New targets of high-density lipoprotein therapy

PURPOSE OF REVIEW: Increasing attention has focused on the development of therapeutic strategies to promote the biologic activity of HDL particles, which possess a number of functional properties that contribute to their role in cardioprotection. Currently available therapies raise levels of HDL-cholesterol by relatively modest amounts. This review describes experimental strategies that promote HDL activity. RECENT FINDINGS: The functional quality of HDL may be more important than the absolute level of HDL-cholesterol found in the systemic circulation. This is supported by the observation that small rises in HDL-cholesterol with current therapies is associated with clinical benefit. This has major implications for the development of new therapies. A number of therapeutic strategies have been developed that promote reverse cholesterol transport, inhibit inflammatory events in the vessel wall, and modify remodeling of HDL particles within the systemic circulation. SUMMARY: A number of emerging therapies appear to promote the biologic activity of HDL. These agents can be administered as acute infusions in the setting of acute ischemic syndromes or as oral therapy for chronic prevention of cardiovascular disease.     

Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks.

In reconstituted high-density lipoproteins, apolipoprotein A-I and phosphatidylcholines combine to form disks in which the amphipathic alpha-helices of apolipoprotein A-1 bind to the edge of a lipid bilayer core, shielding the hydrophic lipid tails from the aqueous environment. We have employed experimental data, sequence analysis, and molecular modeling to construct an atomic model of such a reconstituted high-density lipoprotein disk consisting of two apolipoprotein A-I proteins and 160 palmitoyloleoylphosphatidylcholine lipids. The initial globular domain (1-47) of apolipoprotein A-I was excluded from the model, which was hydrated with an 8-A shell of water molecules. Molecular dynamics and simulated annealing were used to test the stability of the model. Both head-to-tail and head-to-head forms of a reconstituted high-density lipoprotein were simulated. In our simulations the protein contained and adhered to the lipid bilayer while providing good coverage of the lipid tails.     

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10 nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37 °C, but were almost completely denatured around 60 °C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Effects of salt on the thermal stability of human plasma high-density lipoprotein

High-density lipoproteins (HDL) mediate cholesterol removal and thereby protect against atherosclerosis. Mature spherical HDL contain the apolar lipid core and polar surface of proteins and phospholipids. Earlier, we showed that the structural integrity of HDL is modulated by kinetic barriers that prevent spontaneous protein dissociation and lipoprotein fusion and rupture. To determine the role of electrostatic interactions in the kinetic stability of mature HDL, here we analyze the effects of salt and pH on their thermal denaturation. In low-salt buffer at pH 5.7-7.7, HDL are highly thermostable. Increasing the salt concentration from 0 to 0.3 M NaCl causes low-temperature shifts in the calorimetric HDL transitions of up to -14 degrees C. This salt-induced destabilization leads to protein unfolding below 100 degrees C, facilitating the first Arrhenius analysis of HDL denaturation by circular dichroism spectroscopy. In 150 mM NaCl, two kinetic phases in HDL protein unfolding are observed: a faster phase with an activation energy E(a,fast) < or =15 kcal/mol and a slower phase with an E(a,slow) = 50 +/- 7 kcal/mol. Gel electrophoresis and electron microscopic data suggest that the faster phase involves partial protein unfolding but no significant protein dissociation or changes in HDL size, while the slower phase involves complete protein unfolding, partial protein dissociation, and HDL fusion. Hence, the slower phase may resemble HDL remodeling and fusion by plasma enzymes during metabolism. Analysis of the effects of various salts, sucrose, and pH suggests that HDL destabilization by salt results from ionic screening of favorable short-range electrostatic interactions such as salt bridges. Consequently, electrostatic interactions significantly contribute to the high thermostability of HDL in low-salt solutions.     

Proteomic analysis of high-density lipoprotein

Plasma lipoproteins, such as high-density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL-associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1-DE and 2-DE MALDI-TOF, isotope-coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL-associated proteins during isolation, a one-step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high-performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL-associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone-associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.     

Detection of oxidized high-density lipoprotein

This paper reviews working procedures for the separation and detection of oxidized high-density lipoproteins (ox-HDL) and their constituents. It begins with an introductory overview of structural alterations of the HDL particle and its constituents generated during oxidation. The main body of the review delineates various procedures for the isolation and detection of ox-HDL as well as the purification and separation of phosphatidylcholine metabolites and denatured apolipoproteins in the particle. The useful methods published more recently are picked up and the utility of the separation techniques is described. The last section covers a clinical evaluation of changes in these factors in ox-HDL as well as future directions of ox-HDL research.     

