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 α-helical structures and β 2 sheet as well as cysteine oxidation to cysteic acid in β 1 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. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 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. NIH Public Access

LOX-1-Mediated Effects on Vascular Cells in Atherosclerosis

“…In addition, the electronegative LDL fraction that actually represents oxidized lipids and unfolded apoB protein, a major protein component of LDL particles, was found to be extensively bound by LOX-1 but not the LDL receptor [32]. This LDL fraction though LOX-1 induces apoptosis of ECs by up-regulating expression of several proapoptotic proteins such as tumor necrosis factor (TNF-α), Bax, and Bcl-2-associated death promoter (Bad) and down-regulating B-cell lymphoma-extralarge (Bcl-xL) [33].…”

Endothelial GPR124 Exaggerates the Pathogenesis of Atherosclerosis by Activating Inflammation

“…GPR124 not only increases serum cholesterol and lipid deposition in the aortic sinus but also aggravates smooth muscle cell proliferation. Apolipoprotein B-100, the main LDL protein, has found to have tyrosine nitration and cysteine oxidation caused by peroxynitrite, leading to protein unfolding and contributing to atherosclerotic plaque deposition [39, 40]. Thus, we predicted that endothelial-specific GPR124 overexpression will increase peroxynitrite formation and affect several downstream lipids and proteins that are targets for protein tyrosine nitration.…”

Blood flow modulation of vascular dynamics

“…Furthermore, oxLDL activates JNK to promote Mn-SOD ubiquitination and protein degradation [50-52]. Using a targeted proteomic approach, we have gained mechanistic insights into shear-modulated relative ratios of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to ONOO· ;- formation and specific post-translational nitration in the α and β helices of the apoB100 protein [53, 55, 56]; namely, α-1 (Tyr 144 ), α-2 (Tyr 2524 ), β-2 (Tyr 3295 ), α-3 (Tyr 4116 ), and β-2 (Tyr 4211 ) [53]. Nitration leads to Apolipoprotein-B100 unfolding, and the modified particles are endocytosed by the scavenger receptors LOX-1, CD36 and SR-A (scavenger receptor-A), further contributing to the progression of atherosclerosis [57].…”

“…Using a targeted proteomic approach, we have gained mechanistic insights into shear-modulated relative ratios of reactive oxygen species (ROS) and reactive nitrogen species (RNS), leading to ONOO· ;- formation and specific post-translational nitration in the α and β helices of the apoB100 protein [53, 55, 56]; namely, α-1 (Tyr 144 ), α-2 (Tyr 2524 ), β-2 (Tyr 3295 ), α-3 (Tyr 4116 ), and β-2 (Tyr 4211 ) [53]. Nitration leads to Apolipoprotein-B100 unfolding, and the modified particles are endocytosed by the scavenger receptors LOX-1, CD36 and SR-A (scavenger receptor-A), further contributing to the progression of atherosclerosis [57]. Similarly, high-performance liquid chromatography analyses of EC exposed to OSS demonstrates increased expression of the catalytic subunits of NADPH oxidase gp91phox or Nox4 with an ensuing increase in O 2 · - production [3].…”