Phospholipid Efflux Research Articles

Crosstalk Between Lipids and Mitochondria in Diabetic Kidney Disease.

The goal of this review is to review the role that renal parenchymal lipid accumulation plays in contributing to diabetic kidney disease (DKD), specifically contributing to the mitochondrial dysfunction observed in glomerular renal cells in the context of DKD development and progression. Mitochondrial dysfunction has been observed in experimental and clinical DKD. Recently, Ayanga et al. demonstrate that podocyte-specific deletion of a protein involved in mitochondrial dynamics protects from DKD progression. Furthermore, our group has recently shown that ATP-binding cassette A1 (a protein involved in cholesterol and phospholipid efflux) is significantly reduced in clinical and experimental DKD and that genetic or pharmacological induction of ABCA1 is sufficient to protect from DKD. ABCA1 deficiency in podocytes leads to mitochondrial dysfunction observed with alterations of mitochondrial lipids, in particular, cardiolipin (a mitochondrial-specific phospholipid). However, through pharmacological reduction of cardiolipin peroxidation DKD progression is reverted. Lipid metabolism is significantly altered in the diabetic kidney and renders cellular components, such as the podocyte, susceptible to injury leading to worsened DKD progression. Dysfunction of the lipid metabolism pathway can also lead to mitochondrial dysfunction and mitochondrial lipid alteration. Future research aimed at targeting mitochondrial lipids content and function could prove to be beneficial for the treatment of DKD.

Cholesterol Acceptors Regulate the Lipidome of Macrophage Foam Cells.

Arterial foam cells are central players of atherogenesis. Cholesterol acceptors, apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL), take up cholesterol and phospholipids effluxed from foam cells into the circulation. Due to the high abundance of cholesterol in foam cells, most previous studies focused on apoA-I/HDL-mediated free cholesterol (FC) transport. However, recent lipidomics of human atherosclerotic plaques also identified that oxidized sterols (oxysterols) and non-sterol lipid species accumulate as atherogenesis progresses. While it is known that these lipids regulate expression of pro-inflammatory genes linked to plaque instability, how cholesterol acceptors impact the foam cell lipidome, particularly oxysterols and non-sterol lipids, remains unexplored. Using lipidomics analyses, we found cholesterol acceptors remodel foam cell lipidomes. Lipid subclass analyses revealed various oxysterols, sphingomyelins, and ceramides, species uniquely enriched in human plaques were significantly reduced by cholesterol acceptors, especially by apoA-I. These results indicate that the function of lipid-poor apoA-I is not limited to the efflux of cholesterol and phospholipids but suggest that apoA-I serves as a major regulator of the foam cell lipidome and might play an important role in reducing multiple lipid species involved in the pathogenesis of atherosclerosis.

Loss-Of-Function Mutations In Abca1, Hdl-Cholesterol, Metabolomic Profiles And Risk Of Vascular Disease And Dementia – A Cohort Study Of Up To 100,000 Individuals

Background and Aims: The adenosine triphosphate-binding cassette transporter A1 (ABCA1) is a major cholesterol transporter highly expressed in liver and brain. ABCA1 mediates cholesterol and phospholipid efflux to lipid-poor apolipoproteins, and is essential for the biogenesis of high-density lipoprotein (HDL) cholesterol in the circulation and HDL-like particles in the brain. We therefore investigated the impact of two well-known HDL-deficiency loss-of-function mutations in ABCA1 on lipid levels, metabolomic profiles, and on risk of vascular disease and dementia.

