BBA - Molecular and Cell Biology of Lipids (v.1821, #3)

Role of plasma phospholipid transfer protein in lipid and lipoprotein metabolism by John J. Albers; Simona Vuletic; Marian C. Cheung (345-357).
The understanding of the physiological and pathophysiological role of PLTP has greatly increased since the discovery of PLTP more than a quarter of century ago. A comprehensive review of PLTP is presented on the following topics: PLTP gene organization and structure; PLTP transfer properties; different forms of PLTP; characteristics of plasma PLTP complexes; relationship of plasma PLTP activity, mass and specific activity with lipoprotein and metabolic factors; role of PLTP in lipoprotein metabolism; PLTP and reverse cholesterol transport; insights from studies of PLTP variants; insights of PLTP from animal studies; PLTP and atherosclerosis; PLTP and signal transduction; PLTP in the brain; and PLTP in human disease.PLTP's central role in lipoprotein metabolism and lipid transport in the vascular compartment has been firmly established. However, more studies are needed to further delineate PLTP's functions in specific tissues, such as the lung, brain and adipose tissue. Furthermore, the specific role that PLTP plays in human diseases, such as atherosclerosis, cancer, or neurodegenerative disease, remains to be clarified. Exciting directions for future research include evaluation of PLTP's physiological relevance in intracellular lipid metabolism and signal transduction, which undoubtedly will advance our knowledge of PLTP functions in health and disease. 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).Display Omitted► PLTP plays a central role in lipoprotein metabolism. ► PLTP is involved in reverse cholesterol transport. ► Approximately half of human plasma PLTP is inactive in phospholipid transfer. ► Plasma PLTP complexes contain proteins involved in immunity and inflammation. ► PLTP–ABCA1 interaction plays a role in signal transduction.
Keywords: PLTP variant; PLTP animal studies; PLTP and atherosclerosis; PLTP in signal transduction; PLTP in the brain;

Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo. 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).► Acyl-CoA synthetase 1 mediates fatty acid-induced loss of ABCA1 in macrophages. ► Acyl-CoA synthetase 1 thus causes reduced cholesterol efflux to apolipoprotein A-I. ► This study demonstrates a novel link between acyl-CoA synthetase 1 and ABCA1.
Keywords: acyl-CoA synthetase; ATP-binding cassette transporter A1; Cholesterol efflux; High-density lipoprotein; Lipid metabolism; Macrophage;

Increased or decreased hepatic lipase (HL) activity has been associated with coronary artery disease (CAD). This is consistent with the findings that gene variants that influence HL activity were associated with increased CAD risk in some population studies but not in others. In this review, we will explain the conditions that influence the effects of HL on CAD. Increased HL is associated with smaller and denser LDL (sdLDL) and HDL (HDL3) particles, while decreased HL is associated with larger and more buoyant LDL and HDL particles. The effect of HL activity on CAD risk is dependent on the underlying lipoprotein phenotype or disorder. Central obesity with hypertriglyceridemia (HTG) is associated with high HL activity that leads to the formation of sdLDL that is pro-atherogenic. In the absence of HTG, where large buoyant cholesteryl ester-enriched LDL is prominent, elevation of HL does not raise the risk for CAD. In HTG patients, drug therapy that decreases HL activity selectively decreases sdLDL particles, an anti-atherogenic effect. Drug therapy that raises HDL2 cholesterol has not decreased the risk for CAD. In trials where inhibition of cholesterol ester transfer protein (CETP) or HL occurs, the increase in HDL2 most likely is due to inhibition of catabolism of HDL2 and impairment of reverse cholesterol transport (RCT). In patients with isolated hypercholesterolemia, but with normal triglyceride levels and big-buoyant LDL particles, an increase in HL activity is beneficial; possibly because it increases RCT. Drugs that lower HL activity might decrease the risk for CAD only in hypertriglyceridemic patients with sdLDL by selectively clearing sdLDL particles from plasma, which would override the potentially pro-atherogenic effect on RCT. 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).► In hypertriglyceridemia hepatic lipase (HL) hydrolyses LDL and HDL triglyceride. ► In hypertriglyceridemia HL leads to smaller and denser LDL and HDL particles. ► In coronary disease small-dense LDL and decreased HDL2 are due to high HL. ► Drugs that decrease HL are anti-atherogenic due to clearance of small-dense LDL. ► With normal triglyceride levels and big-buoyant LDL, high HL causes increased RCT.
Keywords: Hepatic lipase; Small-dense LDL; Coronary artery disease; GWAS; Triglyceride; Reverse cholesterol transport;

