Lipid-free apoA-I Research Articles

High-Density Lipoproteins Inhibit Vascular Endothelial Inflammation by Increasing 3β-Hydroxysteroid-Δ24 Reductase Expression and Inducing Heme Oxygenase-1

Lipid-free apolipoprotein (apo) A-I and discoidal reconstituted high-density lipoproteins (rHDL) containing apoA-I, (A-I)rHDL, inhibit vascular inflammation by increasing 3β-hydroxysteroid-Δ24 reductase (DHCR24) expression. To determine whether the lipid-free apoA-I-mediated and (A-I)rHDL-mediated increase in DHCR24 expression induces the cytoprotective and potentially cardioprotective enzyme, heme oxygenase-1 (HO-1). In vivo: A single intravenous infusion of lipid-free apoA-I (8 mg/kg) administered 24 hours before inserting a nonocclusive periarterial carotid collar into New Zealand White rabbits decreased collar-induced endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression, reduced intima/media neutrophil infiltration, and increased DHCR24 and HO-1 mRNA levels. Knockdown of vascular DHCR24 and HO-1 and systemic administration of tin-protoporphyrin-IX, an HO inhibitor, abolished these anti-inflammatory effects. In vitro: Preincubation of human coronary artery endothelial cells with (A-I)rHDL before activation with tumor necrosis factor-α increased DHCR24 and HO-1 mRNA levels and inhibited cytokine-induced vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression. Transfection of the cells with DHCR24 and HO-1 small interfering RNA and tin-protoporphyrin-IX treatment abolished these effects. The (A-I)rHDL-mediated induction of HO-1 was reduced in human coronary artery endothelial cells transfected with DHCR24 small interfering RNA. Transfection of human coronary artery endothelial cells with HO-1 small interfering RNA and tin-protoporphyrin-IX treatment did not inhibit the (A-I)rHDL-mediated increase in DHCR24 expression. Inhibition of phosphatidylinositol 3-kinase/Akt reduced the (A-I)rHDL-mediated increase in HO-1, but not DHCR24 expression. The activation of phosphatidylinositol 3-kinase/Akt by (A-I)rHDL was decreased in human coronary artery endothelial cells that were transfected with DHCR24 small interfering RNA. Lipid-free apoA-I and (A-I)rHDL inhibit inflammation by increasing DHCR24 expression, which, in turn, activates phosphatidylinositol 3-kinase/Akt and induces HO-1.

DETERMINING THE FOLDING DOMAIN OF LIPID FREE APOA-I IN SOLUTION BY MUTATION†

DETERMINING THE FOLDING DOMAIN OF LIPID FREE APOA-I IN SOLUTION BY MUTATION†

Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I

HDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a "presentation tunnel" gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain "sticky." Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and "zip-locking" apoA-I into its full LL5/5 registration.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6nm) and small (7.8nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

OS041. Apolipoprotein A-I protects normal integration of the trophoblast into endothelial cellular networks in an in vitro model of preeclampsia

Failure of the trophoblast to appropriately invade uterine spiral arteries is thought to be an initiating event in preeclampsia, a disorder associated with endothelial dysfunction. A dyslipidemia characterised by low plasma levels of high density lipoproteins (HDL) and elevated triglycerides has also been described in preeclampsia. The pro-inflammatory cytokine TNF-α inhibits trophoblast invasion of uterine endothelial cells. Previous work using an in vitro JEG-3 cell/Uterine endothelial cell co-culture model investigated the effect of apoliopoprotein A-I, the main apolipoprotein component of HDL, on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. These effects are now investigated using the human invasive trophoblast cell line HTR-8/SVneo. This study asks if apoA-I, which has established anti-inflammatory properties, can protect against the deleterious effect of TNF-α on trophoblast-endothelial cell interactions. The in vitro trophoblast-uterine endothelial cell co-culture model was used to investigate the effect of apoA-I on trophoblast incorporation into endothelial tubules in the presence and absence of TNF-α. Uterine endothelial cells were pre-incubated with lipid free apoA-I (final apoA-I concentration 1 mg/mL) for 16h prior to seeding on matrigel coated plates. Tubules formed within 4h. Fluorescence-labelled HTR-8/SVneo trophoblast cells were then co-cultured with the endothelial cells±TNF-α (final concentration of 0.2ng/mL). Bright field and fluorescent images were captured after 24h. The effect of TNF-α on HTR-8/SVneo cell invasion was quantified with Image J software. Integration of HTR-8/SVneo trophoblast cells into uterine endothelial tubular networks was also imaged using live cell imaging techniques (Zeiss Axiovert). TNF-α inhibited HTR-8/SVneo (trophoblast) cell integration into endothelial tubular structures by 24.1±3.7% p<0.001. This effect was reversed when the endothelial cells were pre-incubated for 16h with lipid free apoA-I (p<0.001 compared to non-incubated cells). Live cell images of the co-culture clearly demonstrate a disruption to the normal integration of trophoblast into endothelial tubular structures in the presence of TNF-α. The protective effect conferred by pre-incubation of endothelial cells with apoA-I against TNF-α is also clearly visible. Apolipoprotein A-I has been shown to enhance trophoblast-endothelial cell integration in the presence of a pro-inflammatory stimulus. A healthy lipid profile may affect pregnancy outcomes by priming endothelial cells in preparation for trophoblast invasion.