Enhancing effect of taurohyodeoxycholate on ABCB4-mediated phospholipid efflux

Hydrophilic bile salts, ursodeoxycholate and hyodeoxycholate, have choleretic effects. ABCB4, a member of the ABC transporter family, is essential for the secretion of phospholipids from hepatocytes into bile. In this study, we assessed the effects of taurine- or glycine-conjugated cholate, ursodeoxycholate and hyodeoxycholate on the ABCB4-mediated phosphatidylcholine (PC) efflux using Abcb4 knockout mice and HEK293 cells stably expressing ABCB4. To evaluate the effects of bile salts on bile formation in Abcb4+/+ or Abcb4−/− mice, the bile was collected during intravenous infusion of saline or bile salts. The biliary PC secretion in Abcb4+/+ mice was significantly increased by the infusions of all tested bile salts, especially taurohyodeoxycholate. On the other hand, Abcb4−/− mice exhibited extremely low secretion of PC into bile, which was not altered by bile salt infusions. We also showed that the PC efflux from ABCB4-expressing HEK293 cells was stimulated by taurohyodeoxycholate much more strongly than the other tested bile salts. However, taurohyodeoxycholate did not restore the activities of ABCB4 mutants. Furthermore, light scattering measurements demonstrated a remarkable ability of taurohyodeoxycholate to form mixed micelles with PC. Therefore, the enhancing effect of taurohyodeoxycholate on the ABCB4-mediated PC efflux may be due to the strong mixed micelle formation ability.

Selective peroxisome proliferator-activated receptor-α modulator K-877 regulates the expression of ATP-binding cassette transporter A1 in pancreatic beta cells

ATP-binding cassette transporter A1 (ABCA1) protein is a pivotal regulator of cholesterol and phospholipid efflux from cells to high-density lipoprotein (HDL) particles. Pancreatic ABCA1 functions in beta cell cholesterol homeostasis and affects insulin secretion. We investigated the effect of pemafibrate (K-877), a novel selective PPARα modulator (SPPARMα), on pancreatic ABCA1 expression. In vivo experiment, mice were divided into four treatment groups, namely, normal food plus placebo, high fat diet (HFD) plus placebo, normal food plus K-877 (0.3 mg/kg/day), or HFD plus K-877 (0.3 mg/kg/day), and treated for eight weeks. The results in vitro experiment indicate that K-877 treatment increased levels of ABCA1 mRNA, as well as protein, subsequently reduced the cellular cholesterol content in INS-1 cells. PPARα specific antagonist GW6471 attenuate K-877 induced ABCA1 expression in INS-1 cells. ABCA1 promoter activity increased with K-877 treatment at concentration 1 μM and 10 μM. Glucose-stimulated insulin secretion was ameliorated by K-877 treatment in INS-1 cells and isolated mouse islets. Although the expression of ABCA1 was reduced in mice with HFD treatment, both ABCA1 protein and mRNA levels were increased in mice with K-877 treatment. K-877 treatment improved glucose intolerance induced by HFD in mice. These findings raise the possibility that K-877 may affect insulin secretion by controlling ABCA1 expression in pancreatic beta cells.

Factors Influencing the Chaperone-Like Activity of Major Proteins of Mammalian Seminal Plasma, Equine HSP-1/2 and Bovine PDC-109: Effect of Membrane Binding, pH and Ionic Strength.

HSP-1/2 and PDC-109 belong to a family of fibronectin type II proteins, present in high concentrations in bovine and equine seminal plasma, respectively. These proteins act as extracellular small heat shock proteins and protect target/client proteins against various kinds of stress. They also exhibit characteristic binding to choline phospholipids present on the sperm plasma membrane and cause efflux of choline phospholipids and cholesterol, resulting in sperm capacitation. The current study demonstrates that hypersaline conditions decrease the chaperone-like activity (CLA) of HSP-1/2. On the other hand, lipoprotein aggregates formed by the binding of choline phospholipids to this protein exhibit higher CLA than HSP-1/2 alone in vitro; the increased CLA can be correlated to the increased surface hydrophobicity of the lipoprotein aggregates. Presence of cholesterol in the membrane was found to decrease such enhancement in the CLA. We have also observed that salinity of the medium affects the chaperone activity by altering the polydisperse nature of the HSP-1/2. Together these results indicate that hydrophobicity and polydispersity are important for the chaperone-like activity of HSP-1/2 and factors that can alter these properties of HSP-1/2 can modulate its CLA. Further, studies on PDC-109 show that the chaperone-like and membrane-destabilizing activities of this protein are differentially affected by change in pH.

