ABCA1 and ABCG1 as potential therapeutic targets for the prevention of atherosclerosis.

Prevention of atherosclerosis is important because it is a risk factor for cardiovascular diseases globally. One of the causes of atherosclerosis is accumulation of cholesterol and triglycerides in peripheral cells. ATP-binding cassette protein A1 (ABCA1) and G1 (ABCG1) are important in eliminating excess cholesterol from cells including macrophages and forming high-density lipoprotein, which contributes to the prevention and regression of atherosclerosis. Enhanced cholesterol efflux activities of ABCA1 and ABCG1 are expected to prevent the progression of atherosclerosis. ABCA1 and ABCG1 are induced by the LXR/RXR pathway and regulated transcriptionally, post-transcriptionally, and post-translationally. Their mRNAs are destabilized by microRNAs and their cellular localization and degradation are regulated by other proteins and phosphorylation. Furthermore, ABCA1 and ABCG1 suppress the inflammatory responses of macrophages. These proteins are effective targets because their increased activities can suppress cholesterol accumulation and inflammation in macrophages. Moreover, ABCA1 and ABCG1 prevent amyloid ß accumulation; therefore, their increased activity may prevent Alzheimer's disease. Because ABCA1 and ABCG1 are affected by transcriptional, post-transcriptional, and post-translational regulation, the regulatory factors involved could also serve as therapeutic targets. This review highlights that ABCA1 and ABCG1 could be potential therapeutic targets for preventing atherosclerosis by regulating their expression, degradation, and localization.

The distribution of vinculin to lipid rafts plays an important role in sensing stiffness of extracellular matrix.

Extracellular matrix (ECM) stiffness regulates cell differentiation, survival, and migration. Our previous study has shown that the interaction of the focal adhesion protein vinculin with vinexin α plays a critical role in sensing ECM stiffness and regulating stiffness-dependent cell migration. However, the mechanism how vinculin-vinexin α interaction affects stiffness-dependent cell migration is unclear. Lipid rafts are membrane microdomains that are known to affect ECM-induced signals and cell behaviors. Here, we show that vinculin and vinexin α can localize to lipid rafts. Cell-ECM adhesion, intracellular tension, and a rigid ECM promote vinculin distribution to lipid rafts. The disruption of lipid rafts with Methyl-ß-cyclodextrin impaired the ECM stiffness-mediated regulation of vinculin behavior and rapid cell migration on rigid ECM. These results indicate that lipid rafts play an important role in ECM-stiffness regulation of cell migration via vinculin.

Neurite outgrowth stimulation by n-3 and n-6 PUFAs of phospholipids in apoE-containing lipoproteins secreted from glial cells.

PUFAs, which account for 25-30% of the total fatty acids in the human brain, are important for normal brain development and cognitive function. However, it remains unclear how PUFAs are delivered to neurons and exert their effects. In this study, we demonstrated that n-3 and n-6 PUFAs added to the medium are incorporated into membrane phospholipids of primary glial cells from rat cortices, and then secreted as the fatty acid moiety of phospholipids in apoE-containing lipoproteins (LpEs). Tandem mass spectrometry analysis further showed that LpEs secreted from glial cells contain a variety of metabolites of PUFAs produced in glial cells by elongation and unsaturation. LpEs are absorbed by endocytosis into neurons via LDL receptor-related protein 1. LpE-containing n-3 and n-6 PUFAs exhibit a strong effect on neurite outgrowth of hippocampal neurons by increasing the number of branches. This study sheds light on the novel role of LpEs in the central nervous system and also a novel pathway in which PUFAs act on neurons.

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner.

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.

Position 834 in TM6 plays an important role in cholesterol and phosphatidylcholine transport by ABCA1.

ATP-binding cassette protein A1 (ABCA1) plays a key role in eliminating excess cholesterol from peripheral cells by generating nascent high-density lipoprotein (HDL). However, it remains unclear whether both phospholipids and cholesterol are directly loaded onto apolipoprotein A-I (apoA-I) by ABCA1. To identify the amino acid residues of ABCA1 involved in substrate recognition and transport, we applied arginine scan mutagenesis to residues L821-E843 of human ABCA1 and predicted the environment to which each residue is exposed. The relative surface expression of each mutant suggested that residues L821-E843 pass through the plasma membrane as TM6, and the four residues (S826, F830, L834, and V837) of TM6 are exposed to the hydrophilic internal cavity of ABCA1. Furthermore, we showed that L834 is critical for the function of ABCA1.

ATPase activity of nucleotide binding domains of human MDR3 in the context of MDR1.