Inhibition of human and rat lipoprotein lipase by high-density lipoprotein

The hydrolysis in vitro of preactivated Intralipid (an artificial triacylglycerol-phospholipid emulsion) by rat adipose tissue lipoprotein lipase is inhibited by rat high-density lipoprotein (HDL). The aim of this work was to investigate whether human lipoprotein lipase was also inhibited, the mechanism of inhibition of the rat enzyme by HDL, and the role of the various individual apolipoproteins. Both human and rat lipoprotein lipase from post-heparin plasma are inhibited by HDL. This inhibition is considerably decreased if the HDL is first made 'apolipoprotein poor' by removal of some transferable apolipoproteins. In contrast, both native and apolipoprotein poor HDL inhibit the hydrolysis of Intralipid by rat hepatic lipase. Apolipoproteins C and E, either free in solution or attached to lipid vesicles, inhibit the hydrolysis of activated Intralipid by rat lipoprotein lipase to a maximum of 85% and 50%, respectively. Apolipoprotein A attached to vesicles gives little inhibition. HDL apolipoprotein and apolipoprotein C compete with the substrate for binding to lipoprotein lipase with apolipoprotein C having a higher affinity for the enzyme than HDL apolipoprotein. The inhibition of lipoprotein lipase by HDL can be explained by the association of the constituent apolipoproteins, in particular apolipoprotein C, with the enzyme so that there is less enzyme available to act on substrate.     

The interplay between size, morphology, stability, and functionality of high-density lipoprotein subclasses

High-density lipoprotein (HDL) mediates reverse cholesterol transport (RCT), wherein excess cholesterol is conveyed from peripheral tissues to the liver and steroidogenic organs. During this process HDL continually transitions between subclass sizes, each with unique biological activities. For instance, RCT is initiated by the interaction of lipid-free/lipid-poor apolipoprotein A-I (apoA-I) with ABCA1, a membrane-associated lipid transporter, to form nascent HDL. Because nearly all circulating apoA-I is lipid-bound, the source of lipid-free/lipid-poor apoA-I is unclear. Lecithin:cholesterol acyltransferase (LCAT) then drives the conversion of nascent HDL to spherical HDL by catalyzing cholesterol esterification, an essential step in RCT. To investigate the relationship between HDL particle size and events critical to RCT such as LCAT activation and lipid-free apoA-I production for ABCA1 interaction, we reconstituted five subclasses of HDL particles (rHDL of 7.8, 8.4, 9.6, 12.2, and 17.0 nm in diameter, respectively) using various molar ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, free cholesterol, and apoA-I. Kinetic analyses of this comprehensive array of rHDL particles suggest that apoA-I stoichiometry in rHDL is a critical factor governing LCAT activation. Electron microscopy revealed specific morphological differences in the HDL subclasses that may affect functionality. Furthermore, stability measurements demonstrated that the previously uncharacterized 8.4 nm rHDL particles rapidly convert to 7.8 nm particles, concomitant with the dissociation of lipid-free/lipid-poor apoA-I. Thus, lipid-free/lipid-poor apoA-I generated by the remodeling of HDL may be an essential intermediate in RCT and HDL's in vivo maturation.     

Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.     

The paradox of dysfunctional high-density lipoprotein

PURPOSE OF REVIEW: This review addresses how, in atherosclerosis or systemic inflammation, HDL can lose its usual atheroprotective characteristics and even paradoxically assume proinflammatory properties. RECENT FINDINGS: Specific chemical and structural changes within HDL particles can impede reverse cholesterol transport, enhance oxidation of LDL, and increase vascular inflammation. HDL may be viewed as a shuttle that can be either anti-inflammatory or proinflammatory, depending on its cargo of proteins, enzymes, and lipids. Some therapeutic approaches that reduce coronary risk, such as statins and therapeutic lifestyle changes, can favorably moderate the characteristics of proinflammatory HDL. In addition, apolipoprotein A-I mimetic peptides and other compounds that target functional aspects of HDL may offer novel approaches to reduction in cardiovascular risk. SUMMARY: Current data suggest that under some conditions HDL can become dysfunctional and even proinflammatory, but this characterization can change with resolution of systemic inflammation or use of certain treatments.     