Abstract 21122: Contribution of ABCA1 Genetic Variation to Plasma Lipid Levels in Non-Hispanic White Americans

Introduction: Dyslipidemia [i.e., elevated levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG); decreased level of high-density lipoprotein cholesterol (HDL-C)] is one of the risk factors for cardiovascular disease (CVD). Genetics play an important role in inter-individual variation of lipid levels. ATP-binding cassette A1 (ABCA1), a membrane transporter, mediates cellular phospholipid and cholesterol efflux. Mutations in ABCA1 gene cause Tangier’s disease (OMIM #205400) presented with HDL-C deficiency, as well as, increased risk for developing CVD. In this study, we aimed to identify both common [minor allele frequency (MAF)≥5%] and uncommon (MAF<5%) variants of ABCA1 associated with inter-individual variation in major lipid traits. Methods: We resequenced 50 exons and exon-intron boundaries of ABCA1 in a selected set of 95 individuals with extreme HDL-C levels (≤10 th and ≥90 th %tile), and subsequently genotyped selected variants in the entire sample of 623 US Non-Hispanic Whites. With resequencing, we identified 404 variants, and for genotyping we selected 250 variants (216 from sequencing data, 34 from HapMap-CEU data). A total of 182 successfully genotyped and QC-passed bi-allelic variants were tested for association with plasma lipid levels. Results: Gene-based test showed evidence of association between ABCA1 and TG ( p =0.0108). Single-site association analyses demonstrated 26 common variants nominally associated ( p <0.05) with at least one lipid trait. After multiple testing correction, two variants [rs4743763 with TC levels, p =0.0005; rs2066716 (Thr1427Thr) with TG levels, p =0.0016] remained significant. Furthermore, rs2066716 (Thr1427Thr) was among 11 out of 26 lipid-associated ABCA1 variants for which the replication data were available in an independent sample ( n =744) and it remained significantly associated with TG levels in the meta-analysis (meta- p =0.0088). Rare variant analyses revealed association of multiple sets of rare variants with lipid traits. Conclusions: Our findings support the genetic contribution of ABCA1 , both common and rare variants, to lipid levels in humans.

Spermine and spermidine act as chemical chaperones and enhance chaperone-like and membranolytic activities of major bovine seminal plasma protein, PDC-109

The major bovine seminal plasma protein, PDC-109, binds to choline phospholipids of the sperm plasma membrane and induces an efflux of cholesterol and choline phospholipids (cholesterol efflux), which is crucial for sperm capacitation. PDC-109 also exhibits chaperone-like activity and protects target proteins against various kinds of stress. Here we show that the polyamines spermine and spermidine, present in high concentration in the seminal plasma of various mammals, increase the ability of PDC-109 to perturb membrane structure as well as its chaperone-like activity. Interestingly, spermine/spermidine alone did not perturb membrane structure but exhibited chaperone-like activity by protecting target proteins against thermal and oxidative stress. When spermine/spermidine was used along with PDC-109, the observed chaperone-like activity was considerably higher than that expected for a simple additive effect, suggesting that PDC-109 and the polyamines act in a synergistic fashion. These results indicate that at the high concentrations present in the seminal plasma spermine/spermidine exhibit a positive modulatory effect on the chaperone-like activity of PDC-109 and may also function as chemical chaperones and protect other seminal plasma proteins from various kinds of stress.