Abstract 4: Apolipoprotein (apo) A-I Increases Insulin-Dependent Glucose Disposal in Skeletal Muscle by Stimulating the PI3K/Akt/AS160 Signaling Pathway

Introduction: Inhibition of CETP increases plasma high density lipoprotein (HDL) levels and improves glycaemic control in people with type 2 diabetes. Recent in vitro studies have also established that the main HDL apolipoprotein, apoA-I, increases insulin secretion from pancreatic β-cells. However, the effect of apoA-I on glucose uptake by skeletal muscle, the main site of glucose disposal, is not understood. Hypothesis: Lipid-free apoA-I increases insulin-dependent glucose disposal in skeletal muscle. Methods: Primary human skeletal muscle cells were differentiated into myotubes, then incubated for 16 h with lipid-free apoA-I (final concentration 1 mg/ml). Glucose uptake was initiated by adding a tracer amount of [3H]2-deoxy-glucose to the myotubes and continuing the incubation for up to 5 h with or without insulin (0.1 μM final conc.). IRS-1, PI3K, Akt and AS160 protein levels were quantified by subjecting total cell lysates to SDS-PAGE and immunoblotting. Results: Incubation of skeletal muscle myotubes in the absence or presence of insulin resulted in [3H]2-deoxy-glucose uptake of 1000 and 1103±68.9 pmol/mg protein/h, respectively (p&lt;0.1). Pre-incubation of the myotubes with lipid-free apoA-I, followed by incubation in the absence or presence of insulin resulted in [3H]2-deoxy-glucose uptake of 1089±51.35 and 1358±57.9 pmol/mg protein/h (p&lt;0.01), respectively. The apoA-I-mediated increase in insulin-dependent glucose uptake into the myotubes was time- and concentration-dependent. Incubation of myotubes in the presence of apoA-I and insulin increased IRS-1 phosphorylation and total IRS-1 protein by 27.8±5% and 30.8±1.3% (p&lt;0.01 for both), respectively. PI3K, Akt and AS160 phosphorylation increased by 55.1±22%, 52.5±20.9%, and 32±8%, respectively (p&lt;0.01 for all) without any change in total protein. Conclusion: Lipid-free apoA-I increases insulin-dependent glucose uptake into human skeletal muscle cells by stimulating the PI3K/Akt/AS160 insulin signaling pathway. These results suggest that HDL-raising therapies may improve glucose disposal in skeletal muscle, and thus be beneficial in type 2 diabetes.

Abstract 408: Essential Domain Transition Involving Central Helical Repeats Prevent Lipid-Free ApoA-I from Acquiring Lipid

