Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux.

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69-99% of control by double deletion mutants apoA-I[Δ(1-41)Δ(185-243)] and apoA-I[Δ(1-59)Δ(185-243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I

Transcriptional Regulation of the Human ApoA-I Gene in Cell Culture and in ApoA-I Transgenic Mice Regulation of ApoA-I Gene Expression and Prospects to Increase Plasma ApoA-I and HDL Levels

Abbreviations there is a linkage and common regulatory mechanism of the apoA-I/apoCIII/apoA-IV gene cluster The receptor specificity of the HREs of the apoA-I promoter and the apoCIII enhancer were determined by DNA binding gel electrophoresis assays. These analyses established that both HREs present in the proximal apoA-I promoter bind HNF-4, other orphan receptors, and a variety of liganddependent nuclear receptors with different affinities Transcriptional regulation of the human apoA-I gene in transgenic mice. To validate the conclusions drawn by the in vitro experiments, we generated a variety of transgenic mouse lines which express the WT A-I/CIII cluster or the same cluster with the mutations in the HREs and the binding sites of SP1 and other transcription factors. In these constructs, the apoCIII gene was replaced by the CAT gen

Effect of amino-, carboxyl-terminal deletions or double deletions of both the amino- and carboxy-terminal regions of apoA-I bound to rHDL particles on ABCG1-mediated cholesterol efflux from HEK293 cells transfected with an ABCG1-expressing plasmid.

<p>HEK293 cells were transfected with empty vector (mock) or with ABCG1-expressing plasmid, labeled with [¹⁴C]cholesterol (A, B, D, E) or [¹⁴C]cholesterol and 30 µg/ml acLDL (C) for 24 h and then incubated with rHDL containing WT or mutants apoA-I forms, at a concentration of 1 µM apoA-I (A-E) or 3 µM apoA-I (B), for 4 h. rHDL particles have various lipid (POPC, SM, C):apoA-I ratios as indicated in each panel. The net ABCG1-mediated [¹⁴C]cholesterol efflux is calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067993#pone-0067993-g001" target="_blank">Figure 1</a>. (A) Values are the means ± SD from six (A, E) or three (B) independent experiments performed in duplicate or two (C, D) independent experiments performed in triplicate. **, p < 0.01 vs WT apoA-I; ***, p < 0.001 vs WT apoA-I; ****, p ≤ 0.0001 vs WT apoA-I. POPC, 1-palmitoyl-2-oleoyl-L-phosphatidylcholine; C, cholesterol; SM, sphingomyelin.</p

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential.In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent.Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis

Effect of the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] bound to rHDL particles on ABCG1-mediated 7-ketocholesterol efflux from HEK293 cells transfected with an ABCG1-expressing plasmid and from J774 mouse macrophages following treatment with AICAR.

<p>(A) HEK293 cells were transfected with empty vector (mock) or with ABCG1 plasmid, labeled with [³H]7-ketocholesterol for 24 h and then incubated with rHDL containing WT apoA-I or apoA-I[Δ(185–243)], at a concentration of 1 µM apoA-I, for 4 h. (B, C) J774 mouse macrophages were labeled with [³H]7-ketocholesterol (B) or [³H]7-ketocholesterol and 30 µg/ml acLDL (C) for 24 h and then incubated with 1 mM AICAR for 24 h. At the end of this incubation period the cells were incubated with rHDL containing WT apoA-I or apoA-I[Δ(185–243)], at a concentration of 1 µM apoA-I, for 4 h. The net ABCG1-mediated [³H]7-ketocholesterol efflux is calculated as the difference in percent of 7-ketocholesterol efflux between ABCG1-transfected and mock-transfected cells (A) or between untreated and AICAR-treated cells (B, C). Values are the means ± SD from three independent experiments performed in duplicate.</p

Plasma membrane fluidity, probed by 1-pyrenedodecanoic acid, of HEK293 cells labeled with cholesterol or 7-ketocholesterol.

<p>HEK293 cells were transfected with empty vector (mock) or with ABCG1-expressing plasmid, incubated with cholesterol or 7-ketocholestrol at the same concentrations and conditions used for the sterol efflux experiments and then suspended in PBS and mixed with the probe 1-pyrenedodecanoic acid as described in “Materials and Methods”. (A) Fluorescence emission spectra of 1-pyrenedodecanoic acid in the presence or absence of HEK293 cells. The fluorescence spectrum of the probe exhibits two major peaks, one at 475 nm and one at 397 nm, corresponding to the excimer and monomer state of the probe. The monomer peak is low when the probe is in aqueous buffer, but is significantly enhanced when the probe is added onto cell suspension. Characteristic spectra of 6–9 separate experiments are shown. (B) The ratio of excimer to monomer peak was measured in the presence of mock or ABCG1-transfected HEK293 cells labeled with cholesterol or 7-ketocholestrol. Values are the means ± SD of 6–9 separate experiments. Chol: cholesterol; 7 KC: 7-ketocholesterol. *, p<0.05; ****, p<0.0001.</p

Effect of internal deletion or point mutations of apoA-I bound to rHDL particles on ABCG1-mediated cholesterol efflux from HEK293 cells transfected with an ABCG1-expressing plasmid.

<p>(A, B) HEK293 cells were transfected with empty vector (mock) or with ABCG1-expressing plasmid, labeled with [¹⁴C]cholesterol for 24 h and then incubated with rHDL containing WT or mutants apoA-I forms, at various concentrations (A) or 1 µM of apoA-I (B), for 4 h. The net ABCG1-mediated [¹⁴C]cholesterol efflux is calculated as the difference in percent of cholesterol efflux between ABCG1-transfected and mock-transfected cells. The numbers on top of the bars represent the net ABCG1-mediated cholesterol efflux relative to the WT control set to 100% (B). Values are the means ± SD from six independent experiments performed in duplicate. **, p < 0.01 vs WT apoA-I; ***, p < 0.001 vs WT apoA-I.</p

Effect of the carboxyl-terminal deletion mutant apoA-I[Δ(185–243)] bound to rHDL particles on ABCG1-mediated cholesterol efflux from J774 mouse macrophages following treatment with AICAR.

<p>J774 mouse macrophages were labeled with [¹⁴C]cholesterol (A) or [¹⁴C]cholesterol and 30 µg/ml acLDL (B) for 24 h and then incubated with 1 mM AICAR for 24 h. At the end of this incubation period the cells were incubated with rHDL containing WT apoA-I or apoA-I[Δ(185–243)], at a concentration of 1 µM apoA-I, for 4 h. The net AICAR-induced (ABCG1-mediated) [¹⁴C]cholesterol efflux is calculated as the difference in percent of cholesterol efflux between untreated and AICAR-treated cells. Values are the means ± SD from three independent experiments performed in duplicate. **, p < 0.01 vs WT apoA-I; ***, p < 0.001 vs WT apoA-I. (C) Western blot analysis, performed as described in “Materials and Methods”, showing the increase in ABCG1 protein levels after AICAR treatment of J774 macrophages. ABCG1 protein levels of HEK293 cells transfected with empty vector (mock) or with ABCG1-expressing plasmid are also shown for comparison.</p