High-density Lipoprotein Components Research Articles

Real-Time Detection of Apolipoprotein A-I's Lipidation State by Fluorescence Resonance Energy Transfer

Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is the principal facilitator of cholesterol efflux from cholesterol-laden macrophages. Lipid-free apoA-I is the preferred substrate over lipid-associated apoA-I for cholesterol mobilization by the membrane transporter ATP binding cassette A1, which is responsible for more than 60% of cellular cholesterol efflux from cholesterol-laden macrophages. However, more than 95% of apoA-I is lipid-bound and associated with plasma mature HDL. To study the mechanisms that promote the production of lipid-free/lipid-poor (cholesterol-efflux capable) apoA-I in the arterial walls, we developed an apoA-I variant capable of reporting the lipidation-state of apoA-I in real-time.We employed fluorescence resonance energy transfer (FRET) to generate an apoA-I reporter with lipidation-state specific fluorescence. ApoA-I's four endogenous tryptophans (Trp) were substituted with phenylalanines and a single Trp was substituted in at position 19, as the FRET donor. A cysteine residue substituted in at position 136 was labeled with the fluorophore AEDANS, as the FRET acceptor. The resultant apoA-I variant, apoA-I:W19-AED136, was lipidated to varying degrees producing rHDL of different sizes. The fluorescence emission spectrum of lipid free apoA-I:W19-AED136 and each of the rHDL particles was collected. Structural differences in the conformation of lipid-free apoA-I and apoA-I associated with different rHDL sizes altered the relative positions of the FRET donor-acceptor pair, leading to lipidation state specific fluorescence “fingerprints”. Lipid-free apoA-I:W19-AED136 showed the highest degree of energy transfer (E=0.571), and apoA-I exhibited decreasing levels of energy transfer with increasing rHDL particle size (7.8 nm (E=0.387), 8.4 nm (E=0.0780), and 9.6 nm (E=0.0334)).ApoA-I:W19-AED136 was successfully used to measure the transition rate of apoA-I between lipid-associated and lipid-free states, potentially, the rate limiting step of macrophage cholesterol efflux in the atherosclerotic plaque.

Double Superhelix Model of High Density Lipoprotein

High density lipoprotein (HDL), the carrier of so-called "good" cholesterol, serves as the major athero-protective lipoprotein and has emerged as a key therapeutic target for cardiovascular disease. We applied small angle neutron scattering (SANS) with contrast variation and selective isotopic deuteration to the study of nascent HDL to obtain the low resolution structure in solution of the overall time-averaged conformation of apolipoprotein AI (apoA-I) versus the lipid (acyl chain) core of the particle. Remarkably, apoA-I is observed to possess an open helical shape that wraps around a central ellipsoidal lipid phase. Using the low resolution SANS shapes of the protein and lipid core as scaffolding, an all-atom computational model for the protein and lipid components of nascent HDL was developed by integrating complementary structural data from hydrogen/deuterium exchange mass spectrometry and previously published constraints from multiple biophysical techniques. Both SANS data and the new computational model, the double superhelix model, suggest an unexpected structural arrangement of protein and lipids of nascent HDL, an anti-parallel double superhelix wrapped around an ellipsoidal lipid phase. The protein and lipid organization in nascent HDL envisages a potential generalized mechanism for lipoprotein biogenesis and remodeling, biological processes critical to sterol and lipid transport, organismal energy metabolism, and innate immunity.

Abstract 5673: Engagement of the Sphingosine 1 Phosphate Receptor 1 Induces Macrophage Egression From Atherosclerotic Lesions in Apolipoprotein E Deficient Mice