The mammalian ABC transporter ABCA1 induces lipid-dependent drug sensitivity in yeast by Tomasz Bocer; Ana Zarubica; Annie Roussel; Krzysztof Flis; Tomasz Trombik; Andre Goffeau; Stanislaw Ulaszewski; Giovanna Chimini (373-380).
ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).► Successful expression and targeting in yeast of the murine ABCA1 transporter. ► Conservation of ABCA1 functional features across species (yeast to mouse). ► Definition of a novel ABCA1-specific compartment in yeast membrane. ► Induction of drug sensitivity by the expression of wild type ABCA1 in yeast. ► Interplay between ABCA1 function and membrane lipids in yeast and mammalian cells.
Keywords: ABC transporter; Lipid; Membrane composition; Yeast; Cholesterol; Phosphatidylserine;

Genetic variation of GPLD1 associates with serum GPI-PLD levels: A preliminary study by Mark A. Deeg; Xiaoling Xuei; George Eckert; Robert V. Considine; Ying Grace Li; J. Howard Pratt (381-385).
HDL is a heterogeneous mixture of lipoprotein particles varying in composition, size, and function. We and others have described a small (7.0 nm), minor (0.1% of total apolipoprotein AI) particle containing apolipoprotein AI, AIV and glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in humans the function of which is not entirely known. Circulating GPI-PLD levels are regulated by multiple factors including genetics. To determine if genetic variation in GPLD1 affects circulating GPI-PLD levels, we examined the relationship between 32 SNPS upstream, within, and downstream of GPLD1 and circulating GPI-PLD levels in Caucasians (n = 77) and African-Americans (n = 99). The genotype distribution among races differed at 13 SNPs. Nine SNPS were associated with circulating GPI-PLD levels in Caucasians but not African-Americans. These results suggest that genetic variation of GPLD1 appears to associate with circulating GPI-PLD levels. 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).
Keywords: Apolipoprotein AI; HDL; Glycosylphosphatidylinositol-phospholipase D;

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. 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).► Cellular cholesterol homeostasis is a tightly regulated process. ► Mutations in many proteins involved in cholesterol homeostasis result in disease. ► Specific role and substrate of ABCG1 are still unknown. ► This review explores new insights into ABCG1 structure and function.
Keywords: Sterols; Cholesterol; ABC transporter; Lipoprotein receptor; microRNA;

Niemann–Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1−/− and NPC2−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. 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).► ABCA1 expression is impaired in both NPC1- and NPC2-deficient human fibroblasts. ► NPC1 and NPC2 cells expressed ABCA1 following infection with adenoviral ABCA1-EGFP. ► Increased ABCA1 expression reduced lysosomal cholesterol in NPC1 but not NPC2 cells. ► Increased ABCA1 expression corrected HDL formation by NPC1 but not NPC2 cells. ► ABCA1 stimulates removal of lysosomal membrane but not lysosomal lumen cholesterol.
Keywords: ABCA1; Lysosome; Cholesterol; Niemann–Pick disease type C; High density lipoprotein; HDL;