Although human MDR1 and MDR3 share 86% similarity in their amino acid sequences and are predicted to share conserved domains for drug recognition, their physiological transport substrates are quite different: MDR1 transports xenobiotics and confers multidrug resistance, while MDR3 exports phosphatidylcholine into bile. Although MDR1 shows high ATPase activity, attempts to demonstrate the ATPase activity of human MDR3 have not succeeded. Therefore, it is possible that the difference in the functions of these proteins is caused by their different ATPase activities. To test this hypothesis, a chimera protein containing the transmembrane domains (TMDs) of MDR1 and the nucleotide binding domains (NBDs) of MDR3 was constructed and analyzed. The chimera protein was expressed on the plasma membrane and conferred resistance against vinblastine and paclitaxel, indicating that MDR3 NBDs can support drug transport. Vanadate-induced ADP trapping of MDR3 NBDs in the chimera protein was stimulated by verapamil as was MDR1 NBDs. The purified chimera protein showed drug-stimulated ATPase activity like MDR1, while its Vmax was more than 10-times lower than MDR1. These results demonstrate that the low ATPase activity of human MDR3 cannot account for the difference in the functions of these proteins, and furthermore, that TMDs determine the features of NBDs. To our knowledge, this is the first study analyzing the features of human MDR3 NBDs.

ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin.

ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-ß-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.

24(S)-hydroxycholesterol is actively eliminated from neuronal cells by ABCA1.

High cholesterol turnover catalyzed by cholesterol 24-hydroxylase is essential for neural functions, especially learning. Because 24(S)-hydroxycholesterol (24-OHC), produced by 24-hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH-SY5Y neuron-like cells as a model, we examined whether 24-OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24-OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24-OHC efflux was stimulated in the presence of high-density lipoprotein (HDL), whereas apolipoprotein A-I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24-OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A-I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24-OHC. These results suggest that ABCA1 actively eliminates 24-OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.

ABCG5 and ABCG8 Are Involved in Vitamin K Transport.

ATP-binding cassette protein G5 (ABCG5)/ABCG8 heterodimer exports cholesterol from cells, while Niemann-Pick C1-like 1 (NPC1L1) imports cholesterol and vitamin K. We examined whether ABCG5/ABCG8 transports vitamin K similar to NPC1L1. Since high concentrations of vitamin K3 show cytotoxicity, the cytoprotective effects of ABCG5/ABCG8 were examined. BHK cells expressing ABCG5/ABCG8 were more resistant to vitamin K3 cytotoxicity than control cells, suggesting that ABCG5/ABCG8 transports vitamin K3 out of cells. The addition of vitamin K1 reversed the effects of ABCG5/ABCG8, suggesting that vitamin K1 competitively inhibits the transport of vitamin K3. To examine the transport of vitamin K1 by ABCG5/ABCG8, vitamin K1 levels in the medium and cells were measured. Vitamin K1 levels in cells expressing ABCG5/ABCG8 were lower than those in control cells, while vitamin K1 efflux increased in cells expressing ABCG5/ABCG8. Furthermore, the biliary vitamin K1 concentration in Abcg5/Abcg8-deficient mice was lower than that in wild-type mice, although serum vitamin K1 levels were not affected by the presence of Abcg5/Abcg8. These findings suggest that ABCG5 and ABCG8 are involved in the transport of sterols and vitamin K. ABCG5/ABCG8 and NPC1L1 might play important roles in the regulation of vitamin K absorption and excretion.

ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding.

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high-density lipoprotein (HDL) metabolism. Although it is predicted that apolipoprotein A-I (apoA-I) directly binds to ABCA1, the physiological importance of this interaction is still controversial and the conformation required for apoA-I binding is unclear. In this study, the role of the two nucleotide-binding domains (NBD) of ABCA1 in apoA-I binding was determined by inserting a TEV protease recognition sequence in the linker region of ABCA1. Analyses of ATP binding and occlusion to wild-type ABCA1 and various NBD mutants revealed that ATP binds equally to both NBDs and is hydrolyzed at both NBDs. The interaction with apoA-I and the apoA-I-dependent cholesterol efflux required not only ATP binding but also hydrolysis in both NBDs. NBD mutations and cellular ATP depletion decreased the accessibility of antibodies to a hemagglutinin (HA) epitope that was inserted at position 443 in the extracellular domain (ECD), suggesting that the conformation of ECDs is altered by ATP hydrolysis at both NBDs. These results suggest that ATP hydrolysis at both NBDs induces conformational changes in the ECDs, which are associated with apoA-I binding and cholesterol efflux.

Involvement of low-density lipoprotein receptor-related protein and ABCG1 in stimulation of axonal extension by apoE-containing lipoproteins.