Nanobiotechnology applications of reconstituted high density lipoprotein

High-density lipoprotein (HDL) plays a fundamental role in the Reverse Cholesterol Transport pathway. Prior to maturation, nascent HDL exist as disk-shaped phospholipid bilayers whose perimeter is stabilized by amphipathic apolipoproteins. Methods have been developed to generate reconstituted (rHDL) in vitro and these particles have been used in a variety of novel ways. To differentiate between physiological HDL particles and non-natural rHDL that have been engineered to possess additional components/functions, the term nanodisk (ND) is used. In this review, various applications of ND technology are described, such as their use as miniature membranes for solubilization and characterization of integral membrane proteins in a native like conformation. In other work, ND harboring hydrophobic biomolecules/drugs have been generated and used as transport/delivery vehicles. In vitro and in vivo studies show that drug loaded ND are stable and possess potent biological activity. A third application of ND is their use as a platform for incorporation of amphiphilic chelators of contrast agents, such as gadolinium, used in magnetic resonance imaging. Thus, it is demonstrated that the basic building block of plasma HDL can be repurposed for alternate functions.     

Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.     

Aging affects high-density lipoprotein composition and function

Most coronary deaths occur in patients older than 65 years. Age associated alterations in the composition and function of high-density lipoproteins (HDL) may contribute to cardiovascular mortality. The effect of advanced age on the composition and function of HDL is not well understood.     

Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments

The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on the X-ray crystal structure of a large, lipid-free fragment of apoA-I, a "belt model" was recently proposed. In this model, the helices of two antiparallel apoA-I molecules are extended in a circular arrangement and lie perpendicular to the phospholipid acyl chains. To obtain conclusive information on the spatial organization of apoA-I in discoidal HDL, we engineered three separate cysteine mutants of apoA-I (D9C, A124C, A232C) for specific labeling with the fluorescence probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). The labeled apoA-I was reconstituted into well-defined HDL complexes containing two molecules of protein and dipalmitoylphosphatidylcholine, and the complexes were used in three quantitative fluorescence resonance energy transfer (FRET) experiments to determine the distances between two specific sites in an HDL particle. Comparison of the distances measured by FRET (4.7-7.8 nm) with those predicted from the existing models indicated that neither the picket fence nor the belt model can account for the experimental results; rather, a hairpin folding of each apoA-I monomer with most helices perpendicular to the phospholipid acyl chains and a random head-to-tail and head-to-head arrangement of the two apoA-I molecules in the HDL particles are strongly suggested by the distance and lifetime data.     

Apolipoproteins as the basis for heterogeneity in high-density lipoprotein2 and high-density lipoprotein3. Studies by isoelectric focusing on agarose films

A method is described for the isoelectric focusing (IEF) of lipoproteins on thin films of agarose. Within a pH gradient of 4.60-5.30 both high-density lipoproteins 2 and 3 (HDL2 and HDL3) are resolved into more than 10 fractions which could be stained either for protein or for lipids. The isoelectric focusing patterns for HDL2 and HDL3 are similar although HDL2 appears richer in the more alkaline bands. Narrow film strips from the IEF separation of HDL2 and HDL3 were interfaced with various agarose plates containing antisera against apolipoproteins apoAI, apoAII and apoCIII either alone or in combination, to provide two-dimensional IEF immunoelectrophoresis patterns. This technique demonstrated that apoAI and apoAII were present throughout the IEF gel for both subclasses of HDL. It also provided evidence for the existence of lipoproteins containing both apoAI and apoAII and other lipoproteins present in the alkaline region of the gel which contained apoAI but no apoAII. ApoCIII was found mostly in acidic lipoproteins and was not distributed identically in HDL2 and HDL3. The lipoproteins separated by IEF on agarose were also analysed by two-dimensional IEF-SDS electrophoresis and the individual apolipoproteins were identified by reaction with antibodies to apolipoproteins AI, AII, CI, CII, CIII, D, and E. This technique confirmed that in IEF of HDL, apoAI extended throughout the spectrum of lipoproteins whereas apoE was only present in alkaline lipoproteins and apoD was only present in acidic lipoproteins. IEF on agarose of either HDL2 or HDL3 allowed us to collect eight different fractions, which have the same pI in either lipoprotein class. The apolipoprotein composition of each isolated band was analysed by electroimmuno-assays for apolipoproteins AI, AII, CI, CII, CIII, D, and E and the results expressed as the ratio of the measured apolipoprotein to measured apoAI. In both HDL2 and HDL3, acidic lipoprotein fractions were enriched in apoAII, apoCIII and apoD. ApoCII and apoCII were not similarly distributed in HDL2 and HDL3 subfractions whereas the apoCI distribution was similar in both classes. Noteworthy in all experiments was the difference in the distributions of apoCI, apoCII, and apoCIII in HDL2 and HDL3, which indicated that the existence of a lipoprotein containing simultaneously CI, CII and CIII can only account for a small fraction of these apolipoproteins. Therefore these experiments substantiate the theory of the protein basis of HDL heterogeneity and suggest that the majority of apolipoproteins are present in complexes which upon IEF result in lipoprotein fractions of identical pI for both HDL2 or HDL3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of obesity on high-density lipoprotein metabolism