Background: The regulation of macrophage (MØ) and T-cell content is crucial to atherosclerotic plaque stability. MØ have the potential to rapidly egress from plaques. Sphingosine 1-Phosphate (S1P) - an integral component of high density lipoproteins (HDL) - reduces leukocyte counts at sites of inflammation. We here tested if short-term treatment with the synthetic S1P analogue FTY720 or the selective S1P receptor 1 (S1P1) agonist AUY954 affects MØ and T-cells in plaques of ApoE deficient (ApoE −/− ) mice. Methods: ApoE −/− and wild type (WT) mice were fed a 16wk western diet. For 3D in vivo Fluorescence-Mediated-Tomography (FMT), 5×10 6 peritoneal eGFP-MØ labeled with near infrared fluorescent dye (DiR) were given IV 2d prior to 2 or 4d FTY720 (1 μ g/gBW/d), AUY954 (10 μ g/gBW twice/d), or vehicle (CTR) treatment. Baseline MØ content was assessed by FMT in a region of interest covering aortic-valve and -arch. On d2 and 4 MØ influx and egress from plaques into the blood were quantified. MØ egress into aortic lymphnodes (LN) was quantified by flow cytometry. T-cell (Thy1.2), MØ (CD68) and lipid content (Oil Red O) were assessed in plaques. Results: FMT revealed net MØ recruitment during 2 days in CTR (487±151%, d2 vs. d0, p<0.05, n=9) but in neither treatment group (FTY720: 67±11.9%, AUY954: 103±26.5%, p=n.s., n=9/5). After 4 days MØ related signal was reduced consistent with MØ egress from plaques in both treatment groups (FTY720: 40±9.5%, AUY954: 38±10.5%, d4 vs. d0, p<0.05, n=5) but not in CTR (14922.9%, p<n.s., n=5). CD68- and Oil-Red O-pos lesion area decreased by 33% and 26% (FTY720 vs. CTR, n=8, p<0.05). FTY720 but not AUY954 promoted MØ relocation into LN (FTY720: 7.21.9, AUY954: 1.270.73, CTR: 2.20.7, eGFP-MØ/10 5 cells, FTY720 vs. CTR & AUY954, n=5, p<0.05;). 2d treatment with FTY720 reduced plaque T-cell content (10±2.6 vs. 19±4.4 cells/mm 2 , FTY720 vs. CTR, p<0.05, n=5). Conclusions: Both FTY720 and AUY954 rapidly reduce macrophage content in atherosclerotic lesions indicating S1P1 involvement in this process. However, only FTY720 induces MØ relocation into regional LN and reduces plaque T-cell content. Reducing MØ and T-cells in the plaque through engagement of various S1P receptors (FTY720) vs. S1P1 represents a valid strategy for plaque stabilization.

Increased Apolipoprotein AI Production Rate and Redistribution of High-Density Lipoprotein Size Induced by Estrogen plus Progestin as Oral Contraceptive

The impact of estrogen plus progestin as an oral contraceptive on high density lipoprotein (HDL) apolipoprotein (apo) AI metabolism in humans is poorly understood. This study was designed to measure the in vivo effect of Moneva (30 microg ethinylestradiol, 75 microg gestodene) on HDL apoAI production rate and fractional catabolic rate. Using (13)C-leucine, we performed two kinetic studies in the fed state in 10 normolipidemic young women, before and 3 months after beginning Moneva. On Moneva, serum triglycerides increased by 12% (P = 0.03) in the fed state, whereas low-density lipoprotein and HDL cholesterol remained unchanged. HDL apoAI pool size and production rate were increased by 9.2% (67.3 +/- 7.1 vs. 61.6 +/- 6.7 mg x kg(-1); P = 0.05) and 26.5% (14.3 +/- 2.7 vs. 11.3 +/- 2.2 mg x kg(-1) x d(-1); P = 0.02), respectively. HDL apoAI fractional catabolic rate was not significantly modified. Three-month treatment by Moneva induced a shift of HDL size distribution from HDL2 toward HDL3 (HDL3 = 51.5 +/- 8.1 vs. 46.5 +/- 9.2% of total HDL; P = 0.02) and an increase in the proportion of apoAI among HDL components (38.8 +/- 4.3 vs. 34.4 +/- 2.8%; P = 0.01). Oral contraception by estrogen plus progestin induces changes in HDL apoAI metabolism characterized by an increase in production rate and pool size, with a higher proportion of HDL3 particles. Whether or not these changes are beneficial to prevent atherosclerosis has to be explored further.