Plasma high density lipoprotein (HDL) concentration is negatively correlated with the occurrence of coronary heart disease in the human population. Because apoA-I is the main protein constituent of HDL, a thorough understanding of apoA-I structural topology is essential for elucidating its ability to package and mobilize cholesterol for catabolism. To determine which of the 10 helical repeats within apoA-I participates in the structural transitions that drive unfolding of the 4-helix bundle, we created several loss of function mutations. Published three-dimensional coordinates of full-length lipid-free apoA-I were used to predict amino acids having spatial separations of 3-5Å within the 4-helix bundle. Based on these predictions, we proposed that specific targeted double cysteine residue substitutions could form disulfide linkages and prevent “opening” of a critical domain required for unfolding of the apoA-I 4-helix bundle when exposed to lipid. To test the importance of helical repeats 4, 5, 6 and 7, double cysteine mutants D103C-R177C apoA-I and F104C-H162C apoA-I were created, expressed and purified using established procedures. Mass spectrometry combined with MS/MS sequencing was used to verify the “locked” disulfide form of each double cysteine substitution mutants. Using 20% SDS-PAGE we show that electrophoretic mobility-shift distinguishes between “locked” or -DTT versus “unlocked” or +DTT form for each of the mutant apoA-I proteins. Particle formation was tested for each mutant by measuring the formation of recombinant HDL (rHDL) using cholate dialysis, as well as, the formation of nascent HDL (nHDL) from ABCA1 expressing cells. Examination of the size of rHDL and nHDL particles formed suggests that unfolding of lipid-free apoA-I to acquire lipid involves the “locked” or restricted helical repeats 4-7. In conclusion, when both double cysteine apoA-I mutants exist in their “locked” conformation evidence of impaired particle formation was observed confirming the existence of the apoA-I 4 helix bundle in lipid free state and the role of central helical repeats 4-7 in lipid binding and particle formation.

Abstract 34: Apolipoprotein A-I Increases Insulin Secretion from β-cells via a Protein Kinase A Dependent Mechanism

Introduction: The progression to hyperglycaemia in type 2 diabetes is marked by β-cell insulin secretory dysfunction and cell loss. We have previously demonstrated that apolipoprotein (apo) A-I, the major protein constituent of high density lipoproteins (HDL) increases insulin expression and secretion from β-cells. Clinical data also suggests that pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes. With the current interest in HDL raising therapeutics, defining the mechanism by which apoA-I acts on insulin secretion is of importance. Objective: To elucidate the cell signalling events responsible for increasing insulin secretion from pancreatic β-cells treated with lipid-free apoA-I. Methods: Ins-1E (rat insulinoma) cells were pre-treated for 30 min with the Protein kinase A (PKA) specific inhibitor H89 (20 μM), soluble and transmembrane adenyl cyclase specific inhibitors (KH7, 30 μM and 2’5’ dideoxyadenosine, 50 μM, respectively) or vehicle control, then incubated for 1 h with lipid-free apoA-I (final concentration 1 mg/mL) under both basal (2.8 mM) and high (25 mM) glucose conditions. The insulin concentration in the culture supernatants was determined by radioimmunoassay and the cells were either lysed for protein analysis by western blotting or treated with 0.1 M HCl for determining cAMP by enzyme immunoassay. Results: Incubation of Ins-1E cells with apoA-I increased insulin secretion up to 3-fold. This increase was no longer apparent when the cells were pre-treated with H89. Incubation with apoA-I increased cAMP accumulation in Ins-1E cells 2.5-fold. This increase was totally inhibited when the cells were pre-incubated with 2’5’ dideoxyadenosine but not by KH7, indicating that transmembrane adenyl cyclase(s) are responsible for this response. ApoA-I also activated the small GTPase Cdc42, which may link cell surface apoA-I receptors with transmembrane adenyl cyclases. Conclusion: ApoA-I increases insulin secretion from pancreatic β-cells via a PKA-dependent mechanism involving transmembrane, but not soluble, adenyl cyclases and possibly Cdc42. This provides a possible explanation of the clinical observations that increased HDL may be beneficial in type 2 diabetes.

Abstract 3: Site-Specific Chlorination of ApoA-I, the Major HDL Protein, Is Associated with Impaired Sterol Efflux and Is Markedly Increased in Subjects with Coronary Artery Disease