The HDL proteome in acute coronary syndromes shifts to an inflammatory profile by Khalid Alwaili; Dana Bailey; Zuhier Awan; Swneke D. Bailey; Isabelle Ruel; Anouar Hafiane; Larbi Krimbou; Sylvie Laboissiere; Jacques Genest (405-415).
Inflammation is a major factor underlying acute coronary syndromes (ACS). HDL particles may be remodeled, becoming functionally defective, under the inflammatory conditions seen in ACS. Shotgun proteomics was used to monitor changes in the HDL proteome between male age-matched control, stable CAD, and ACS subjects (n  = 10/group). HDL was isolated by ultracentrifugation and separated by 1D-gel followed by LC–MS/MS. We identified 67 HDL-associated proteins, 20 of which validated recently identified proteins including vitronectin and complement C4B, and 5 of which were novel. Using gene ontology analysis, we found that the HDL-proteome consisted of proteins involved in cholesterol homeostasis (~ 50%), with significant contributions by proteins involved in lipid binding, antioxidant, acute-phase response, immune response, and endopeptidase/protease inhibition. Importantly, levels of apoA-IV were significantly reduced in ACS patients, whereas levels of serum amyloid A (SAA) and complement C3 (C3) were significantly increased (spectral counting; t-test p ≤ 0.05), as confirmed by immunoblot or ELISA. Despite differences in protein composition, ABCA1, ABCG1, and SR-BI mediated cholesterol efflux assays did not indicate that HDL from ACS patients is functionally deficient as compared to controls, when corrected for apoA-I mass. Our results support that the HDL proteome differs between control, CAD and ACS patients. Increased abundance of SAA, C3, and other inflammatory proteins in HDL from ACS patients suggests that HDL reflects a shift to an inflammatory profile which, in turn, might alter the protective effects of HDL on the atherosclerotic plaque. 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).► HDL proteome differs in ACS vs. controls. ► SAA and complement C3 are increased in ACS. ► ApoA-IV is reduced in ACS. ► HDL efflux function remains unaltered.
Keywords: Acute coronary syndrome; Lipid; Atherosclerosis; High-density lipoprotein; Proteomics;

Increased risk of coronary artery disease in Caucasians with extremely low HDL cholesterol due to mutations in ABCA1, APOA1, and LCAT by Ian Tietjen; G. Kees Hovingh; Roshni Singaraja; Chris Radomski; Jason McEwen; Elden Chan; Maryanne Mattice; Annick Legendre; John J.P. Kastelein; Michael R. Hayden (416-424).
Mutations in ABCA1, APOA1, and LCAT reduce HDL cholesterol (HDLc) in humans. However, the prevalence of these mutations and their relative effects on HDLc reduction and risk of coronary artery disease (CAD) are less clear. Here we searched for ABCA1, APOA1, and LCAT mutations in 178 unrelated probands with HDLc < 10th percentile but no other major lipid abnormalities, including 89 with ≥ 1 first-degree relative with low HDLc (familial probands) and 89 where familial status of low HDLc is uncertain (unknown probands). Mutations were most frequent in LCAT (15.7%), followed by ABCA1 (9.0%) and APOA1 (4.5%), and were found in 42.7% of familial but only 14.6% of unknown probands (p = 2.44 ∗ 10− 5). Interestingly, only 16 of 24 (66.7%) mutations assessed in families conferred an average HDLc < 10th percentile. Furthermore, only mutation carriers with HDLc < 5th percentile had elevated risk of CAD (odds ratio (OR) = 2.26 for 34 ABCA1 mutation carriers vs. 149 total first-degree relative controls, p = 0.05; OR = 2.50 for 26 APOA1 mutation carriers, p = 0.04; OR = 3.44 for 38 LCAT mutation carriers, p = 1.1 ∗ 10− 3). These observations show that mutations in ABCA1, APOA1, and LCAT are sufficient to explain > 40% of familial hypoalphalipoproteinemia in this cohort. Moreover, individuals with mutations and large reductions in HDLc have increased risk of CAD. 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).► ABCA1, APOA1, and LCAT mutations are in 42.7% of patients with familial low HDLc. ► However, mutations are in 14.6% of patients with unknown family history of low HDLc. ► 41.2% of mutations are novel and segregate with reduced HDLc in families. ► However, only two-thirds of mutations on average confer HDLc < 10th percentile. ► Mutation carriers with HDLc < 5th percentile have significantly increased CAD.
Keywords: Coronary artery disease; Genetics; Lipid; Family-based study;

Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice by Kimberly A. Edgel; Timothy S. McMillen; Hao Wei; Nathalie Pamir; Barbara A. Houston; Mark T. Caldwell; Phuong-Oanh T. Mai; John F. Oram; Chongren Tang; Renée C. LeBoeuf (425-434).
The prevalence of obesity has reached epidemic proportions and is associated with several co-morbid conditions including diabetes, dyslipidemia, cancer, atherosclerosis and gallstones. Obesity is associated with low systemic inflammation and an accumulation of adipose tissue macrophages (ATMs) that are thought to modulate insulin resistance. ATMs may also modulate adipocyte metabolism and take up lipids released during adipocyte lipolysis and cell death. We suggest that high levels of free cholesterol residing in adipocytes are released during these processes and contribute to ATM activation and accumulation during obesity and caloric restriction. Db/db mice were studied for extent of adipose tissue inflammation under feeding conditions of ad libitum (AL) and caloric restriction (CR). The major finding was a marked elevation in epididymal adipose ABCG1 mRNA levels with obesity and CR (6-fold and 16-fold, respectively) over that seen for lean wild-type mice. ABCG1 protein was also elevated for CR as compared to AL adipose tissue. ABCG1 is likely produced by cholesterol loaded ATMs since this gene is not highly expressed in adipocytes and ABCG1 expression is sterol mediated. Our data supports the concept that metabolic changes in adipocytes due to demand lipolysis and cell death lead to cholesterol loading of ATMs. Based on finding cholesterol-loaded peritoneal leukocytes with elevated levels of ABCG1 in CR as compared to AL mice, we suggest that pathways for cholesterol trafficking out of adipose tissue involve ATM egress as well as ABCG1 mediated cholesterol efflux. 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).► ABCG1 expression is markedly elevated by obesity and caloric restriction (CR). ► Adipose tissue macrophage (MO) phenotypes are altered by acute caloric restriction. ► CR leads to cholesterol loaded peritoneal MO and elevations in ABCG1, ABCA1 and apoE.
Keywords: ABCG1; ABCA1; Cholesterol efflux; Obesity; Caloric restriction; Mouse;

A systems genetic analysis of high density lipoprotein metabolism and network preservation across mouse models by Peter Langfelder; Lawrence W. Castellani; Zhiqiang Zhou; Eric Paul; Richard Davis; Eric E. Schadt; Aldons J. Lusis; Steve Horvath; Margarete Mehrabian (435-447).
We report a systems genetic analysis of high density lipoprotein (HDL) levels in an F2 intercross between inbred strains CAST/EiJ and C57BL/6J. We previously showed that there are dramatic differences in HDL metabolism in a cross between these strains, and we now report co-expression network analysis of HDL that integrates global expression data from liver and adipose with relevant metabolic traits. Using data from a total of 293 F2 intercross mice, we constructed weighted gene co-expression networks and identified modules (subnetworks) associated with HDL and clinical traits. These were examined for genes implicated in HDL levels based on large human genome-wide associations studies (GWAS) and examined with respect to conservation between tissue and sexes in a total of 9 data sets. We identify genes that are consistently ranked high by association with HDL across the 9 data sets. We focus in particular on two genes, Wfdc2 and Hdac3, that are located in close proximity to HDL QTL peaks where causal testing indicates that they may affect HDL. Our results provide a rich resource for studies of complex metabolic interactions involving HDL. 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).► We investigate genetic factors affecting HDL in a CASTxB6 F2 mouse cross. ► Network analysis identifies gene co-expression modules associated with HDL. ► Studies across independent data sets confirm robustness of identified modules. ► Using meta-analysis techniques we identify genes consistently associated with HDL. ► Causal testing implicates Wfdc2 and Hdac3 as novel genes affecting HDL levels.
Keywords: HDL; Genetics; Network; Co-expression;

The “beta-clasp” model of apolipoprotein A-I — A lipid-free solution structure determined by electron paramagnetic resonance spectroscopy by Jens O. Lagerstedt; Madhu S. Budamagunta; Grace S. Liu; Nicole C. DeValle; John C. Voss; Michael N. Oda (448-455).
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).► Electron paramagnetic resonance spectroscopy was used to map apoA-I's structure. ► ApoA-I bears 4 beta strands, which may stabilize its lipid-free structure. ► The beta strands may organize the overall structure of apoA-I.
Keywords: Apolipoprotein A-I; High density lipoprotein; Reverse cholesterol transport; ABCA1;

