HDL Assembly Research Articles

Proteomic Analysis of Follicular Fluid in Polycystic Ovary Syndrome: Insights into Protein Composition and Metabolic Pathway Alterations.

This study explores the proteomic composition of follicular fluid (FF) from women undergoing oocyte retrieval for in vitro fertilisation (IVF), with a focus on the effects of polycystic ovary syndrome (PCOS). FF samples were collected from 74 patients, including 34 with PCOS and 40 oocyte donors. Proteomic profiling using machine learning identified significant differences in protein abundance between the PCOS and control groups. Of the 484 quantified proteins, 20 showed significantly altered levels in the PCOS group. Functional annotation and pathway enrichment analysis pointed to the involvement of protease inhibitors and immune-related proteins in the pathophysiology of PCOS, suggesting that inflammation and immune dysregulation may play a key role. Additionally, HDL assembly was identified as a significant pathway, with apolipoprotein-AI (APOA1) and alpha-2-macroglobulin (A2M) as the major proteins involved. Notably, myosin light polypeptide 6 was the most downregulated protein, showing the highest absolute fold change, and may serve as a novel independent biomarker for PCOS.

1888-P: Activation of Myocardial Mitochondrial Akt1 Remotely Modulated Body Composition and Adiposity through LXR/RXR Signaling

Insulin resistance contributed to the development of type 2 diabetes (T2D) and is a major risk factor for diabetic cardiomyopathy (DCM). We have previously reported that insulin resistance led to impaired Akt1 translocation into myocardial mitochondria in DCM. To study the role of mitochondrial Akt1 in DCM, we have generated two cardiac-specific inducible Cre-lox transgenic mice models that expresses (1) a mitochondria-targeting dominant negative Akt (CAMDAKT) or (2) a mitochondria-targeting constitutively active Akt (CAMCAKT) upon Tamoxifen induction (T). T-CAMDAKT mice showed LV dysfunction with increased heart failure markers, while T-CAMCAKT mice restored LV dysfunction to normal in the T2D model. In a T2D model induced by high fat/high fructose diet with relative insulin deficiency, T-CAMCAKT mice showed lower body fat mass (16.6% vs. 26.5%, p<0.001) and higher body lean mass (77.1% vs. 68.1%, p<0.001). To study the mechanism underlying the changes of body composition, we performed transcriptomic profiling and analysis in gonadal white adipose tissues (WAT) by bulk RNA sequencing. When compared to the control T2D mice, 544 genes were upregulated and 1077 genes were downregulated in the WAT of T-CAMCAKT T2D mice. The result indicated enhanced lipid catabolism, triglyceride catabolism, HDL assembly, and downregulated critical nuclear transcription. Pathway analysis revealed the Liver X receptor/Retinoid X receptor signaling was activated in WAT of T-CAMCAKT mice as compared to the controls. This was accompanied by increased fatty acid oxidation and apolipoproteins expression. These findings suggest that impaired mitochondrial Akt signaling in DCM could remotely increase adiposity by reducing lipid catabolism in WAT through modulating LXR/RXR signaling. Disclosure Y. Chen: None. A. Ta: None. E. Tom: None. Y. Chen: None. P.H. Wang: Board Member; Self; Dianavi. Funding National Institutes of Health (R01HL096987)

Abstract 397: Cotinine, but Not Nicotine, Increased ApoA-I Mediated HDL Synthesis in THP-1 Cells