3-Chlorotyrosine is a specific product of myeloperoxidase (MPO), a heme protein expressed by macrophages in human atherosclerotic lesions. We have previously shown in vitro that chlorination of Tyr192 (3-chloroTyr192) together with methionine oxidation of lipid-free apoA-I impairs the apolipoprotein’s ability to promote cholesterol efflux from macrophages by the ABCA1 pathway. Using a sensitive and quantitative LC-MS/MS approach with selected reaction monitoring (SRM), we have recently demonstrated that Tyr192 is the major site of chlorination in apoA-I of HDL isolated from human atherosclerotic lesions. In a prospective study, we determined if: i) MPO contributes to the chlorination of apoA-I in circulating HDL; and ii) 3-chloroTyr192 serves as a marker for clinically significant cardiovascular disease. We quantified HDL oxidation and sterol efflux in three groups: i) stable CAD subjects, ii) acute coronary syndrome (ACS) subjects, and iii) healthy control subjects (N=20 per group). SRM analysis demonstrated that levels of 3-chloroTyr192 were markedly elevated in HDL isolated from subjects with CAD or ACS (p=0.009 and p=0.01 vs. control subjects, respectively). A key cardioprotective effect of HDL is its ability to promote cholesterol efflux from macrophages by the ABCA1 pathway. We found that the ABCA1-dependent sterol efflux capacity of HDL isolated from CAD and ACS subjects was significantly impaired. Importantly, sterol efflux capacity associated inversely with levels of 3-chloroTyr192 (R=-0.30, p=0.02), suggesting that oxidation of HDL by MPO contributes to the impaired cholesterol efflux capacity of HDL. Receiver operating characteristic curve analysis revealed that levels of 3-chloroTyr192 distinguished between CAD or ACS subjects and control subjects (AUC 0.76, p=0.0008 and AUC 0.73, p=0.004, respectively). These observations strongly suggest that MPO contributes to the generation of dysfunctional HDL in subjects with clinically significant atherosclerosis. They further indicate that quantification of 3-chloroTyr192 levels in HDL may provide a new approach to the diagnosis and treatment of CAD and ACS.

The fibrillogenic L178H variant of apolipoprotein A-I forms helical fibrils

A number of amyloidogenic variants of apoA-I have been discovered but most have not been analyzed. Previously, we showed that the G26R mutation of apoA-I leads to increased β-strand structure, increased N-terminal protease susceptibility, and increased fibril formation after several days of incubation. In vivo, this and other variants mutated in the N-terminal domain (residues 26 to ∼90) lead to renal and hepatic accumulation. In contrast, several mutations identified within residues 170 to 178 lead to cardiac, laryngeal, and cutaneous protein deposition. Here, we describe the structural changes in the fibrillogenic variant L178H. Like G26R, the initial structure of the protein exhibits altered tertiary conformation relative to wild-type protein along with decreased stability and an altered lipid binding profile. However, in contrast to G26R, L178H undergoes an increase in helical structure upon incubation at 37°C with a half time (t1/2) of about 12 days. Upon prolonged incubation, the L178H mutant forms fibrils of a diameter of 10 nm that ranges in length from 30 to 120 nm. These results show that apoA-I, known for its dynamic properties, has the ability to form multiple fibrillar conformations, which may play a role in the tissue-specific deposition of the individual variants.

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.

Lipid-bound apolipoproteins in tyrosyl radical-oxidized HDL stabilize ABCA1 like lipid-free apolipoprotein A-I

BackgroundATP-binding cassette transporter A1 (ABCA1) mediates the lipidation of exchangeable apolipoproteins, the rate-limiting step in the formation of high density lipoproteins (HDL). We previously demonstrated that HDL oxidized ex vivo by peroxidase-generated tyrosyl radical (tyrosylated HDL, tyrHDL) increases the availability of cellular cholesterol for efflux and reduces the development of atherosclerosis when administered to apolipoprotein E-deficient mice as compared to treatment with control HDL.ResultsIn the current study we determined that tyrHDL requires functional ABCA1 for this enhanced activity. Like lipid-free apolipoprotein A-I (apoA-I), tyrHDL increases total and cell surface ABCA1, inhibits calpain-dependent and -independent proteolysis of ABCA1, and can be bound by cell surface ABCA1 in human skin fibroblasts. Additionally, tyrHDL apoproteins are susceptible to digestion by enteropeptidase like lipid-free apoA-I, but unlike lipid-bound apoA-I on HDL, which is resistant to proteolysis.ConclusionsThese results provide the first evidence that lipid-bound apolipoproteins on the surface of spherical HDL particles can behave like lipid-free apoA-I to increase ABCA1 protein levels and activity.

The “beta-clasp” model of apolipoprotein A-I — A lipid-free solution structure determined by electron paramagnetic resonance spectroscopy

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. 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).

