Distribution Of Cholesterol Research Articles

Molecular mechanisms of STARD3 addiction in HER2 tumors

Human Epidermal growth factor Receptor 2 (HER2) is overexpressed and amplified in approximately 15–20% of breast cancers. HER2 is a tyrosine kinase receptor belonging to the EGFR family. HER2 amplification and overexpression play a central role in initiation, progression and metastasis of common cancers like lung, breast and gastric cancer (Hynes and MacDonald, 2009). HER2 amplification is a marker of poor prognosis, especially in breast and gastric cancer (Slamon and Clark, 1988). Although anti‐HER2‐targeted therapies have shown significant clinical benefits, de novo and acquired resistance remain a considerable issue (Garrett and Arteaga, 2011). Genomic profiling has demonstrated that HER2 positive‐tumors have additional alterations, and more importantly they co‐amplify and co‐express several genes on the 17q12 locus (Bièche et al., 1996; Kauraniemi et al., 2001; Tomasetto et al., 1995). Among these genes co‐expressed with HER2, our team identified and characterized STARD3, a gene belonging to the HER2 amplicon (Alpy and Tomasetto, 2014).STARD3 is a lipid transfer protein belonging to the START domain protein family (Alpy et al., 2001). This protein has two domains: the MENTAL domain anchors the protein in late endosomes and the cytoplasmic START domain allows cholesterol transport. Moreover, an FFAT motif present at the interface between the two domains of STARD3 interacts with VAP or MOSPD2 proteins localized in the endoplasmic reticulum (ER) (Alpy et al., 2013; Mattia et al., 2018). Thus, this interaction creates a membrane contact site between late endosomes and the ER allowing then the transfer of cholesterol towards late endosomes (Alpy et al., 2013; Wilhelm et al., 2017). Also, STARD3 has been functionally implicated in the survival of HER2‐amplified cells. Indeed, inhibition of STARD3 expression by RNA interference limits cell growth only in HER2‐amplified cells (Alpy and Tomasetto, 2014; Sahlberg et al., 2013). Thus, STARD3 belongs to the HER2 smallest region of amplification and its expression suggests that it contributes to HER2 tumor growth. We think that STARD3 cooperates with HER2 for tumor growth through an indirect molecular mechanism. Our results suggest that STARD3 acts on cancer cells by altering the normal cholesterol distribution and modifying the lipid composition of cellular membranes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proline residues in scavenger receptor-BI's C-terminal region support efficient cholesterol transport.

High-density lipoproteins (HDLs) facilitate reverse cholesterol transport, a process in which HDL removes cholesterol from circulation and carries it to the liver for biliary excretion. Reverse cholesterol transport is also facilitated by HDL's high-affinity receptor, scavenger receptor-BI (SR-BI), by mechanisms that are not fully understood. To improve our understanding of SR-BI function, we previously solved the NMR (nuclear magnetic resonance) structure of a peptide encompassing amino acids 405–475 of SR-BI. This segment of SR-BI, that includes the functionally critical C-terminal transmembrane domain and part of the extracellular domain, also contains four conserved proline (Pro) residues. We hypothesized that these proline residues support SR-BI in a conformation that allows for efficient cholesterol transport. To test this, we generated individual Pro-to-alanine mutations in full-length SR-BI and transiently expressed the mutant receptors in COS-7 cells to measure the effects on SR-BI-mediated cholesterol transport functions. Our findings reveal that HDL cell association and uptake of HDL-cholesteryl esters are impaired by mutation of Pro-412, Pro-438, or the transmembrane proline kink residue (Pro-459). In addition, SR-BI-mediated cholesterol efflux and membrane cholesterol distribution are impaired by mutation of Pro-412 or Pro-438, indicating that these residues are essential for a fully functional SR-BI receptor. Furthermore, we demonstrate that Pro-408 is necessary for proper SR-BI expression, but mutation of Pro-408 does not cause SR-BI to become misfolded or rapidly degraded by the proteasome or the lysosome. We conclude that key proline residues play an important role in SR-BI function by allowing for the efficient transport of cholesterol between cells and HDL.

