in β-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. |
| Keywords: | LDL Nitration Oxidation Endothelial cells |
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