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality by Nicholas N. Lyssenko; Mami Hata; Padmaja Dhanasekaran; Margaret Nickel; David Nguyen; Palaniappan Sevugan Chetty; Hiroyuki Saito; Sissel Lund-Katz; Michael C. Phillips (456-463).
The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (Vmax/Km) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. 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).► Apolipoprotein (apo) A-I structure and function. ► ApoA-I interacts with ABCA1 to mediate efflux of cellular cholesterol. ► ApoA-I solubilizes lipids to form HDL particles. ► Increased apoA-I α-helix hydrophobicity enhances formation of smaller HDL particles.
Keywords: ATP binding cassette transporter A1 (ABCA1); Amphipathic α-helix; Apolipoprotein A-I; Cellular cholesterol efflux; High density lipoprotein (HDL); Lipid solubilization;

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux by Ginny L. Weibel; Michelle R. Joshi; W. Gray Jerome; Sandra R. Bates; Kevin J. Yu; Michael C. Phillips; George H. Rothblat (464-472).
Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30–40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. 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 cytoskeleton effects lipid droplet formation and lipid mobilization. ► We employ cytoskeleton disrupting agents. ► Cytochalasin D alters cholesterol efflux to HDL. ► The cytoskeleton impacts on the size, composition and metabolism of lipid droplets. ► The cytoskeleton plays an integral role in lipid droplet metabolism.
Keywords: Lipid droplet; Cytoskeleton; Cholesteryl ester; Foam cell; Hydrolysis;

The inverse relationship between plasma HDL levels and the risk of developing coronary heart disease is well established. The underlying mechanisms of this relationship are poorly understood, largely because HDL consist of several functionally distinct subpopulations of particles that are continuously being interconverted from one to another. This review commences with an outline of what is known about the origins of individual HDL subpopulations, how their distribution is regulated, and describes strategies that are currently available for isolating them. We then summarise what is known about the functionality of specific HDL subpopulations, and how these findings might impact on cardiovascular risk. The final section highlights major gaps in existing knowledge of HDL functionality, and suggests how these deficiencies might be addressed. 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).► HDL levels are inversely correlated to cardiovascular risk. ► HDL consist of multiple subpopulations of particles. ► Plasma factors interconvert HDL subpopulations. ► HDL subpopulations are not all equally cardioprotective.
Keywords: High density lipoproteins; HDL function; HDL subpopulations;

To develop a detailed double belt model for discoidal HDL, we previously scored inter-helical salt bridges between all possible registries of two stacked antiparallel amphipathic helical rings of apolipoprotein (apo) A-I. The top score was the antiparallel apposition of helix 5 with 5 followed closely by appositions of helix 5 with 4 and helix 5 with 6. The rationale for the current study is that, for each of the optimal scores, a pair of identical residues can be identified in juxtaposition directly on the contact edge between the two antiparallel helical belts of apoA-I. Further, these residues are always in the ‘9th position’ in one of the eighteen 11-mer repeats that make up the lipid-associating domain of apoA-I. To illustrate our terminology, 129j (LL5/5) refers to the juxtaposition of the Cα atoms of G129 (in a ‘9th position’) in the pairwise helix 5 domains. We reasoned that if identical residues in the double belt juxtapositions were mutated to a cysteine and kept under reducing conditions during disc formation, we would have a precise method for determining registration in discoidal HDL by formation of a disulfide-linked apoA-I homodimer. Using this approach, we conclude that 129j (LL5/5) is the major rotamer orientation for double belt HDL and propose that the small ubiquitous gap between the pairwise helix 5 portions of the double belt in larger HDL discoidal particles is significantly dynamic to hinge off the disc edge under certain conditions, e.g., in smaller particles or perhaps following binding of the enzyme LCAT. 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).► Interhelical salt bridges for the HDL belt model maximize at wheel position (wp) 9. ► Only wp9 lies directly in the contact edge between helical rings. ► Wp9 was mutated to Cys and kept under reducing conditions during disc formation. ► We show that helix 5, under certain conditions, progresses to a full hinged domain. ► We hypothesize that, upon binding of LCAT, helix 5 forms a full hinged domain.
Keywords: ApoA-I; Site-directed mutagenesis; Crosslinking; Molecular dynamics simulation; Molecular modeling; LCAT;