Leukocyte ABCA1 impedes progression of established atherosclerotic lesions after dietary cholesterol lowering in LDLr-/- mice

The ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of cholesterol and phospholipids to lipid-free apolipoprotein A1 and small dense high-density lipoproteins. Various studies have shown that leukocyte ABCA1 is an anti-atherogenic factor. Dietary cholesterol lowering stabilizes atherosclerotic lesions, however the role of ABCA1 in this process is unknown. Therefore, this study aims to investigate the effect of leukocyte ABCA1 on diet-induced atherosclerotic lesions after withdrawal of the atherogenic diet . Leukocyte ABCA1 was studied by transplanting bone marrow cells from donor mice that were either knock-out (ABCA1-/-) or wild-type for ABCA1 or that overexpressed ABCA1 (ABCA1Tg) into low-density lipoprotein receptor knock-out (LDLr-/-) mice. All three groups of chimeric mice were fed a Western-type diet (WTD: 0.25%cholesterol, 15Ecao butter) for 6 weeks to induce atherosclerotic lesion development. After this period, a baseline group was sacrificed (ABCA1-/- >LDLr-/- n=12; wild-type >LDLr-/- n=14; ABCA1Tg >LDLr-/- n=9) for lesion assessment. The remainder of chimeric mice was switched to a chow diet (low fat, no added cholesterol) for 3 weeks to lower plasma cholesterol levels (ABCA1-/- >LDLr-/- n=14; wild-type >LDLr-/- n=14; ABCA1Tg >LDLr-/- n=12).Withdrawal of the atherogenic diet normalized plasma cholesterol levels in all three groups. As a result, lesion development was stabilized in both the wild-type >LDLr-/- and ABCA1Tg >LDLr-/- mice, however not in the ABCA1-/- >LDLr-/- mice, which displayed a 1.5-fold increase (p Leukocyte ABCA1 is required to halt atherosclerotic lesion progression after dietary cholesterol lowering. Overexpression of ABCA1 did not result in any additional beneficial effects.

Structure of the Human Lipid Exporter ABCA1

ABCA1, an ATP-binding cassette (ABC) subfamily A exporter, mediates the cellular efflux of phospholipids and cholesterol to the extracellular acceptor apolipoprotein A-I (apoA-I) for generation of nascent high-density lipoprotein (HDL). Mutations of human ABCA1 are associated with Tangier disease and familial HDL deficiency. Here, we report the cryo-EM structure of human ABCA1 with nominal resolutions of 4.1Å for the overall structure and 3.9Å for the massive extracellular domain. The nucleotide-binding domains (NBDs) display a nucleotide-free state, while the two transmembrane domains (TMDs) contact each other through a narrow interface in the intracellular leaflet of the membrane. In addition to TMDs and NBDs, two extracellular domains of ABCA1 enclose an elongated hydrophobic tunnel. Structural mapping of dozens of disease-related mutations allows potential interpretation of their diverse pathogenic mechanisms. Structural-based analysis suggests a plausible "lateral access" mechanism for ABCA1-mediated lipid export that may be distinct from the conventional alternating-access paradigm.

Evaluating the Role of Multidrug Resistance Protein 3 (MDR3) Inhibition in Predicting Drug-Induced Liver Injury Using 125 Pharmaceuticals.

