Tyrosine 192 in apolipoprotein A-I is the major site of nitration and chlorination by myeloperoxidase, but only chlorination markedly impairs ABCA1-dependent cholesterol transport

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO(-)). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO(-). Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr(192) of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO(-), suggesting that solvent accessibility accounted in part for the reactivity of Tyr(192). The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) is the predominant site of nitration and chlorination when MPO or ONOO(-) oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease.     

Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dependent cholesterol efflux from macrophages

We recently reported that apolipoprotein A-I (apoA-I), the major protein component of high density lipoprotein, is a selective target for myeloperoxidase (MPO)-catalyzed nitration and chlorination in both and serum of subjects with cardiovascular disease. We further showed that the extent of both apoA-I nitration and chlorination correlated with functional impairment in reverse cholesterol transport activity of the isolated lipoprotein. Herein we used tandem mass spectrometry to map the sites of MPO-mediated apoA-I nitration and chlorination in vitro and in vivo and to relate the degree of site-specific modifications to loss of apoA-I lipid binding and cholesterol efflux functions. Of the seven tyrosine residues in apoA-I, Tyr-192, Tyr-166, Tyr-236, and Tyr-29 were nitrated and chlorinated in MPO-mediated reactions. Site-specific liquid chromatography-mass spectrometry quantitative analyses demonstrated that the favored modification site following exposure to MPO-generated oxidants is Tyr-192. MPO-dependent nitration and chlorination both proceed with Tyr-166 as a secondary site and with Tyr-236 and Tyr-29 modified only minimally. Parallel functional studies demonstrated dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid binding activities with apoA-I modification by MPO. Finally tandem mass spectrometry analyses showed that apoA-I in human atherosclerotic tissue is nitrated at the MPO-preferred sites, Tyr-192 and Tyr-166. The present studies suggest that site-specific modifications of apoA-I by MPO are associated with impaired lipid binding and ABCA1-dependent cholesterol acceptor functions, providing a molecular mechanism that likely contributes to the clinical link between MPO levels and cardiovascular disease risk.     

Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.     

Oxidation of apolipoprotein A-I by myeloperoxidase impairs the initial interactions with ABCA1 required for signaling and cholesterol export

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.     

Impact of HDL oxidation by the myeloperoxidase system on sterol efflux by the ABCA1 pathway

Protein oxidation by phagocytic white blood cells is implicated in tissue injury during inflammation. One important target might be high-density lipoprotein (HDL), which protects against atherosclerosis by removing excess cholesterol from artery wall macrophages. In the human artery wall, cholesterol-laden macrophages are a rich source of myeloperoxidase (MPO), which uses hydrogen peroxide for oxidative reactions in the extracellular milieu. Levels of two characteristic products of MPO-chlorotyrosine and nitrotyrosine-are markedly elevated in HDL from human atherosclerotic lesions. Here, we describe how MPO-dependent chlorination impairs the ability of apolipoprotein A-I (apoA-I), HDL's major protein, to transport cholesterol by the ATP-binding cassette transporter A1 (ABCA1) pathway. Faulty interactions between apoA-I and ABCA1 are involved. Tandem mass spectrometry and investigations of mutated forms of apoA-I demonstrate that tyrosine residues in apoA-I are chlorinated in a site-specific manner by chloramine intermediates on suitably juxtaposed lysine residues. Plasma HDL isolated from subjects with coronary artery disease (CAD) also contains higher levels of chlorinated and nitrated tyrosine residues than HDL from healthy subjects. Thus, the presence of chlorinated HDL might serve as a marker of CAD risk. Because HDL damaged by MPO in vitro becomes dysfunctional, inhibiting MPO in vivo might be cardioprotective.     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Modifying Apolipoprotein A-I by Malondialdehyde,but Not by an Array of Other Reactive Carbonyls,Blocks Cholesterol Efflux by the ABCA1 Pathway

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.     

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO⁻) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN⁻), yielding CNO⁻ and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on “lipid-poor” apoA-I forms via the nonenzymatic CNO⁻ pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.     

