4-oxo-2-nonenal Adducts In HDL Are Elevated In Familial Hypercholesterolemia: Identification Of Modified Sites And Functional Consequences (original) (raw)
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Journal of Biological Chemistry
The lipid aldehyde 4-oxo-2-nonenal (ONE) is a highly reactive protein cross-linker derived from peroxidation of n-6 polyunsaturated fatty acids and generated together with 4-hydroxynonenal (HNE). Lipid peroxidation productmediated cross-linking of proteins in high-density lipoprotein (HDL) causes HDL dysfunction and contributes to atherogenesis. Although HNE is relatively well studied, the role of ONE in atherosclerosis and in modifying HDL is unknown. Here, we found that individuals with familial hypercholesterolemia (FH) had significantly higher ONE-ketoamide (lysine) adducts in HDL (54.6 ± 33.8 pmol/mg) than healthy controls (15.3 ± 5.6 pmol/mg). ONE crosslinked apolipoprotein A-I (apoA-I) on HDL at a concentration of > 3 mol ONE per 10 mol apoA-I (0.3 eq.), which was 100fold lower than HNE, but comparable to the potent protein crosslinker isolevuglandin. ONE-modified HDL partially inhibited HDL's ability to protect against lipopolysaccharide (LPS)-induced tumor necrosis factor α (Tnfα) and interleukin-1β (Il-1β) gene expression in murine macrophages. At 3 eq., ONE dramatically decreased apoA-I exchange from HDL, from ~46.5% to ~18.4% (P < 0.001).
Clinica Chimica Acta, 2011
Background: Genetic factors regulate both high-density lipoprotein (HDL) levels and functionality, thus affecting HDL antiatherogenic properties. We characterized the HDL antioxidant/anti-inflammatory properties and apoA-I-containing subpopulations in families with monogenic low HDL disorders. Methods: Subjects with mutations in apolipoprotein A-I (apoA-I), ATP-binding cassette transporter A1 (ABCA1) or lecithin:cholesterol acyltransferase (LCAT) and family controls were studied. HDL antioxidant/antiinflammatory properties were assayed by an in vitro fluorometric method and HDL-associated paraoxonase-1 (PON1), platelet activating factor-acetylhydrolase (PAF-AH), LCAT, malondialdehyde (MDA), PAF and serum amyloid A (SAA) were measured. ApoA-I-containing HDL subpopulations were analyzed by two-dimensional non-denaturing gel electrophoresis. Results: ApoA-I heterozygotes and subjects with partial or complete ABCA1 or LCAT deficiency had HDL with reduced antioxidant/anti-inflammatory properties and increased MDA levels. HDL-PON1 activity was reduced in apoA-I heterozygotes and in subjects with complete ABCA1 deficiency. HDL-PAF-AH activity was reduced in subjects with partial or complete ABCA1 deficiency or complete LCAT deficiency. HDL-LCAT activity was reduced in all LCAT mutation carriers. HDL-PAF levels were increased in apoA-I heterozygotes. HDL-SAA levels were increased in subjects with complete ABCA1 deficiency. ApoA-I, ABCA1 and LCAT heterozygotes were depleted of the large α1 HDL subpopulation. Subjects with complete LCAT deficiency showed mostly the small α4 HDL subpopulation and subjects with complete ABCA1 deficiency the α4 and preβ HDL subpopulations. Conclusions: This study shows that mutations in apoA-I, ABCA1 and LCAT have direct effect on the antioxidant/ anti-inflammatory properties of HDL. Furthermore, our study shows the effect of specific mutations on the apoA-I-containing HDL subpopulation profiles.
Journal of Biological Chemistry, 2020
Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well established. We have recently demonstrated the highly selective and efficient modification of highdensity lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-oxoalkenal phospholipids. In the current study, we report that γ-oxoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo. Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-oxoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H 2 O 2-NO 2 − system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein crosslinking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR −/− mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.
Biochemical and …, 1997
scarce indirect evidence advances the possibility of its Oxidized lipoproteins have been involved in the occurrence, i.e., it is known that HDL is modified in an pathogenesis of atherosclerosis and atherosclerotic leoxidatively way by human polymorphonuclear leukosions contain oxidized low density lipoprotein. Concytes probably due to the actions of superoxide anions versely, the presence of oxidized high density lipopro-(4). Another potential source of oxidized HDL could be tein (HDL) in vivo has not been clearly established. HDL of the interstitial fluid bathing the epidermis of Oxidation of HDL in vitro models produces an increase uv-exposed skin (5).