The role of bile salt export protein (BSEP) inhibition in drug-induced liver injury (DILI) has been investigated widely, while inhibition of the canalicular multidrug resistant protein 3 (MDR3) has received less attention. This transporter plays a pivotal role in secretion of phospholipids into bile and functions coordinately with BSEP to mediate the formation of bile acid-containing biliary micelles. Therefore, inhibition of MDR3 in human hepatocytes was examined across 125 drugs (70 of Most-DILI-concern and 55 of No-DILI-concern). Of these tested, 41% of Most-DILI-concern and 47% of No-DILI-concern drugs had MDR3 IC50 values of <50 μM. A better distinction across DILI classifications occurred when systemic exposure was considered where safety margins of 50-fold had low sensitivity (0.29), but high specificity (0.96). Analysis of physical chemical property space showed that basic compounds were twice as likely to be MDR3 inhibitors as acids, neutrals, and zwitterions and that inhibitors were more likely to have polar surface area (PSA) values of <100 Å2 and cPFLogD values between 1.5 and 5. These descriptors, with different cutoffs, also highlighted a group of compounds that shared dual potency as MDR3 and BSEP inhibitors. Nine drugs classified as Most-DILI-concern compounds (four withdrawn, four boxed warning, and one liver injury warning in their approved label) had intrinsic potency features of <20 μM in both assays, thereby reinforcing the notion that multiple inhibitory mechanisms governing bile formation (bile acid and phospholipid efflux) may confer additional risk factors that play into more severe forms of DILI as shown by others for BSEP inhibitors combined with multidrug resistance-associated protein (MRP2, MRP3, MRP4) inhibitory properties. Avoiding physical property descriptors that highlight dual BSEP and MDR3 inhibition or testing drug candidates for inhibition of multiple efflux transporters (e.g., BSEP, MDR3, and MRPs) may be an effective strategy for prioritizing drug candidates with less likelihood of causing clinical DILI.

Abstract 180: Chronic Myelogenous Leukemia Cell Viability is Differentially Regulated by LXR Activation, HDL-associated Proteins, and LDL

LXR activation and HDL exert anti-cancer effects, albeit to varying degrees depending on cancer type and model. One of the mechanisms by which LXR and HDL impair cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Thus, the variability in LXR- and HDL-mediated anti-cancer effects may in part be due to differences in 1) HDL functionality and 2) the ability to induce expression of LXR target genes involved in lipid efflux and homeostasis due to adaptive shifts in cancer cell metabolism. Interestingly, serum amyloid A (SAA), a pro-inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity, has recently been shown to differentially increase and decrease cholesterol efflux, despite its ability to displace apoA1 on HDL. Therefore, we investigated the effects of LXR activation, lipoproteins (HDL and LDL) and HDL-associated proteins (apoA1 and SAA) on the viability of chronic myelogenous leukemia cells - a cancer model previously unstudied in regards to cholesterol metabolism. Treatment of cells with the LXR agonist TO901317 (1μM, 5μM, and 10μM) significantly increased ABCA1 mRNA expression at 24hr and 72hr, in addition to dose-dependently decreasing HMG-CoA reductase and anti-apoptotic Bcl-xL mRNA expression. Changes in TO901317-induced gene expression corresponded to time- (24hr, 48hr, 72hr) and dose-dependent decreases in cell viability and average cell size. Interestingly, the addition of HDL to cell cultures with or without TO901317 treatment did not affect K562 cell viability, whereas LDL dose-dependently increased cell viability. Conversely, human-derived apoA1 (25 μg/mL) or HDL + apoA1 drastically reduced TO901317-treated K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA (10 μg/mL) treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that LXR activation, HDL-associated proteins, and LDL differentially regulate chronic myelogenous leukemia cell viability.

Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1

Objectives: ABCA1 encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I). ABCA1 variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known. Methods: In silico analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of ABCA1 missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel ABCA1 variants were generated in vitro through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods. Results: A total of 32 mutations in ABCA1 were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors. Conclusions: These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.

HDL‐Associated Proteins and LDL Differentially Modulate Chronic Myelogenous Leukemia Cell Viability