The Crystal Structure of the C-Terminal Truncated Apolipoprotein A-I Sheds New Light on Amyloid Formation by the N-Terminal Fragment

Apolipoprotein A-I (apoA-I) is the main protein of plasma high-density lipoproteins (HDL, or good cholesterol) that remove excess cell cholesterol and protect against atherosclerosis. In hereditary amyloidosis, mutations in apoA-I promote its proteolysis and the deposition of the 9-11 kDa N-terminal fragments as fibrils in vital organs such as kidney, liver, and heart, causing organ damage. All known amyloidogenic mutations in human apoA-I are clustered in two residue segments, 26-107 and 154-178. The X-ray crystal structure of the C-terminal truncated human protein, Δ(185-243)apoA-I, determined to 2.2 Å resolution by Mei and Atkinson, provides the structural basis for understanding apoA-I destabilization in amyloidosis. The sites of amyloidogenic mutations correspond to key positions within the largely helical four-segment bundle comprised of residues 1-120 and 144-184. Mutations in these positions disrupt the bundle structure and destabilize lipid-free apoA-I, thereby promoting its proteolysis. Moreover, many mutations place a hydrophilic or Pro group in the middle of the hydrophobic lipid-binding face of the amphipathic α-helices, which will likely shift the population distribution from HDL-bound to lipid-poor/free apoA-I that is relatively unstable and labile to proteolysis. Notably, the crystal structure shows segment L44-S55 in an extended conformation consistent with the β-strand-like geometry. Exposure of this segment upon destabilization of the four-segment bundle probably initiates the α-helix to β-sheet conversion in amyloidosis. In summary, we propose that the amyloidogenic mutations promote apoA-I proteolysis by destabilizing the protein structure not only in the lipid-free but also in the HDL-bound form, with segment L44-S55 providing a likely template for the cross-β-sheet conformation.

Nascent HDL formation by hepatocytes is reduced by the concerted action of serum amyloid A and endothelial lipase

Inflammation is associated with significant decreases in plasma HDL-cholesterol (HDL-C) and apoA-I levels. Endothelial lipase (EL) is known to be an important determinant of HDL-C in mice and in humans and is upregulated during inflammation. In this study, we investigated whether serum amyloid A (SAA), an HDL apolipoprotein highly induced during inflammation, alters the ability of EL to metabolize HDL. We determined that EL hydrolyzes SAA-enriched HDL in vitro without liberating lipid-free apoA-I. Coexpression of SAA and EL in mice by adenoviral vector produced a significantly greater reduction in HDL-C and apoA-I than a corresponding level of expression of either SAA or EL alone. The loss of HDL occurred without any evidence of HDL remodeling to smaller particles that would be expected to have more rapid turnover. Studies with primary hepatocytes demonstrated that coexpression of SAA and EL markedly impeded ABCA1-mediated lipidation of apoA-I to form nascent HDL. Our findings suggest that a reduction in nascent HDL formation may be partly responsible for reduced HDL-C during inflammation when both EL and SAA are known to be upregulated.

Apolipoprotein A-I Exerts Bactericidal Activity against Yersinia enterocolitica Serotype O:3

Apolipoprotein A-I (apoA-I), the main protein component of high density lipoprotein (HDL), is well recognized for its antiatherogenic, antioxidant, and antiinflammatory properties. Here, we report a novel role for apoA-I as a host defense molecule that contributes to the complement-mediated killing of an important gastrointestinal pathogen, Gram-negative bacterium Yersinia enterocolitica. We specifically show that the C-terminal domain of apoA-I is the effector site providing the bactericidal activity. Although the presence of the lipopolysaccharide O-antigen on the bacterial surface is absolutely required for apoA-I to kill the bacteria, apoA-I does not interact with the bacteria directly. To the contrary, exposure of the bacteria by serum proteins triggers apoA-I deposition on the bacterial surface. As our data show that both purified lipid-free and HDL-associated apoA-I displays anti-bacterial potential, apoA-I mimetic peptides may be a promising therapeutic agent for the treatment of certain Gram-negative infections.