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine – two characteristic products of MPO – are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. 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).► MPO impairs ABCA1 cholesterol efflux by modifying Met residues and Tyr192 in apoA-I. ► Chlorination of apoA-I by MPO impairs the initial interactions with ABCA1. ► Acrolein impairs cholesterol efflux by modifying Lys226 in apoA-I. ► MDA impairs cholesterol export by cross-linking of C-terminal Lys residues in apoA-I. ► MPO and reactive carbonyls might generate dysfunctional HDL in humans.
Keywords: Myeloperoxidase; Malondialdehyde; Acrolein; Dysfunctional HDL; 3-Chlorotyrosine; Coronary artery disease;

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice by Mary G. Sorci-Thomas; Manal Zabalawi; Manish S. Bharadwaj; Ashley J. Wilhelm; John S. Owen; Bela F. Asztalos; Shaila Bhat; Michael J. Thomas (502-512).
The mutation L159R apoA-I or apoA-IL159R (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL−/−, apoA-I−/−) (double knockout or DKO) were crossed > 9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr−/−, ApoA-I−/− (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12 weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α3-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-IL159R contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta. 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).► Most apoA-IL159R synthesized in mouse hepatocytes was retained and degraded. ► ApoA-IL159R reduced the secretion of apoA-IWT when expressed together. ► ApoA-IL159R had no effect on plasma levels of HDL or apoA-IWT. ► HDL-like particles from FIN-DKO mice contained large amounts of apoE and B. ► FIN-DKO mice had increased aortic cholesterol compared to mice lacking apoA-I.
Keywords: Apolipoprotein A-1; Apolipoprotein A-1FIN; Atherosclerosis; Dysfunctional HDL; L159R ApoA-I;

Anti-atherogenic mechanisms of high density lipoprotein: Effects on myeloid cells by Andrew J. Murphy; Marit Westerterp; Laurent Yvan-Charvet; Alan R. Tall (513-521).
In some settings increasing high density lipoprotein (HDL) levels has been associated with a reduction in experimental atherosclerosis. This has been most clearly seen in apolipoprotein A-I (apoA-I) transgenic mice or in animals infused with HDL or its apolipoproteins. A major mechanism by which these treatments are thought to delay progression or cause regression of atherosclerosis is by promoting efflux of cholesterol from macrophage foam cells. In addition, HDL has been described as having anti-inflammatory and other beneficial effects. Some recent research has linked anti-inflammatory effects to cholesterol efflux pathways but likely multiple mechanisms are involved. Macrophage cholesterol efflux may have a role in facilitating emigration of macrophages from lesions during regression. While macrophages can mediate cholesterol efflux by several pathways, studies in knockout mice or cells point to the importance of active efflux mediated by ATP binding cassette transporter (ABC) A1 and G1. In addition to traditional roles in macrophages, these transporters have been implicated in the control of hematopoietic stem cell proliferation, monocytosis and neutrophilia, as well as activation of monocytes and neutrophils. Thus, HDL and cholesterol efflux pathways may have important anti-atherogenic effects at all stages of the myeloid cell/monocyte/dendritic cell/macrophage lifecycle. 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).► HDL can regulate myelopoiesis through ABCA1 and ABCG1. ► HDL can prevent monocyte entry into the atherosclerotic lesion by inhibiting number and activation. ► HDL attenuates neutrophil activation and adhesion in acute models of inflammation. ► HDL modulates macrophage functions including migration, efferocytosis and TLR signaling.
Keywords: High density lipoprotein; Macrophage; Monocyte; Neutrophil; Hematopoietic stem and multipotent progenitor cell; Atherosclerosis;