HDL has been shown to exert anti‐cancer effects. One of the mechanisms by which HDL impairs cancer cell growth is through mediation of cellular cholesterol and phospholipid efflux, thereby limiting the availability of essential lipids for rapidly proliferating cells. Cholesterol efflux is facilitated through interactions between cellular ATP‐binding cassette transporter A1 (ABCA1) and apolipopotein A1 (apoA1), in addition to efflux via ABCG1, scavenger receptor class B type 1 (SR‐BI), and passive diffusion to lipidated HDL. Efflux can further be regulated serum amyloid A (SAA), a pro‐inflammatory acute phase protein shown to be elevated in cancer, infection, and obesity. Despite the ability of SAA to displace apoA1 on HDL, studies have reported SAA to both increase and decrease cholesterol efflux. In this study, we investigated the effects of lipoproteins (HDL and LDL) and HDL‐associated proteins (apoA1 and SAA) on chronic myelogenous leukemia cell viability. K562 cells were cultured in the presence of human HDL or LDL (10, 25, or 50 ug/mL) for 24 hours, then assessed for viability. While no changes in K562 cell viability were observed with HDL treatment, LDL dose‐dependently increased cell viability. We further examined the effects of lipoproteins on K562 viability in the presence of the liver X receptor (LXR) agonist TO901317 (5 uM), which is known to induce expression of HDL‐related transporters. Accordingly, we observed significantly increased expression of ABCA1 at 24hr and 72hr following TO901317 treatment. Interestingly, the addition of HDL to TO901317‐treated cells did not affect K562 cell viability, whereas LDL dose‐dependently increased viability. Conversely, treatment of cells with human‐derived apoA1 or HDL + apoA1 drastically reduced K562 cell viability after 48hr and 72hr. K562 cell viability was modestly decreased by SAA treatment, with greater decreases observed in cells treated with SAA + HDL. These findings suggest that HDL‐associated proteins and LDL differentially regulate chronic myelogenous leukemia cell viabilitySupport or Funding InformationThe Science Institute of the College of Arts and Sciences and Faculty Research Committee, Fairfield University

Liver X receptor agonist treatment significantly affects phenotype and transcriptome of APOE3 and APOE4 Abca1 haplo-deficient mice.

ATP-binding cassette transporter A1 (ABCA1) controls cholesterol and phospholipid efflux to lipid-poor apolipoprotein E (APOE) and is transcriptionally controlled by Liver X receptors (LXRs) and Retinoic X Receptors (RXRs). In APP transgenic mice, lack of Abca1 increased Aβ deposition and cognitive deficits. Abca1 haplo-deficiency in mice expressing human APOE isoforms, increased level of Aβ oligomers and worsened memory deficits, preferentially in APOE4 mice. In contrast upregulation of Abca1 by LXR/RXR agonists significantly ameliorated pathological phenotype of those mice. The goal of this study was to examine the effect of LXR agonist T0901317 (T0) on the phenotype and brain transcriptome of APP/E3 and APP/E4 Abca1 haplo-deficient (APP/E3/Abca1+/- and APP/E4/Abca1+/-) mice. Our data demonstrate that activated LXRs/RXR ameliorated APOE4-driven pathological phenotype and significantly affected brain transcriptome. We show that in mice expressing either APOE isoform, T0 treatment increased mRNA level of genes known to affect brain APOE lipidation such as Abca1 and Abcg1. In both APP/E3/Abca1+/- and APP/E4/Abca1+/- mice, the application of LXR agonist significantly increased ABCA1 protein level accompanied by an increased APOE lipidation, and was associated with restoration of APOE4 cognitive deficits, reduced levels of Aβ oligomers, but unchanged amyloid load. Finally, using Gene set enrichment analysis we show a significant APOE isoform specific response to LXR agonist treatment: Gene Ontology categories “Microtubule Based Process” and “Synapse Organization” were differentially affected in T0-treated APP/E4/Abca1+/- mice. Altogether, the results are suggesting that treatment of APP/E4/Abca1+/- mice with LXR agonist T0 ameliorates APOE4-induced AD-like pathology and therefore targeting the LXR-ABCA1-APOE regulatory axis could be effective as a potential therapeutic approach in AD patients, carriers of APOEε4.

SAT-210 - Regeneration linked miRNA in acute liver failure can predict clinical recompensation after DAA associated HCV cure: a novel biomarker to inform treatment selection in decompensated HCV cirrhosis?