The β-Chain of Cell Surface F 0 F 1 ATPase Modulates ApoA-I and HDL Transcytosis Through Aortic Endothelial Cells

Both HDLs and their major protein constituent apolipoprotein A-I (apoA-I) are transported through aortic endothelial cells. The knock-down of the ATP-binding cassette transporters A1 (ABCA1), G1 (ABCG1), and of the scavenger receptor-BI (SR-BI) diminishes but does not completely block the transport of apoA-I or HDL, so that other receptors appear to be involved. The ectopic β-chain of F(0)F(1) ATPase has been previously characterized as an apoA-I receptor, triggering HDL internalization in hepatocytes. The ectopic presence of the β-chain of F(0)F(1) ATPase on the surface of endothelial cells was confirmed by cell surface biotinylation. RNA-interference and the F(0)F(1) ATPase inhibitory peptide IF(1) reduced cell binding of apoA-I but not HDL, as well as association and transendothelial transport of both apoA-I and HDL. Furthermore, apoA-I stimulated F(0)F(1) ATPase catalyzed ATP hydrolysis. The generated ADP as well as apoA-I stimulated the binding, cell association, and internalization of HDL. Both in the presence and absence of ADP inhibition of the purinergic receptor P2Y(12) but not P2Y(1) decreased the cell association of apoA-I and HDL. Coinhibition of β-ATPase and ABCA1 had no additive effects on the cell association and transport of apoA-I. Reduced cell association of HDL by β-ATPase inhibition was not further decreased by additional knock-down of ABCG1 or SR-BI. Binding of apoA-I to ectopic F(0)F(1) ATPase triggers the generation of ADP, which via activation of the purinergic receptor P2Y(12) stimulates the uptake and transport of HDL and initially lipid-free apoA-I by endothelial cells.

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.

Apolipoprotein E Mediates Enhanced Plasma High-Density Lipoprotein Cholesterol Clearance by Low-Dose Streptococcal Serum Opacity Factor via Hepatic Low-Density Lipoprotein Receptors In Vivo

Recombinant streptococcal serum opacity factor (rSOF) mediates the in vitro disassembly of human plasma high-density lipoprotein (HDL) into lipid-free apolipoprotein (apo) A-I, a neo-HDL that is cholesterol poor, and a cholesteryl ester-rich microemulsion (CERM) containing apoE. Given the occurrence of apoE on the CERM, we tested the hypothesis that rSOF injection into mice would reduce total plasma cholesterol clearance via apoE-dependent hepatic low-density lipoprotein receptors (LDLR). rSOF (4 μg) injection into wild-type C57BL/6J mice formed neo-HDL, CERM, and lipid-free apoA-I, as observed in vitro, and reduced plasma total cholesterol (-43%, t(1/2)=44±18 minutes) whereas control saline injections had a negligible effect. Similar experiments with apoE(-/-) and LDLR(-/-) mice reduced plasma total cholesterol ≈0% and 20%, respectively. rSOF was potent; injection of 0.18 μg of rSOF produced 50% of maximum reduction of plasma cholesterol 3 hours postinjection, corresponding to a ≈0.5-mg human dose. Most cholesterol was cleared hepatically (>99%), with rSOF treatment increasing clearance by 65%. rSOF injection into mice formed a CERM that was cleared via hepatic LDLR that recognize apoE. This reaction could provide an alternative mechanism for reverse cholesterol transport.

Analysis of apolipoprotein A-I as a substrate for matrix metalloproteinase-14

Substrates for matrix metalloproteinase (MMP)-14 were previously identified in human plasma using proteomic techniques. One putative MMP-14 substrate was apolipoprotein A-I (apoA-I), a major component of high-density lipoprotein (HDL). In vitro cleavage assays showed that lipid-free apoA-I is a more accessible substrate for MMP-14 compared to lipid-bound apoA-I, and that MMP-14 is more prone to digest apoA-I than MMP-3. The 28-kDa apoA-I was cleaved into smaller fragments of 27, 26, 25, 22, and 14-kDa by MMP-14. ApoA-I sites cleaved by MMP-14 were determined by isotope labeling of C-termini derived from the cleavage and analysis of the labeled peptides by mass spectrometry, along with N-terminal sequencing of the fragments. Cleavage of apoA-I by MMP-14 resulted in a loss of ability to form HDL. Our results suggest that cleavage of lipid-free apoA-I by MMP-14 may contribute to reduced HDL formation, and this may be occurring during the development of various vascular diseases as lipid metabolism is disrupted.