Apolipoprotein E (apoE)-containing lipoproteins (LpE) are produced by glial cells in the central nervous system (CNS). When LpE are supplied to distal axons, but not cell bodies, of CNS neurons (retinal ganglion cells) the rate of axonal extension is increased. In this study we have investigated the molecular requirements underlying the stimulatory effect of LpE on axonal extension. We show that enhancement of axonal growth by LpE requires the presence of the low-density lipoprotein receptor-related protein-1 (LRP1) in neurons since RNA silencing of LRP1 in neurons, or antibodies directed against LRP, suppressed the LpE-induced axonal extension. In contrast, an alternative LRP1 ligand, α2-macroglobulin, failed to stimulate axonal extension, suggesting that LpE do not exert their growth-stimulatory effect solely by activation of a LRP1-mediated signaling pathway. In addition, although apoE3-containing LpE enhanced axonal extension, apoE4-containing LpE did not. Over-expression of ABCG1 in rat cortical glial cells resulted in production of LpE that increased the rate of axonal extension to a greater extent than did expression of an inactive, mutant form of ABGC1. Furthermore, reconstituted lipoprotein particles containing apoE3, phosphatidylcholine and sphingomyelin, but not cholesterol, stimulated axonal extension, suggesting that sphingomyelin, but not cholesterol, is involved in the stimulatory effect of LpE. These observations demonstrate that LpE and LRP1 promote axonal extension, and suggest that lipids exported to LpE by ABCG1 are important for the enhancement of axonal extension mediated by LpE.

ATP-binding cassette proteins involved in glucose and lipid homeostasis.

Glucose and lipids are essential to the body, but excess glucose or lipids lead to metabolic syndrome. ATP-binding cassette (ABC) proteins are involved in the homeostasis of glucose and lipid in that they regulate insulin secretion and remove excess cholesterol from the body. Sulfonylurea receptor (SUR) is a subunit of the ATP-sensitive potassium channels, which regulate insulin secretion from pancreatic beta-cells by sensing cellular metabolic levels. ABCG1 removes excess cholesterol from peripheral tissues and functions in reverse cholesterol transport to the liver. ABCG5 and ABCG8 suppress the absorption of cholesterol in the intestine and exclude cholesterol from the liver to the bile duct. ABCG1 and ABCG4, expressed in the central nervous system, play roles in lipid metabolism in the brain. These ABC proteins are targets of drugs and functional foods to cure and prevent diabetes, hyperlipidemia, and neurodegenerative diseases. In this review, recent knowledge of the physiological function and regulation of ABC proteins in the homeostasis of glucose and lipids is discussed.

A common variant of LDL receptor related protein 2 (LRP2) gene is associated with gout susceptibility: a meta-analysis in a Japanese population.

Gout, which results from elevated serum uric acid (SUA), is a common form of arthritis that is induced by urate crystals. A single nucleotide polymorphism, rs2544390, of LDL receptor related protein 2 (LRP2/Megalin), has previously been reported to be associated with SUA by a genome-wide association study in a Japanese population. However, it was controversial as to whether rs2544390 is associated with gout in a Japanese population, since previous studies with Japanese populations have reported an association between gout and rs2544390 both with and without significance. This prompted us to investigate the association between gout and rs2544390 of LRP2. Using 1208 clinically diagnosed gout patients and 1223 controls in a Japanese male population, our results showed that while rs2544390 did not show a significant association with gout susceptibility in the present study (p = 0.0793, odds ratio [OR] with 95% confidential interval [CI] 1.11 [0.99-1.24]). However, a meta-analysis using previous studies on Japanese populations revealed a significant association with gout (pmeta = 0.0314, OR with 95% CI 1.09 [1.01-1.18]). We have therefore for the first time confirmed a positive association between rs2544390 and gout with only a Japanese male population. Our study provides clues to a better understanding of the pathogenesis of gout and has the potential to lead to novel therapeutic strategies against gout using LRP2 as a molecular target.

Improved expression and purification of human multidrug resistance protein MDR1 from baculovirus-infected insect cells.

Multidrug resistance protein MDR1 (P-glycoprotein/ABCB1) is an ATP-dependent efflux pump for various cytotoxic agents, and is implicated in the resistance of human tumors to chemotherapeutic drugs. To achieve the three-dimensional structural analysis for its mechanistic implications, large amounts of high-quality and homogeneous MDR1 protein are essential. Here we report a cost-effective method for large-scale expression of human MDR1 using a baculovirus/insect expressSF+ cell system and an alterative purification method to maintain MDR1 in a monodispersed state. After extensively optimizing the detergent, pH, and additives, a high yield (2.8 mg/L) of purified MDR1 was obtained by immobilized metal chelate affinity and size-exclusion chromatographies with 49% recovery. The purified MDR1 exhibited specific ATP hydrolase activity (1.7 micromol/min/mg) in the presence of a substrate, verapamil. This value was 14-fold greater than the basal activity without the drug. Size-exclusion chromatography analysis of purified MDR1 showed a monodispersed elution profile. The present purification method provides suitable material for structural and functional studies on human MDR1.