Reduced levels of high-density lipoproteins (HDL) in non-obese and obese states are associated with increased risk for the development of coronary artery disease. Therefore, it is imperative to determine the mechanisms responsible for reduced HDL in obese states and, conversely, to examine therapies aimed at increasing HDL levels in these individuals. This paper examines the multiple causes for reduced HDL in obese states and the effect of exercise and diet--two non-pharmacologic therapies--on HDL metabolism in humans. In general, the concentration of HDL-cholesterol is adversely altered in obesity, with HDL-cholesterol levels associated with both the degree and distribution of obesity. More specifically, intra-abdominal visceral fat deposition is an important negative correlate of HDL-cholesterol. The specific subfractions of HDL that are altered in obese states include the HDL2, apolipoprotein A-I, and pre-beta1 subfractions. Decreased HDL levels in obesity have been attributed to both an enhancement in the uptake of HDL2 by adipocytes and an increase in the catabolism of apolipoprotein A-I on HDL particles. In addition, there is a decrease in the conversion of the pre-beta1 subfraction, the initial acceptor of cholesterol from peripheral cells, to pre-beta2 particles. Conversely, as a means of reversing the decrease in HDL levels in obesity, sustained weight loss is an effective method. More specifically, weight loss achieved through exercise is more effective at raising HDL levels than dieting. Exercise mediates positive effects on HDL levels at least partly through changes in enzymes of HDL metabolism. Increased lipid transfer to HDL by lipoprotein lipase and reduced HDL clearance by hepatic triglyceride lipase as a result of endurance training are two important mechanisms for increases in HDL observed from exercise.     

Impact of freezing on high-density lipoprotein functionality

Structural and functional properties of high-density lipoprotein (HDL) after short-term freezing in the presence or absence of 10% sucrose were compared with HDL stored at 4 °C. Freezing did not affect the size of HDL particles or their antiinflammatory and antioxidant properties. Freezing slightly impaired the ability of HDL to support cholesterol efflux from human macrophages, but this property was preserved when HDL was frozen in the presence of sucrose. Freezing also resulted in approximately 10% loss of HDL in the samples. We conclude that freezing HDL in the presence of 10% sucrose preserves its structural and functional properties.     

Structure and function of apolipoprotein A-I and high-density lipoprotein

Structural biology and molecular modeling have provided intriguing insights into the atomic details of the lipid-associated structure of the major protein component of HDL, apo A-I. For the first time, an atomic resolution map is available for future studies of the molecular interactions of HDL in such biological processes as ABC1-regulated HDL assembly, LCAT activation, receptor binding, reverse lipid transport and HDL heterogeneity. Within the context of this paradigm, the current review summarizes the state of HDL research.     

Oxidation of methionine residues affects the structure and stability of apolipoprotein A-I in reconstituted high density lipoprotein particles.

To determine the effect of oxidative damage to lipid-bound apolipoprotein A-I (apo A-I) on its structure and stability that might be related to previously observed functional disorders of oxidized apo A-I in high density lipoproteins (HDL), we prepared homogeneous reconstituted HDL (rHDL) particles containing unoxidized apo A-I and its commonly occurring oxidized form (Met-112, 148 bis-sulfoxide). The size of the obtained discoidal rHDL particles ranged from 9.0 to 10.0 nm and did not depend upon the content of the oxidized protein. Using circular dichroism methods, no change in the secondary structure of lipid-bound oxidized apo A-I was found. Isothermal and thermal denaturation experiments showed a significant destabilization of the oxidized protein to denaturation by guanidine hydrochloride or heat. This effect was observed with and without co-reconstituted apolipoprotein A-II. Limited tryptic digestion indicated that the central region of oxidatively damaged apo A-I becomes exposed to proteolysis in the rHDL particles. Implications of these data for apolipoprotein function are discussed.