Aim: Cotinine is known as a stable metabolite of nicotine, the plasma half-life is over 20-30 hrs. Here, we evaluated its characteristic properties on the apoA-I mediated HDL generation. Method: Cotinine (5.6 to 11.2μM), or nicotine (5 to 150μM) were supplemented to medium and co-incubated with apoA-I (10μg/mL) for 18 hrs with THP-1 cells to determine their effect on efflux activity. Cellular ACAT accessible cholesterol pool size was determined by [ 14 C]oleic acid labeling of cells for 60 min immediately after removal of the culture medium. The culture medium and the cellular lipids were extracted by organic solvents and cholesterol and phospholipids content were determined. Nicotine was administered orally as an aqueous solution (10 μM) to C57BL/6 mice for 10 days and the plasma lipoprotein profiles were measured by gel-HPLC (tandem TSK-lipopropak XL ) with online enzymatic lipid assay system (Skylight biotech, Inc.). Results: Supplementation of nicotine into the medium showed no effect on the apoA-I mediated cellular cholesterol release from THP-1 cells. On the other hand, cotinine increased apoA-I mediated cellular cholesterol efflux 2.8 fold. ACAT accessible cholesterol pool size was dramatically decreased to 80% and 58% of control with 5 and 10 μM cotinine treatment, respectively, indicating that cotinine effectively increased apoA-I/ABCA1 mediated cellular cholesterol efflux. Mice with oral nicotine administration showed increased plasma HDL by 11.4% (50.1±4.4 and 55.8±3.9 mg/dL for control and nicotine treatment, respectively). Conclusions: Cotinine increased apoA-I mediated cellular cholesterol efflux in THP-1 cells. The cellular ACAT accessible cholesterol pool size reduction is one of the early reactions of apoA-I/ABCA1 mediated cellular cholesterol efflux. Cotinine seemingly modifies cellular factor/factors to induce a change in cholesterol distribution. Mice administrated oral nicotine may convert the compound to cotinine by CYP2A6 and aldehyde oxidase activities in their liver, and which likely increased HDL generation. Further experiments are necessary to examine the direct effect of cotinine on the ABCA1 transporter for increasing HDL assembly system.

Abstract 542: Cellular Pip2 is Effluxed By Abca1 to Apoa1 and Pip2 Is Carried on Hdl That Can be Delivered to Target Tissues via Sr-b1

Introduction: apoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport. Cell surface phosphatidylinositol 4,5- bisphosphate (PIP2) mediates this process, but whether PIP2 is effluxed via apoA1-ABCA1 pathway and is carried in plasma is unknown. Objective: To determine if PIP2 is effluxed during ABCA1-dependent nHDL biogenesis, whether PIP2 is found on plasma HDL, and if PIP2 can be delivered to target cells via SRB1. Methods and Results: We previously reported at this meeting that: 1) apoAI binds to PIP2 with a K d of ~100 nM; 2) ABCA1 expression translocates PIP2 from the inner to outer leaflet of the plasma membrane; 3) cell surface PIP2 is required for ABCA1-dependent cellular binding of apoA1 and cholesterol efflux. Here, we report that PIP2 incubation with lipid-free apoA1 promoted monomeric vs. dimeric apoA1. We found that ABCA1 expression increased efflux of PIP2 by ~20-fold in RAW264.7 cells labeled with [3H]myo-inositol, and chased with apoA1 (mean ± SD; ***, p<0.001, two-tailed t-test, n=3). Plasma from apoA1 knockout (A1 KO), wild type (WT), and human apoA1 transgenic (A1-Tg) mice contained apoA1-gene dosage dependent levels of both cholesterol and PIP2, with 64-fold higher PIP2 levels in the A1-Tg vs. A1 KO mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). PIP2 was also reverse transported in vivo from [3H]myo-inositol-labeled macrophages implanted s.c into mice. Labeled PIP2 was recovered in the plasma, with a 4-fold higher of the injected radioactivity found in the WT vs. A1 KO host mice (mean ± SD; **, p<0.01, two-tailed t-test, n=3). FPLC separation of human plasma determined that almost all of the plasma PIP2 was found in the HDL fraction; and, human HDL contained two PIP2 species with either 18:0, 20:4 fatty acids or 16:0, 20:4 fatty acids, as detected by liquid chromatography tandem mass spectrometry. SR-BI-inducible BHK cells exhibited 2-fold higher uptake of [3H]PIP2-labeled HDL after SR-BI induction, indicating that HDL can deliver PIP2 to target cells (mean ± SD; **, p<0.01, two-tailed t-test, n=3). Conclusions: PIP2 is effluxed from cells via the apoA1-ABCA1 pathway and is carried on HDL. HDL can deliver PIP2 to target tissues via SR-B1.