Recent Advances on the Antiatherogenic Effects of HDL-Derived Proteins and Mimetic Peptides

The plasma levels of high-density lipoprotein (HDL) cholesterol are inversely correlated with the risk of atherosclerosis and cardiovascular disease (CVD) in humans. One of the major mechanisms whereby HDL particles protect against atherosclerosis is that of reverse cholesterol transport from atherosclerotic lesion macrophages to the liver. HDL particles also exhibit various antiatherogenic and cardioprotective effects by modulating the function of various cells including the cells of the artery wall and by expressing antioxidant, anti-inflammatory, antithrombotic and antiapoptotic effects. Most these effects are mediated by various lipid and protein HDL components. A plethora of studies have been conducted in order to shed light on the mechanisms by which each HDL component contributes to the functionality of this lipoprotein. The complete elucidation of these mechanisms will significantly contribute to current efforts focused on the development of therapeutic strategies to promote the antiatherogenic potency of HDL. The present review discusses current knowledge on the biological activities of the major apolipoproteins and enzymes associated with HDL, which may significantly contribute to the overall antiatherogenic and cardioprotective effects of this lipoprotein.

High-density lipoprotein metabolism and the human embryo

High-density lipoprotein (HDL) appears to be the dominant lipoprotein particle in human follicular fluid (FF). The reported anti-atherogenic properties of HDL have been attributed in part to reverse cholesterol transport. The discoveries of the scavenger receptor class B type I (SR-BI) and the ATP-binding cassette A1 lipid (ABCA1) transporter have generated studies aimed at unraveling the pathways of HDL biogenesis, remodeling and catabolism. The production of SR-BI and ABCA1 knockout mice as well as other lipoprotein metabolism-associated mutants has resulted in reduced or absent fertility, leading us to postulate the existence of a human hepatic-ovarian HDL-associated axis of fertility. Here, we review an evolving literature on the role of HDL metabolism on mammalian fertility and oocyte development. An extensive online search was conducted of published articles relevant to the section topics discussed. All relevant English language articles contained in Pubmed/Medline, with no specific time frame for publication, were considered for this narrative review. Cardiovascular literature was highly cited due to the wealth of relevant knowledge on HDL metabolism, and the dearth thereof in the reproductive field. Various vertebrate models demonstrate a role for HDL in embryo development and fertility. In our clinical studies, FF levels of HDL cholesterol and apolipoprotein AI levels were negatively associated with embryo fragmentation, but not with embryo cell cleavage rate. However, the HDL component, paraoxonase 1 arylesterase activity, was positively associated with embryo cell cleavage rate. HDL contributes to intra-follicular cholesterol homeostasis which appears to be important for successful oocyte and embryo development.

Dysfunctional proinflammatory high‐density lipoproteins confer increased risk of atherosclerosis in women with systemic lupus erythematosus

Women with systemic lupus erythematosus (SLE) have an increased risk of atherosclerosis. Identification of at-risk patients and the etiology underlying atherosclerosis in SLE remain elusive. The antioxidant capacity of normal high-density lipoproteins (HDLs) is lost during inflammation, and these dysfunctional HDLs might predispose individuals to atherosclerosis. The aim of this study was to determine whether dysfunctional proinflammatory HDL (piHDL) is associated with subclinical atherosclerosis in SLE. Carotid artery ultrasound was performed in 276 women with SLE to identify carotid plaques and measure intima-media thickness (IMT). The antioxidant function of HDL was measured as the change in oxidation of low-density lipoprotein after the addition of HDL cholesterol. Two antiinflammatory HDL components, paraoxonase 1 and apolipoprotein A-I, were also measured. Among the SLE patients, 48.2% were determined to have piHDL on carotid ultrasound, while 86.7% of patients with plaque had piHDL compared with 40.7% of those without plaque (P<0.001). Patients with piHDL also had a higher IMT (P<0.001). After multivariate analysis, the only factors found to be significantly associated with plaque were the presence of piHDL (odds ratio [OR] 16.1, P<0.001), older age (OR 1.2, P<0.001), hypertension (OR 3.0, P=0.04), dyslipidemia (OR 3.4, P=0.04), and mixed racial background (OR 8.3, P=0.04). Factors associated with IMT measurements in the highest quartile were the presence of piHDL (OR 2.5, P=0.02), older age (OR 1.1, P<0.001), a higher body mass index (OR 1.07, P=0.04), a cumulative lifetime prednisone dose>or=20 gm (OR 2.9, P=0.04), and African American race (OR 8.3, P=0.001). Dysfunctional piHDL greatly increases the risk of developing subclinical atherosclerosis in SLE. The presence of piHDL was associated with an increased prevalence of carotid plaque and with a higher IMT. Therefore, determination of piHDL may help identify patients at risk for atherosclerosis.