Myeloperoxidase-mediated oxidation of high-density lipoproteins: fingerprints of newly recognized potential proatherogenic lipoproteins

Substantial evidence supports the notion that oxidative processes participate in the pathogenesis of atherosclerotic heart disease. Major evidence for myeloperoxidase (MPO) as enzymatic catalyst for oxidative modification of lipoproteins in the artery wall has been suggested in numerous studies performed with low-density lipoprotein. In contrast to low-density lipoprotein, plasma levels of high-density lipoprotein (HDL)-cholesterol and apoAI, the major apolipoprotein of HDL, inversely correlate with the risk of developing coronary artery disease. These antiatherosclerotic effects are attributed mainly to HDL's capacity to transport excess cholesterol from arterial wall cells to the liver during 'reverse cholesterol transport'. There is now strong evidence that HDL is a selective in vivo target for MPO-catalyzed oxidation impairing the cardioprotective and antiinflammatory capacity of this antiatherogenic lipoprotein. MPO is enzymatically active in human lesion material and was found to be associated with HDL extracted from human atheroma. MPO-catalyzed oxidation products are highly enriched in circulating HDL from individuals with cardiovascular disease where MPO concentrations are also increased. The oxidative potential of MPO involves an array of intermediate-generated reactive oxygen and reactive nitrogen species and the ability of MPO to generate chlorinating oxidants-in particular hypochlorous acid/hypochlorite-under physiological conditions is a unique and defining activity for this enzyme. All these MPO-generated reactive products may affect structure and function of HDL as well as the activity of HDL-associated enzymes involved in conversion and remodeling of the lipoprotein particle, and represent clinically useful markers for atherosclerosis.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.     

Differential Stability of High-density Lipoprotein Subclasses: Effects of Particle Size and Protein Composition

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL₂ (large) and HDL₃ (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL₂. Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.     

Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Crystal structure of peroxynitrite-modified bovine Cu,Zn superoxide dismutase.

The crystal structure of bovine Cu,Zn superoxide dismutase modified with peroxynitrite (ONOO-) was determined by X-ray diffraction, utilizing the existing three-dimensional model of the native structure deposited in the Brookhaven Protein Data Bank (J. A. Tainer et al., J. Mol. Biol. 160, 181-217, 1982). The native structure and the modified derivative were refined to R factors of 19.0 and 18.7% respectively using diffraction data from 6.0 to 2.5 A. The major result after reaction with peroxynitrite was the appearance of electron density 1.45 A from a single epsilon carbon of Tyr-108, the only tyrosine residue in the sequence. Tyr-108 is a solvent-exposed residue 18 A from the copper atom in the active site. The electron density was consistent with nitration of Tyr-108 at one of the epsilon carbons to form 3-nitrotyrosine. We propose that the nitration occurs in solution by transfer of a nitronium-like species from the active site on one superoxide dismutase dimer to the Tyr-108 of a second dimer.     

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1

Apolipoprotein A-I (apoA-I) and an apoA-I peptide mimetic removed seeding molecules from human low density lipoprotein (LDL) and rendered the LDL resistant to oxidation by human artery wall cells. The apoA-I-associated seeding molecules included hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE). LDL from mice genetically susceptible to fatty streak lesion formation was highly susceptible to oxidation by artery wall cells and was rendered resistant to oxidation after incubation with apoA-I in vitro. Injection of apoA-I (but not apoA-II or murine serum albumin) into mice rendered their LDL resistant to oxidation within 3 h. Infusion of apoA-I into humans rendered their LDL resistant to oxidation within 6 h.We conclude that 1) oxidation of LDL by artery wall cells requires seeding molecules that include HPODE and HPETE; 2) LDL from mice genetically susceptible to atherogenesis is more readily oxidized by artery wall cells; and 3) normal HDL and its components can remove or inhibit the activity of lipids in freshly isolated LDL that are required for oxidation by human artery wall cells.     

Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function

We performed alignment of apolipoprotein A-I (apoA-I) sequences from 31 species of animals. We found there is specific conservation of salt bridge-forming residues in the first 30 residues of apoA-I and general conservation of a variety of residue types in the central domain, helix 2/3 to helix 7/8. In the lipid-associating domain, helix 7 and helix 10 are the most and least conserved helixes, respectively. Furthermore, eight residues are completely conserved: P66, R83, P121, E191, and P220, and three of seven Tyr residues in human apoA-I, Y18, Y115, and Y192, are conserved. Residue Y18 appears to be important for assembly of HDL. E191-Y192 represents the only completely conserved pair of adjacent residues in apoA-I; Y192 is a preferred target for site-specific oxidative modification within atheroma, and molecular dynamic simulations suggest that the conserved pair E191-Y192 is in a solvent-exposed loop-helix-loop. Molecular dynamics testing of human apoA-I showed that M112 and M148 interact with Y115, a microenvironment unique to human apoA-I. Finally, conservation of Arg residues in the α11/3 helical wheel position 7 supports several possibilities: interactions with adjacent phospholipid molecules and/or oxidized lipids and/or binding of antioxidant enzymes through cation-π orbital interactions. We conclude that sequence alignment of apoA-I provides unique insights into apoA-I structure-function relationship.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.