Abstract 5: ApoAI Binds to Phosphatidylinositol 4,5 Bisphosphate (PIP2), Which is Exposed on the Cell Surface by Novel PIP2 Floppase Activity of ABCA1, and Promotes Cholesterol Efflux

Introduction: ApoAI-ABCA1 mediated nascent HDL assembly promotes cholesterol efflux and reverse cholesterol transport, but the mechanism of HDL assembly is unknown. Objective: To determine role of PIP2 in ApoAI-ABCA1 mediated nascent HDL assembly. Methods and Results: To determine which lipid species are directly bound by apoAI, a lipid-protein overlay assay was performed using PIPstrips and sphingostrips from Echelon Biosciences. ApoAI bound directly to phosphatidylinositol phosphates (PIPs) rather than PC, PS, PE, SM or cholesterol. This interaction was not solely electrostatic, as PIP3, the most negative charged PIP had less apoAI binding than various PIP2 species. ApoAI did not bind to other charged lipids such as sphingosine 1-P, PI(3)-P, PI(4)-P, and PI(5)-P. Surface plasmon resonance assays using immobilized apoAI or PIP2 were performed, showing that apoAI interacted reversibly with PI(4,5)P2 in a dose dependent manner at the nM concentration range. The central domain of apoAI (residues 44-185) was sufficient for PIP2 binding. In RAW264.7 macrophages, depletion of cellular PIPs by treatments with PI3K inhibitor or PTEN inhibitor decreased cholesterol efflux by 45±1.4% and 41±6.4% respectively (p<0.005). Degradation of cell surface PIP2 via treatment with PI specific PLC decreased cell surface binding of apoAI by 37±6% and cholesterol efflux by 48±8% (p<0.005). In mouse bone-marrow derived macrophages, cholesterol efflux to apoAI was increased ~47% by GRIP (PIP2 binding PH domain of PLCδ) and decreased 41±8% by PTEN inhibition (p<0.005). Liposomes made with POPC:POPS:FC:PIP2 (65:20:10:5 mole ratio) were solubilized by apoAI at 37 o C, pH5, but apoAI had little activity using similar liposomes without PIP2 . ABCA1 expression led to 2-fold higher cell surface PIP2, assayed by flow cytometry using a PIP2 specific antibody. RAW cells stably transfected with PIP2 binding domain 2X PH-PLCδ-eGFP showed enrichment of this PIP2 reporter on the plasma membrane, while expression of ABCA1 led to partial redistribution to cytoplasm, consistent with ABCA1 having PIP2 floppase activity. Conclusions: ApoAI has novel PIP2 binding activity, ABCA1 is a novel PIP2 floppase, and cell surface PIP2 is required for optimal cholesterol acceptor activity of apoAI.

Free Cholesterol Determines Reassembled High-Density Lipoprotein Phospholipid Phase Structure and Stability

Reassembled high-density lipoproteins (rHDL) of various sizes and compositions containing apo A-I or apo A-II as their sole protein, dimyristoylphosphatidylcholine (DMPC), and various amounts of free cholesterol (FC) have been isolated and analyzed by differential scanning calorimetry (DSC) and by circular dichroism to determine their stability and the temperature dependence of their helical content. Our data show that the multiple rHDL species obtained at each FC mole percent usually do not have the same FC mole percent as the starting mixture and that the size of the multiple species increases in a quantized way with their respective FC mole percent. DSC studies reveal multiple phases or domains that can be classified as virtual DMPC, which contains a small amount of DMPC that slightly reduces the melting temperature (Tm), a boundary phase that is adjacent to the apo A-I or apo A-II that circumscribes the discoidal rHDL, and a mixed FC/DMPC phase that has a Tm that increases with FC mole percent. Only the large rHDL contain virtual DMPC, whereas all contain boundary phase and various amounts of the mixed FC/DMPC phase according to increasing size and FC mole percent. As reported by others, FC stabilizes the rHDL. For rHDL (apo A-II) compared to rHDL (apo A-I), this occurs in spite of the reduced number of helical regions that mediate binding to the DMPC surface. This effect is attributed to the very high lipophilicity of apo A-II and the reduction in the polarity of the interface between DMPC and the aqueous phase with an increasing FC mole percent, an effect that is expected to increase the strength of the hydrophobic associations with the nonpolar face of the amphipathic helices of apo A-II. These data are relevant to the differential effects of FC and apolipoprotein species on intracellular and plasma membrane nascent HDL assembly and subsequent remodeling by plasma proteins.