Cardiovascular diseases (CVD) are the major cause of morbidity and mortality in Malaysia, and dyslipidemia is a known contributing factor to the development of CVD. MicroRNAs (miRNAs) have been identified as potent post-transcriptional regulators of lipid metabolism, making it an important area of research to be explored in lipid disorders. Until today, no published data was reported on circulating miRNA among young men with dyslipidemia.To determine the differentially expressed miRNA between with dyslipidemia and nondyslipidemia in young men.Fourteen young males (32 ± 0.45 years) were recruited at UKMMC after obtaining written consent and were divided into dyslipidemia and non-dyslipidemia. Mean values for TC and LDL were significantly higher in dyslipidemia men compared to non-dyslipidemia (4.20 ± 0.8 mg/dl vs. 3.24 ± 0.33 mg/dl and 5.9 ± 0.67 mg/dl vs. 4.9 ± 0.31 mg/dl) (p<0.05), respectively. TG and HDL levels were not significant between both groups. Ten ml of blood was collected for total RNA extraction which then subjected to quantitative PCR using miRCURY LNA™ Universal RT microRNA PCR Serum/Plasma Focus Panel. All data was analysed using GenEx software (reference gene: miR-423-5p) and SPSS v.16 with p<0.05 was considered significant.Our results revealed six miRNAs were significantly upregulated and two were downregulated in dyslipidemia men compared to non-dyslipidemia (p<0.05). miR-144 is the most significant upregulated miRNA. This miRNA plays an important role in lipid metabolism as it targeted Liver X Receptor (LXR), an established key modulators of lipid metabolism. Eventually, it led to accumulation of cholesterol and attenuated cholesterol efflux by suppressing ABCA1 expression. ABCA1 is known to facilitate the efflux of cholesterol and phospholipids to lipid-poor lipoproteins.The study has successfully discovered a list of miRNA in circulating blood of young men with dyslipidemia. Further validation of these candidate miRNA is required before they are used as biomarkers for the current health problem in our community.

A pH Switch Regulates the Inverse Relationship between Membranolytic and Chaperone-like Activities of HSP-1/2, a Major Protein of Horse Seminal Plasma.

HSP-1/2, a major protein of horse seminal plasma binds to choline phospholipids present on the sperm plasma membrane and perturbs its structure by intercalating into the hydrophobic core, which results in an efflux of choline phospholipids and cholesterol, an important event in sperm capacitation. HSP-1/2 also exhibits chaperone-like activity (CLA) in vitro and protects target proteins against various kinds of stress. In the present study we show that HSP-1/2 exhibits destabilizing activity toward model supported and cell membranes. The membranolytic activity of HSP-1/2 is found to be pH dependent, with lytic activity being high at mildly acidic pH (6.0-6.5) and low at mildly basic pH (8.0-8.5). Interestingly, the CLA is also found to be pH dependent, with high activity at mildly basic pH and low activity at mildly acidic pH. Taken together the present studies demonstrate that the membranolytic and chaperone-like activities of HSP-1/2 have an inverse relationship and are regulated via a pH switch, which is reversible. The higher CLA observed at mildly basic pH could be correlated to an increase in surface hydrophobicity of the protein. To the best of our knowledge, this is the first study reporting regulation of two different activities of a chaperone protein by a pH switch.

Functional Rescue of Trafficking-Impaired ABCB4 Mutants by Chemical Chaperones.