Molecular mechanisms of subcellular localization of ABCG5 and ABCG8.

Human ABCG subfamily proteins ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8 are half-type ATP-binding cassette (ABC) proteins that transport sterols or xenobiotics. ABCG1, ABCG2, and ABCG4 function as homodimers on the plasma membrane. In contrast, ABCG5 and ABCG8 function as heterodimers on the plasma membrane, and the homodimer of either ABCG5 or ABCG8 is retained in the endoplasmic reticulum (ER). To examine the molecular mechanisms of the regulated trafficking of ABCG5 and ABCG8, the subcellular localizations of chimeric proteins, fused with ABCG1 or ABCG2, were analyzed. Homodimers of chimeric proteins, in which the N-terminal cytosolic domain of ABCG1 or ABCG2 was fused to the C-terminal transmembrane domain of ABCG5 or ABCG8 localized to the plasma membrane, whereas chimeric proteins in which the N-terminal cytosolic domain of ABCG5 or ABCG8 was fused to the C-terminal transmembrane domain of ABCG1 or ABCG2 localized to the ER. Mutations in ER-retrieval motif-like sequences in ABCG5 or ABCG8 did not affect their subcellular localization. This suggests that the N-terminal cytosolic domains of ABCG5 and ABCG8 are involved in ER retention of their homodimers, and that novel ER-retention or -retrieval motifs exist within these domains.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux.

ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/ßI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

Niemann-Pick C1-like 1 Promotes Intestinal Absorption of Siphonaxanthin.

Siphonaxanthin is a carotenoid found in certain green algae, and its promising beneficial properties, such as its anti-obesity effect, have recently been demonstrated. However, there is little information about the molecular mechanisms underlying intestinal absorption of siphonaxanthin. In this study, we aimed to elucidate how siphonaxanthin is transported across the intestinal epithelium using differentiated Caco-2 cells (dCaco-2 cells), recombinant proteins, and an animal model. Siphonaxanthin was taken up by dCaco-2 cells, a model of intestinal epithelial cells, and its uptake linearly increased up to at least 6 h. Pharmacological inhibition of Nieman-Pick C1-like 1 (NPC1L1), but not that of scavenger receptor class B type 1 (SR-B1), significantly suppressed siphonaxanthin uptake by dCaco-2 cells. Results from an in vitro binding assay suggested that the N-terminal domain of NPC1L1, which is an extracellular domain of NPC1L1, binds with siphonaxanthin. Moreover, pretreatment with ezetimibe, an inhibitor of NPC1L1, significantly decreased the plasma level of siphonaxanthin following oral administration in mice. Considered together, we concluded that NPC1L1 promotes siphonaxanthin transport across the intestinal epithelium.

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol.

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.

ABCG1 and ABCG4 Suppress γ-Secretase Activity and Amyloid ß Production.

ATP-binding cassette G1 (ABCG1) and ABCG4, expressed in neurons and glia in the central nervous system, mediate cholesterol efflux to lipid acceptors. The relationship between cholesterol level in the central nervous system and Alzheimer's disease has been reported. In this study, we examined the effects of ABCG1 and ABCG4 on amyloid precursor protein (APP) processing, the product of which, amyloid ß (Aß), is involved in the pathogenesis of Alzheimer's disease. Expression of ABCG1 or ABCG4 in human embryonic kidney 293 cells that stably expressed Swedish-type mutant APP increased cellular and cell surface APP levels. Products of cleavage from APP by α-secretase and by ß-secretase also increased. The levels of secreted Aß, however, decreased in the presence of ABCG1 and ABCG4, but not ABCG4-KM, a nonfunctional Walker-A lysine mutant. In contrast, secreted Aß levels increased in differentiated SH-SY5Y neuron-like cells in which ABCG1 and ABCG4 were suppressed. Furthermore, Aß42 peptide in the cerebrospinal fluid from Abcg1 null mice significantly increased compared to the wild type mice. To examine the underlying mechanism, we analyzed the activity and distribution of γ-secretase. ABCG1 and ABCG4 suppressed γ-secretase activity and disturbed γ-secretase localization in the raft domains where γ-secretase functions. These results suggest that ABCG1 and ABCG4 alter the distribution of γ-secretase on the plasma membrane, leading to the decreased γ-secretase activity and suppressed Aß secretion. ABCG1 and ABCG4 may inhibit the development of Alzheimer's disease and can be targets for the treatment of Alzheimer's disease.