HDL and Cardiovascular Risk

High-density lipoprotein cholesterol (HDL-C) has been dubbed the good cholesterol because it is thought to reflect the ability of HDL particles to remove excess cholesterol molecules from peripheral cells (including those in atherosclerotic plaques) for return to the liver. Not surprisingly, then, HDL-C has frequently been assumed to be a biomarker of HDL function, consistent with the inverse relationship in observational studies between plasma levels of HDL-C and risk of coronary artery disease. Recently, Voight et al have challenged this assumption by showing that genetically elevated HDL-C did not protect against myocardial infarction. This finding has fueled a lively discussion in the lay, scientific, and medical press about the relationship between HDL-C and HDL function, and the potential effectiveness of various HDL-C raising strategies.

C-Terminus of Apolipoprotein A-I Removes Phospholipids from a Triolein/Phospholipids/Water Interface, but the N-Terminus Does Not: A Possible Mechanism for Nascent HDL Assembly

Apolipoprotein A-I (ApoA-I) is the principle protein component of HDL, also known as “good cholesterol,” which is an inverse marker for cardiovascular disease. The N-terminal 44 amino acids of ApoA-I (N44) are predicted to be responsible for stabilization of soluble ApoA-I, whereas the C-terminal 46 amino acids (C46) are predicted to initiate lipid binding and oligomerization. In this work, we apply what we believe to be a novel application of drop tensiometry to study the adsorption and desorption of N44 and C46 at a triolein/POPC/water (TO/POPC/W) interface. The amount of peptide that adsorbed to the surface was dependent on the surface concentration of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and pressure (Π) before adsorption. At a TO/POPC/W interface, the exclusion pressure (ΠEX) of C46 was 25.8 mN/m, and was 19.3 mN/m for N44. Once adsorbed, both peptides formed a homogeneous surface with POPC but were progressively ejected from the surface by compression. During a compression, C46 removed POPC from the surface whereas N44 did not. Repeated compressions caused C46 to deplete entirely the surface of phospholipid. If full-length ApoA-I could also remove phospholipid, this could provide a mechanism for the transfer of surface components of chylomicrons and very low density lipoprotein to high density lipoprotein with the assistance of phospholipid transfer protein.

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

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

Apolipoprotein M expression increases the size of nascent preβ HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.

Niemann-Pick C1-Like 1 Is Required for an LXR Agonist to Raise Plasma HDL Cholesterol in Mice

Activation of liver x receptor (LXR) raises plasma HDL-cholesterol (HDL-C) in mice. Interestingly, the LXR agonist GW3965 fails to raise plasma HDL-C in mice lacking intestinal ABCA1, indicating that intestinal ABCA1 plays a predominant role in GW3965-mediated HDL production. How this is coupled to intestinal function remains elusive. Because cholesterol is essential for HDL assembly and directly regulates intestinal ABCA1 expression via activating LXR, we hypothesized that cholesterol absorption, a major function of intestine, modulates LXR-dependent HDL formation. Mice lacking Niemann-Pick C1-Like 1 (NPC1L1) (L1-KO mice), a gene that is essential for cholesterol absorption, were treated with LXR agonist T0901317 for 7 days. Intriguingly, this treatment failed to significantly raise plasma HDL-C but caused a much greater fecal cholesterol excretion in L1-KO mice. The intestinal ABCA1 mRNA level was about 4-fold lower in L1-KO versus wild-type mice, and increased 3.9-fold and 8.8-fold after T0901317 treatment in wild-type and L1-KO mice, respectively. Hepatic ABCA1 failed to respond to T0901317 in mice of both genotypes, although hepatic mRNAs for many LXR target genes were higher in the T0901317-treated versus untreated wild-type animals. NPC1L1 is required for an LXR agonist to increase plasma HDL-C in mice.

The C-Terminal Lipid-Binding Domain of Apolipoprotein E Is a Highly Efficient Mediator of ABCA1-Dependent Cholesterol Efflux that Promotes the Assembly of High-Density Lipoproteins

This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.

PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes oxidative cleavage of the sterol side chain in the bile acid biosynthetic pathway in the liver and 27-hydroxylation of cholesterol in most tissues. Recent studies suggest that 27-hydroxycholesterol (27-HOC) activates liver orphan receptor alpha (LXRalpha) and induces the cholesterol efflux transporters ABCA1 and ABCG1 in macrophages. The steroid- and bile acid-activated pregnane X receptor (PXR) plays critical roles in the detoxification of bile acids, cholesterol metabolites, and xenobiotics. The role of CYP27A1 in the intestine is not known. This study investigated PXR and CYP27A1 regulation of cholesterol metabolism in the human intestinal cell lines Caco2 and Ls174T. A human PXR ligand, rifampicin, induced CYP27A1 mRNA expression in intestine cells but not in liver cells. Rifampicin induced CYP27A1 gene transcription, increased intracellular 27-HOC levels, and induced ABCA1 and ABCG1 mRNA expression only in intestine cells. A functional PXR binding site was identified in the human CYP27A1 gene. Chromatin immunoprecipitation assays revealed that rifampicin induced the PXR recruitment of steroid receptor coactivator 1 to CYP27A1 chromatin. Cholesterol loading markedly increased intracellular 27-HOC levels in intestine cells. Rifampicin, 27-HOC, and a potent LXRalpha agonist, T0901317, induced ABCA1 and ABCG1 protein expression and stimulated cholesterol efflux from intestine cells to apolipoprotein A-I and HDL. This study suggests an intestine-specific PXR/CYP27A1/LXRalpha pathway that regulates intestine cholesterol efflux and HDL assembly.

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.

Assembly of high density lipoprotein by the ABCA1/apolipoprotein pathway

Mammalian somatic cells do not catabolize cholesterol and therefore need to export it for sterol homeostasis at the levels of cells and whole bodies. This mechanism may reduce intracellularly accumulated cholesterol in excess, and thereby would contribute to the prevention or cure of the initial stage of atherosclerotic vascular lesions. HDL is thought to play a main role in this reaction on the basis of epidemiological evidence and in-vitro experimental data. Two independent mechanisms have been identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from the cell surface, and cholesterol is trapped by various extracellular acceptors including lipoproteins. Extracellular cholesterol esterification on HDL provides a driving force for the net removal of cell cholesterol, and some cellular factors may enhance this reaction. The other mechanism is an apolipoprotein-mediated process to generate HDL by removing cellular phospholipid and cholesterol. This reaction is mediated by a membrane protein ABCA1, and lipid-free or lipid-poor helical apolipoproteins recruit cellular phospholipid and cholesterol to assemble HDL particles. The reaction is composed of two elements: the assembly of HDL particles with phospholipid by apolipoprotein, and cholesterol enrichment in HDL. ABCA1 is essential for the former step, and the latter step requires further intracellular events. ABCA1 is a rate-limiting factor of HDL assembly and is regulated by transcriptional factors and posttranscriptional factors. Posttranscriptional regulation of ABCA1 involves the modulation of its calpain-mediated degradation.

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.

The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger.

Exchangeable apolipoproteins can convert between lipid-free and lipid-associated states. The C-terminal domain of human apolipoprotein A-I (apoA-I) plays a role in both lipid binding and self-association. Site-directed spin-label electron paramagnetic resonance spectroscopy was used to examine the structure of the apoA-I C terminus in lipid-free and lipid-associated states. Nitroxide spin-labels positioned at defined locations throughout the C terminus were used to define discrete secondary structural elements. Magnetic interactions between probes localized at positions 163, 217 and 226 in singly and doubly labeled apoA-I gave inter- and intramolecular distance information, providing a basis for mapping apoA-I tertiary and quaternary structure. Spectra of apoA-I in reconstituted HDL revealed a lipid-induced transition of defined random coils and beta-strands into alpha-helices. This conformational switch is analogous to triggered events in viral fusion proteins and may serve as a means to overcome the energy barriers of lipid sequestration, a critical step in cholesterol efflux and HDL assembly.

1P-0229∗ HDL assembly in hepatocytes

1P-0229∗ HDL assembly in hepatocytes

3FS06-5 Cellular events in ABCA1-mediated HDL assembly

3FS06-5 Cellular events in ABCA1-mediated HDL assembly