Circulation Research, 2004
Apolipoprotein A-II (apoA-II), the second major high-density lipoprotein (HDL) apolipoprotein, has been linked to familial combined hyperlipidemia. Human apoA-II transgenic mice constitute an animal model for this proatherogenic disease. We studied the ability of human apoA-II transgenic mice HDL to protect against oxidative modification of apoB-containing lipoproteins. When challenged with an atherogenic diet, antigens related to low-density lipoprotein (LDL) oxidation were markedly increased in the aorta of 11.1 transgenic mice (high human apoA-II expressor). HDL from control mice and 11.1 transgenic mice were coincubated with autologous very LDL (VLDL) or LDL, or with human LDL under oxidative conditions. The degree of oxidative modification of apoB lipoproteins was then evaluated by measuring relative electrophoretic mobility, dichlorofluorescein fluorescence, 9-and 13hydroxyoctadecadienoic acid content, and conjugated diene kinetics. In all these different approaches, and in contrast to control mice, HDL from 11.1 transgenic mice failed to protect LDL from oxidative modification. A decreased content of apoA-I, paraoxonase (PON1), and platelet-activated factor acetyl-hydrolase activities was found in HDL of 11.1 transgenic mice. Liver gene expression of these HDL-associated proteins did not differ from that of control mice. In contrast, incubation of isolated human apoA-II with control mouse plasma at 37°C decreased PON1 activity and displaced the enzyme from HDL. Thus, overexpression of human apoA-II in mice impairs the ability of HDL to protect apoB-containing lipoproteins from oxidation. Further, the displacement of PON1 by apoA-II could explain in part why PON1 is mostly found in HDL particles with apoA-I and without apoA-II, as well as the poor antiatherogenic properties of apoA-II-rich HDL. (Circ Res. 2004;95:789-797.)
Journal of Biological Chemistry, 1997
15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15lipoxygenase (rhLO) can be ascribed largely to ␣-tocopherol (␣-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A 2 (PLA 2) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE-O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu 2؉ ions. Analyses of the regio-and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. ␣-Tocopheroxyl radical (␣-TO ⅐) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA 2. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing ␣-TO ⅐ and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.
Characterization of covalent modifications of HDL apoproteins by endogenous oxidized phospholipids
Free Radical Biology and Medicine, 2017
High density lipoprotein (HDL) is cardioprotective, unless it is pathologically modified under oxidative stress. Covalent modifications of lipid-free apoA-I, the most abundant apoprotein in HDL, compromise its atheroprotective functions. HDL is enriched in oxidized phospholipids (oxPL) in vivo in oxidative stress. Furthermore, oxidized phospholipids can covalently modify HDL apoproteins. We have now carried out a systematic analysis of modifications of HDL apoproteins by endogenous oxPL. Human HDL or plasma were oxidized using a physiologically relevant MPO-H 2 O 2-NO 2 − system or AIPH, or were exposed to synthetic oxPL. Protein adduction by oxPL was assessed using LC-MS/MS and MALDI-TOF MS. The pattern of HDL apoprotein modification by oxPL was independent of the oxidation systems used. ApoA-I and apoA-II were the major modification targets. OxPL with a γ-hydroxy (or oxo)-alkenal were mostly responsible for modifications, and the Michael adduct was the most abundant adduct. Histidines and lysines in helices 5-8 of apoA-I were highly susceptible to oxPL modifications, while lysines in helices 1, 2, 4 and 10 were resistant to modification by oxPL. In plasma exposed to oxidation or synthetic oxPL, oxPL modification was highly selective, and four histidines (H155, H162, H193 and H199) in helices 6-8 of apoA-I were the main modification target. H710 and H3613 in apoB-100 of LDL and K190 of human serum albumin were also modified by oxPL but to a lesser extent. Comparison of oxPL with short chain aldehyde HNE using MALDI-TOF MS demonstrated high selectivity and efficiency of oxPL in the modification of HDL apoproteins. These findings provide a novel insight into a potential mechanism of the loss of atheroprotective function of HDL in conditions of oxidative stress.
Journal of Lipid Research, 2004
For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/antiinflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential. -Navab, M.,