Low- and High-Density Lipoproteins Modulate Function, Apoptosis, and Proliferation of Primary Human and Murine Pancreatic β-Cells

A low high-density lipoprotein (HDL) plasma concentration and the abundance of small dense low-density lipoproteins (LDL) are risk factors for developing type 2 diabetes. We therefore investigated whether HDL and LDL play a role in the regulation of pancreatic islet cell apoptosis, proliferation, and secretory function. Isolated mouse and human islets were exposed to plasma lipoproteins of healthy human donors. In murine and human beta-cells, LDL decreased both proliferation and maximal glucose-stimulated insulin secretion. The comparative analysis of beta-cells from wild-type and LDL receptor-deficient mice revealed that the inhibitory effect of LDL on insulin secretion but not proliferation requires the LDL receptor. HDL was found to modulate the survival of both human and murine islets by decreasing basal as well as IL-1beta and glucose-induced apoptosis. IL-1beta-induced beta-cell apoptosis was also inhibited in the presence of either the delipidated protein or the deproteinated lipid moieties of HDL, apolipoprotein A1 (the main protein component of HDL), or sphingosine-1-phosphate (a bioactive sphingolipid mostly carried by HDL). In murine beta-cells, the protective effect of HDL against IL-1beta-induced apoptosis was also observed in the absence of the HDL receptor scavenger receptor class B type 1. Our data show that both LDL and HDL affect function or survival of beta-cells and raise the question whether dyslipidemia contributes to beta-cell failure and hence the manifestation and progression of type 2 diabetes mellitus.

Identification of DNA-dependent Protein Kinase as a Cofactor for the Forkhead Transcription Factor FoxA2

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear factor-4alpha. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2. We show that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se.

Age-Associated Decrease of High-Density Lipoprotein-Mediated Reverse Cholesterol Transport Activity

High-density lipoproteins (HDL) are considered atheroprotective in contrast to low-density lipoproteins (LDL), which are atherogenic in their oxidized form. A growing body of evidence suggests that HDL exert part of their antiatherogenic effect by counteracting LDL oxidation as well as their proinflammatory effect. However, a number of studies, carried over the past 30 years, have shown that cholesterol efflux plays a major role in the atheroprotective effects of HDL and cholesterol homeostasis. These studies have further identified the scavenger receptor type B-I (SR-BI), the adenosine triphosphate (ATP)-binding cassette transporters ATP-binding cassette subfamily A1 (ABCA1), ATP-binding cassette subfamily G1 (ABCG1) and ABCG4, the liver X receptor/retinoid X receptor (LXR/RXR) and peroxisome proliferator-activated receptorgamma(PPAR gamma) transcription factors, the HDL components apolipoprotein A-I (apoA-I), lecithin-cholesterol acyltransferase (LCAT), and phospholipids as additional mediators of cholesterol transport. Cholesterol efflux occurs via three independent pathways: (1) aqueous diffusion, (2) nonspecific efflux via SR-BI receptors, and (3) specific efflux via cholesterol-responsive members of the ABC superfamily. Whereas aqueous diffusion and scavenger receptor class B, type I (SR-BI)-mediated efflux transport free cholesterol to a wide variety of cholesterol acceptors (particles containing phospholipids, HDL, and lipidated apo-lipoproteins; LDL, etc), the ABCA1 pathway mediates the transport of cholesterol in a unidirectional manner, mainly to lipid-poor apoA-I. In contrast, the ABCG1 pathway is responsible for the transport of cholesterol to all the subfamily members of HDL. Although HDL-mediated cholesterol efflux is apoA-I-dependent, recent studies have suggested an involvement of the enzyme paraoxonase 1 (PON1). Cholesterol efflux is carried on by a number of factors such as genetic mutations, smoking, stress, and high-fat diets. It is attenuated with aging due to changes in the composition and structure of HDL, especially the phosphatidylcholine/sphingomyelin ratio, the fluidity of the phospholipidic layer, the concentration of apoA-I, and the activity of PON1. This review summarizes the findings that cholesterol homeostasis is disrupted with aging as a consequence of dysfunctional cholesterol efflux and the impairment of physiological functions.