Regulation of ABCA1 functions by signaling pathways by Yuhua Liu; Chongren Tang (522-529).
ATP-binding cassette transporter A1 (ABCA1) is an integral cell membrane protein that protects cardiovascular disease by at least two mechanisms: by export of excess cholesterol from cells and by suppression of inflammation. ABCA1 exports cholesterol and phospholipids from cells by multiple steps that involve forming cell surface lipid domains, binding of apolipoproteins to ABCA1, activating signaling pathways, and solubilizing these lipids by apolipoproteins. ABCA1 executes its anti-inflammatory effect by modifying cell membrane lipid rafts and directly activating signaling pathways. The interaction of apolipoproteins with ABCA1 activates multiple signaling pathways, including Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), protein kinase A, Rho family G protein CDC42 and protein kinase C. Activating protein kinase A and Rho family G protein CDC42 regulates ABCA1-mediated lipid efflux, activating PKC stabilizes ABCA1 protein, and activating JAK2/STAT3 regulates both ABCA1-mediated lipid efflux and anti-inflammation. Thus, ABCA1 behaves both as a lipid exporter and a signaling receptor. Targeting ABCA1 receptor-like property using agonists for ABCA1 protein could become a promising new therapeutic target for increasing ABCA1 function and treating cardiovascular disease. 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).► ABCA1 functions are regulated by multiple signaling pathways. ► Activating PKA and Rho family G protein CDC42 regulates ABCA1-mediated lipid efflux. ► Activating PKC stabilizes ABCA1 protein. ► Activating JAK2/STAT3 regulates both ABCA1-mediated lipid efflux and anti-inflammation.
Keywords: ATP-binding cassette transporter A1; Signaling pathway; Cholesterol efflux; Lipid metabolism;

Lysine residues of ABCA1 are required for the interaction with apoA-I by Kohjiro Nagao; Yasuhisa Kimura; Kazumitsu Ueda (530-535).
ATP-binding cassette protein A1 (ABCA1) plays a pivotal role in cholesterol homeostasis by generating high-density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), a lipid acceptor for ABCA1, reportedly interacts with ABCA1. However, it has also been proposed that apoA-I interacts with ABCA1-generated special domains on the plasma membrane, but apart from ABCA1, and solubilizes membrane lipids. To determine the importance of the apoA-I–ABCA1 interaction in HDL formation, the electrostatic interaction between apoA-I and ABCA1, which mediates the interaction between apoB100 in low-density lipoprotein particles (LDL) and LDL receptor, was analyzed. The apoA-I binding to ABCA1 and the cross-linking between them were inhibited by the highly charged molecules heparin and poly-L-lysine. Treating cells with membrane impermeable reagents that specifically react with primary amino groups abolished the interaction between apoA-I and ABCA1. However, these reagents did not affect the characteristic tight ATP binding to ABCA1. These results suggest that lysine residues in the extracellular domains of ABCA1 contribute to the interaction with apoA-I. The electrostatic interaction between ABCA1 and apoA-I is predicted to be the first step in HDL formation. 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 importance of ABCA1–apoA-I interaction in HDL formation is controversial. ► The ABCA1–apoA-I interaction is mediated via the electrostatic interaction. ► Lysine residues in the extracellular domains of ABCA1 contribute to the interaction. ► The direct ABCA1–apoA-I interaction is the first step in HDL formation.
Keywords: High-density lipoprotein; ABCA1; LDL receptor; Apolipoprotein A-I;

The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1 by Sophie Stukas; Sharon May; Anna Wilkinson; Jeniffer Chan; James Donkin; Cheryl L. Wellington (536-546).
Lipoprotein metabolism in the central nervous system (CNS) is based on high-density lipoprotein-like particles that use apoE as their predominant apolipoprotein rather than apoA-I. Although apoA-I is not expressed in astrocytes and microglia, which produce CNS apoE, apoA-I is reported to be expressed in porcine brain capillary endothelial cells and also crosses the blood–brain barrier (BBB). These mechanisms allow apoA-I to reach concentrations in cerebrospinal fluid (CSF) that are approximately 0.5% of its plasma levels. Recently, apoA-I has been shown to enhance cognitive function and reduce cerebrovascular amyloid deposition in Alzheimer's Disease (AD) mice, raising questions about the regulation and function of apoA-I in the CNS. Peripheral apoA-I metabolism is highly influenced by ABCA1, but less is known about how ABCA1 regulates CNS apoA-I. We report that ABCA1 deficiency leads to greater retention of apoA-I in the CNS than in the periphery. Additionally, treatment of symptomatic AD mice with GW3965, an LXR agonist that stimulates ABCA1 expression, increases apoA-I more dramatically in the CNS compared to the periphery. Furthermore, GW3965-mediated up-regulation of CNS apoA-I is independent of ABCA1. Our results suggest that apoA-I may be regulated by distinct mechanisms on either side of the BBB and that apoA-I may serve to integrate peripheral and CNS lipid metabolism. 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).► ApoA-I is present in the CNS and influences cognitive function. ► ABCA1 deficiency reduces apoA-I levels more extensively in plasma than in the CNS. ► GW3965 treatment elevates apoA-I levels in the CNS independent of ABCA1. ► ApoA-I may be regulated by to distinct pathways in peripheral and CNS compartments. ► ApoA-I may be a point of integration between peripheral and CNS lipid metabolism.
Keywords: Apo-A-I; apoE; ABCA1; LXR; Brain lipid metabolism;