Multidrug resistance protein 3 (MDR3, ABCB4) is a hepatocellular membrane protein that mediates biliary secretion of phosphatidylcholine. Null mutations in ABCB4 gene give rise to severe early-onset cholestatic liver disease. We have previously shown that the disease-associated mutations p.G68R, p.G228R, p.D459H, and p.A934T resulted in retention of ABCB4 in the endoplasmic reticulum, thus failing to target the plasma membrane. In the present study, we tested the ability of two compounds with chaperone-like activity, 4-phenylbutyrate and curcumin, to rescue these ABCB4 mutants by assessing their effects on subcellular localization, protein maturation, and phospholipid efflux capability. Incubation of transfected cells at a reduced temperature (30°C) or exposure to pharmacological doses of either 4-PBA or curcumin restored cell surface expression of mutants G228R and A934T. The delivery of these mutants to the plasma membrane was accompanied by a switch in the ratio of mature to inmature protein forms, leading to a predominant expression of the mature protein. This effect was due to an improvement in the maturation rate and not to the stabilization of the mature forms. Both mutants were also functionally rescued, displaying bile salt-dependent phospholipid efflux activity after addition of 4-PBA or curcumin. Drug-induced rescue was mutant specific, given neither 4-PBA nor curcumin had an effect on the ABCB4 mutants G68R and A934T. Collectively, these data indicate that the functionality of selected trafficking-defective ABCB4 mutants can be recovered by chemical chaperones through restoration of membrane localization, suggesting a potential treatment for patients carrying such mutations.

ABCA1 agonist peptides for the treatment of disease.

The review summarizes information pertaining to the preclinical development of new apolipoprotein (apo) E mimetic peptides that stimulate cellular cholesterol efflux. Small α-helical peptides based on the C-terminal domain of apoE have been developed for therapeutic applications. These peptides stimulate cellular cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1) with high potency, like native apolipoproteins on a molar basis. This potent activity has been related to the unique ability of these peptides to maintain α-helix structure upon dilution. Recent structure-activity studies improving the safety features of these mimetic peptides have greatly improved their potential for clinical use. These studies have identified structural features of the class A α-helix motif that induce muscle toxicity and hypertriglyceridemia, which may have implications for the design of other HDL mimetic peptides. ABCA1 is an integral membrane protein that plays a central role in biology. Its principal function is to mediate the efflux of cholesterol and phospholipid from cells to extracellular apo, preventing a build-up of excess cholesterol in membranes. This process generates HDL particles that perform a variety of functions to protect against disease. A number of these functions can be viewed as directly or indirectly supporting ABCA1 activity, thus constituting a positive feedback system to optimize cellular lipid efflux responses and disease prevention. Consequently, therapeutic approaches that mimic the activities of apos may prove highly effective to combat disease. One such approach involves the use of peptides. The broad biological relevance of ABCA1 suggests these apo mimetic peptides may be useful for the treatment of a number of diseases, such as atherosclerosis, diabetes, and Alzheimer's disease.

Lipidated APOE has effects on cognitive function that are independent of amyloid-β pathology.

This scientific commentary refers to ‘Opposing effects of Apoe/Apoa1 double deletion on amyloid-β pathology and cognitive performance in APP mice’, by Fitz et al. (doi:10.1093/brain/awv293). Since the discovery of the apolipoprotein E e4 allele ( APOE4 ) as the strongest genetic risk factor for Alzheimer’s disease (reviewed by Verghese et al. , 2011), there has been a dramatic increase in research on the intersection of lipid biology and Alzheimer pathology. APOE is the major cholesterol and lipid carrier in the brain, while apolipoprotein A1 (APOA1) is the major lipid carrier in the periphery. Efflux of cholesterol and phospholipids from cells onto lipid-poor APOE and APOA1 is regulated by the ATP binding cassette transporter A1 (ABCA1), which is essential for the generation of the vast majority of high density lipoprotein (HDL) particles. The importance of APOE in Alzheimer pathology is highlighted by the fact that crossing a transgenic Alzheimer’s disease mouse model with APOE-deficient mice produced a marked decrease in amyloid-β pathology in the offspring (Bales et al. , 1997). By contrast, crossing ABCA1-deficient mice with an Alzheimer’s disease model generated offspring with increased amyloid-β pathology, despite vastly reduced levels of APOE and APOA1 (reviewed by Koldamova et al. , 2014). This apparent contradiction is currently unresolved. However, in this issue of Brain , Fitz and co-workers provide substantial insight into the complex role of apolipoproteins and their lipidation state in the pathophysiology of Alzheimer’s disease (Fitz et al. , 2015 …