Small, dense HDL 3 particles attenuate apoptosis in endothelial cells: pivotal role of apolipoprotein A‐I

Plasma high-density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low-density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC-1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC-1 against oxLDL-induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase-3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis-inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti-oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A-I (apoA-I) predominated, accounted for ∼70% of HDL anti-apoptotic activity. Furthermore, HDL reconstituted with apoA-I, cholesterol and phospholipid potently protected HMEC-1 from apoptosis. By contrast, modification of the content of sphingosine-1-phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid-poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA-I is pivotal to such protection.

Anionic Phospholipids Lose Their Procoagulant Properties When Incorporated into High Density Lipoproteins

Blood coagulation involves a series of enzymatic protein complexes that assemble on the surface of anionic phospholipid. To investigate whether apolipoproteins affect coagulation reactions, they were included during the preparation of anionic phospholipid vesicles using a detergent solubilization-dialysis method. Apolipoprotein components of high density lipoproteins, especially apolipoprotein A-I, had a pronounced anticoagulant effect. The anionic phospholipids lost their procoagulant effect when the vesicle preparation method was performed in the presence of apolipoprotein A-I. The anionic phospholipid-apolipoprotein A-I particles were 8-10 nm in diameter and contained around 60-80 phospholipid molecules, depending on the phospholipid composition. The phospholipids of these particles were unable to support the activation of prothrombin by factor Xa in the presence of factor Va and unable to support binding of factor Va, whereas binding of prothrombin and factor Xa were efficient. Phospholipid transfer protein was shown to mediate transfer of phospholipids from liposomes to apolipoprotein A-I-containing reconstituted high density lipoprotein. In addition, serum was also shown to neutralize the procoagulant effect of anionic liposomes and to efficiently mediate transfer of phospholipids from liposomes to either apolipoprotein A-I- or apolipoprotein B-containing particles. In conclusion, apolipoprotein A-I was found to neutralize the procoagulant properties of anionic phospholipids by arranging the phospholipids in surface areas that are too small to accommodate the prothrombinase complex. This anionic phospholipid scavenger function may be an important mechanism to control the exposure of such phospholipids to circulating blood and thereby prevent inappropriate stimulation of blood coagulation.

ApolipoproteinA-I, the major protein component of high density lipoproteins, inhibits tubulogenesis in vitro and neovascularisation in vivo

ApolipoproteinA-I, the major protein component of high density lipoproteins, inhibits tubulogenesis in vitro and neovascularisation in vivo

Impaired antioxidant activity of high-density lipoprotein in chronic kidney disease

Chronic kidney disease (CKD) is associated with accelerated atherosclerosis and increased mortality from cardiovascular disease. CKD results in oxidative stress, inflammation, and high-density lipoprotein (HDL) deficiency, which work in concert to promote atherosclerosis. Normal HDL confers protection against atherosclerosis by inhibiting the oxidation of lipids and lipoproteins and by retrieving surplus cholesterol and phospholipids from lipid-laden cells in the artery wall for disposal in the liver (reverse cholesterol transport). The plasma level of oxidized low-density lipoprotein (LDL) is increased, plasma HDL-cholesterol is reduced, and HDL maturation is impaired in CKD. This study was designed to examine the antioxidant properties of HDL in patients with CKD. In all, 32 stable hemodialysis-dependent patients and 13 age-matched controls were studied. HDL was isolated and used for determination of in vitro antioxidant activity. In addition, the plasma level of key components of HDL, namely paraoxonase (PON), glutathione peroxidase (GPX), platelet activating factor acetylhydrolase (PAF-AH), lecithin cholesterol acyltransferase (LCAT), and apolipoprotein A-I (ApoA-I), were measured. The end-stage renal disease (ESRD) patients exhibited significant reductions of HDL-cholesterol, ApoA-I (-41%), GPX (-50%), and LCAT (-60%) concentrations, and a decrease in PON (-30%) and GPX (-50%) activities. These results were accompanied by a marked reduction of antioxidant activity of HDL (-127%), which was unaffected by the hemodialysis procedure. Thus, in addition to diminished plasma HDL concentration, the composition and antioxidant activity of HDL are altered in CKD; these events can contribute to a heightened risk of atherosclerosis.