Calpain-mediated ABCA1 degradation: Post-translational regulation of ABCA1 for HDL biogenesis by Shinji Yokoyama; Reijiro Arakawa; Cheng-ai Wu; Noriyuki Iwamoto; Rui Lu; Maki Tsujita; Sumiko Abe-Dohmae (547-551).
Helical apolipoproteins remove cellular phospholipid and cholesterol to generate nascent HDL and this reaction is the major source of plasma HDL. ABCA1 is mandatory and rate-limiting for this reaction. Besides regulation of the gene expression by transcriptional factors including LXR, AP2 and SREBP, the ABCA1 activity is regulated post-translationally by calpain-mediated proteolytic degradation of ABCA1 protein that occurs in the early endosome after its endocytosis. When the HDL biogenesis reaction is ongoing as helical apolipoproteins interact with ABCA1, ABCA1 becomes resistant to calpain and is recycled to cell surface after endocytosis. Biogenesis of HDL is most likely to take place on cell surface. Clearance rate of ABCA1 by this mechanism is also retarded by various factors that interact with ABCA1, such as α1-syntrophin, LXRβ and calmodulin. Physiological relevance of the retardation by these factors is not entirely clear. Pharmacological inhibition of the calpain-mediated ABCA1 degradation results in the increase of the ABCA1 activity and HDL biogenesis in vitro and in vivo, and potentially suppresses atherogenesis. 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).► Calpain-mediated degradation is one of the major regulations for the ABCA1 activity. ► ABCA1 recycling between surface and endosome is enhanced by HDL biogenesis reaction. ► Inhibiting ABCA1 degradation increases HDL and potentially decreases atherogenesis.
Keywords: ABCA1; Calpain; HDL; Cholesterol; Atherosclerosis;

The ATP-binding cassette transporter G1 (ABCG1) mediates free cholesterol efflux onto lipidated apolipoprotein A-I (apoA-I) and plays an important role in macrophage reverse cholesterol transport thereby reducing atherosclerosis. However, how ABCG1 mediates the efflux of cholesterol onto lipidated apoA-I is unclear. Since the crystal structure of ABCG family is not available, other approaches such as site-directed mutagenesis have been widely used to identify amino acid residues important for protein functions. We noticed that ABCG1 contains a single cysteine residue in its putative transmembrane domains. This cysteine residue locates at position 514 (Cys514) within the third putative transmembrane domain and is highly conserved. Replacement of Cys514 with Ala (C514A) essentially abolished ABCG1-mediated cholesterol efflux onto lipidated apoA-I. Substitution of Cys514 with more conserved amino acid residues, Ser or Thr, also significantly decreased cholesterol efflux. However, mutation C514A had no detectable effect on protein stability and trafficking. Mutation C514A also did not affect the dimerization of ABCG1. Our findings demonstrated that the sulfhydryl group of Cys residue located at position 514 plays a critical role in ABCG1-mediated cholesterol efflux. 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).► Cys514 is critical for ABCG1-mediated cholesterol efflux. ► Mutating Cys514 to Ala, Thr and Ser impaired ABCG1-mediated cholesterol efflux. ► Mutation of Cys514 has no effect on ABCG1 trafficking and dimerization.
Keywords: ATP-binding cassette transport; ABCG1; Cholesterol efflux; Transmembrane domain; Cysteine residue; Atherosclerosis;