Apolipoprotein A-I, a hyperosmotic adaptation-related protein in ayu (Plecoglossus altivelis)

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum and participates in the reverse transport of cholesterol from tissues to the liver for excretion. 2DE gel and protein identification by MALDI-TOF-MS revealed that an apoA-I-like molecule was significantly decreased in the liver of ayu transferred from freshwater (FW) to brackish water (BW). A full-length cDNA clone of this protein was subsequently isolated. It contains 1209 bp with an open reading frame of 825 bp, coding for 275 amino acids with MW 31.1 kDa and pI 5.25. Ayu apoA-I had highest similarity (88.6% amino acid identity) to that of rainbow smelt. Phylogenetic analysis showed that vertebrate apoA-Is formed two major groups, representing mammalian-bird-amphibian (M-A) and fish apoA-Is respectively. Ayu apoA-I was most closely related to rainbow smelt apoA-I. In FW ayu, apoA-I transcripts were present in all tested tissues including brain, spleen, liver, kidney, gill, muscle, heart and intestine, and highest in liver, brain and intestine. In BW ayu, the expression levels of apoA-I gene were significantly decreased and almost totally inhibited in spleen, kidney, gill, heart and muscle. Significant down-regulation of proteins and mRNA in ayu transferred from FW to BW suggests an involvement in hyperosmotic regulation in this species.

Effect of Leucine to Phenylalanine Substitution on the Nonpolar Face of a Class A Amphipathic Helical Peptide on Its Interaction with Lipid: HIGH RESOLUTION SOLUTION NMR STUDIES OF 4F-DIMYRISTOYLPHOSPHATIDYLCHOLINE DISCOIDAL COMPLEX

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Apolipoprotein A-II augments monocyte responses to LPS by suppressing the inhibitory activity of LPS-binding protein

Lipopolysaccharide (LPS) binding protein (LBP) plays an important role in regulating leukocyte responses to LPS. Remarkably, it may either augment these responses at low LBP concentrations or inhibit them at high concentrations. We previously reported that native high-density lipoprotein (HDL) augments human monocyte responses to LPS by suppressing the inhibitory activity of high concentrations of LBP, a process that occurs before HDL can inhibit the response by subsequently binding and neutralizing LPS. We now show that this novel activity is conferred largely by an HDL component protein, apolipoprotein (apo)A-II. Purified apoA-II was highly active in our assays. We also found that HDL from apoA-II-deficient mice was almost completely inactive, whereas the activities of HDLs that lacked apoA-I, apoC-I, apoE, or apoC-III were similar to that of wild-type HDL. Decreased activity was also observed in rabbit HDL, which is naturally deficient in apoA-II. Incorporating human apoA-II into rabbit HDL increased its activity to levels found in human HDL. Our investigation of the mechanism of apoA-II activity revealed that LBP promoted the formation of large LPS aggregates with low bioactivity and that apoA-II inhibited the formation of these aggregates without binding and directly inhibiting LPS bioactivity. Our results suggest a novel pro-inflammatory activity of apoA-II that may help maintain sensitive host responses to LPS by suppressing LBP-mediated inhibition. Our findings also raise the possibility that the decline of plasma apoA-II during sepsis may help control the response to LPS.

Hepatic siRNA delivery using recombinant human apolipoprotein A-I in mice

Apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein (HDL), plays a key role in reverse cholesterol transport from peripheral tissues to liver or steroidogenic organs. Class B, type 1 scavenger receptor (SR-BI) is abundantly expressed in these target tissues and recognizes apo A-I of HDL for selective cholesteryl ester uptake. Recently, we reported the liver-targeting potential of plasma-derived apo A-I and the efficient delivery of therapeutic small interfering RNAs (siRNA) assembled with cationic liposome and apo A-I. In this study, we expressed and purified recombinant human apo A-I (rhapo A-I), low endotoxin grade, from an Escherichia coli expression system. The liver-targeting property of rhapo A-I was compared to that of plasma-derived apo A-I. Using a hepatitis C virus mouse model, intravenous administration of virus-specific siRNA with liposome and rhapo A-I significantly inhibited viral protein expression, demonstrating great promise for its use in clinical applications.

Identification of Proteins Adducted by Lipid Peroxidation Products in Plasma and Modifications of Apolipoprotein A1 with a Novel Biotinylated Phospholipid Probe

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallmanet al.Chem. Res. Toxicol.2007, 7, 227−23417305406), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS−MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS−MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10(E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.

Lysosulfatide Regulates the Motility of a Neural Precursor Cell Line Via Calcium-mediated Process Collapse

Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.