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

Changes in the asymmetric distribution of cholesterol in the plasma membrane influence streptolysin O pore formation

ATP-binding cassette A1 (ABCA1) plays a key role in generating high-density lipoprotein (HDL) and preventing atherosclerosis. ABCA1 exports cholesterol and phospholipid to apolipoprotein A-I (apoA-I) in serum to generate HDL. We found that streptolysin O (SLO), a cholesterol-dependent pore-forming toxin, barely formed pores in ABCA1-expressing cells, even in the absence of apoA-I. Neither cholesterol content in cell membranes nor the amount of SLO bound to cells was affected by ABCA1. On the other hand, binding of the D4 domain of perfringolysin O (PFO) to ABCA1-expressing cells increased, suggesting that the amount of cholesterol in the outer leaflet of the plasma membrane (PM) increased and that the cholesterol dependences of these two toxins differ. Addition of cholesterol to the PM by the MβCD–cholesterol complex dramatically restored SLO pore formation in ABCA1-expressing cells. Therefore, exogenous expression of ABCA1 causes reduction in the cholesterol level in the inner leaflet, thereby suppressing SLO pore formation.

Oral glucose effectiveness and metabolic risk in obese children and adolescents.

To investigate whether GE is affected in children/adolescents with obesity and abnormalities of the metabolic syndrome (MetS). Cross-sectional study of oral GE (oGE), insulin sensitivity and secretion (calculated on 5 time-points oral glucose tolerance test) and metabolic abnormalities in 1012 patients with overweight/obesity (aged 6.0-17.9 years old). A MetS risk score was calculated on the basis of distribution of fasting glucose, triglycerides, HDL-cholesterol, total cholesterol, systolic and diastolic blood pressure. Non-alcoholic fatty liver disease (NAFLD) was suspected based on thresholds of alanine aminotransferases. Four-hundred and eighty patients (47.73%) had low-MetS risk score, 488 medium (48.22% with 1-2 risk factors) and 41 (4.05% with ≥ 3 factors) high risk. oGE was significantly lower in subjects with obesity [3.81 (1.46) mg/dl/min- 1] than in those with overweight [4.98 (1.66) mg/dl/min- 1; p value < 0.001]. oGE was negatively correlated with BMI (ρ = - 0.79; p < 0.001) and BMI z score (ρ = - 0.56; p < 0.001) and decreased significantly among MetS risk classes (p = 0.001). The median difference of oGE from low to medium risk was estimated to be as - 4.9%, from medium to high as - 13.38% and from low to high as - 17.62%. oGE was not statistically different between NAFLD+ and NAFLD- cases. In children and adolescents with obesity oGE decreases. Noteworthy, it decreases as the Met score increases. Therefore, reduced oGE may contribute to the higher risk of these individuals to develop type 2 diabetes.

A Single Meal Containing Phytosterols Does Not Affect the Uptake or Tissue Distribution of Cholesterol in Zebrafish (Danio rerio)

Increased plant oil inclusion in aquaculture feeds has led to higher dietary phytosterol concentrations and speculation about whether this affects the metabolism and health of the fish. The mechanisms of cholesterol absorption and how phytosterols may affect this is unknown in fish. Zebrafish (Danio rerio) were used to study the effects of phytosterols on the uptake and organ distribution of dietary cholesterol in fish. One meal of diets containing a constant addition of cholesterol (cold and [4-14C] cholesterol) and varying types and concentrations of phytosterols were fed to fish in individual compartments. The fish were not previously conditioned on the experimental diets. Activity of 14C was then measured in water and fish tissues to quantify the tissue distribution and excretion of cholesterol. There were no effects of the moderate dietary concentrations of phytosterols on the excretion or tissue distribution of dietary cholesterol 24 hours after the meal.

Cell migration is negatively modulated by ABCA1

Temporal and spatial changes of membrane lipid distribution in the plasma membrane are thought to be important for various cellular functions. ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for the generation of high density lipoprotein. Recently, we reported that ABCA1 maintains an asymmetric distribution of cholesterol in the plasma membrane. Here we report that ABCA1 suppresses cell migration by modulating signal pathways. ABCA1 knockdown in mouse embryonic fibroblasts accelerated cell migration and increased activation of Rac1 and its localization to detergent-resistant membranes. Phosphorylation of MEK and ERK also increased. Inhibition of Rac1 or MEK-ERK signals suppressed cell migration in ABCA1 knockdown cells. Because our experimental conditions for cell migration did not contain cholesterol or lipid acceptors for ABCA1, cellular cholesterol content was not changed. These data suggest that ABCA1 modulates cell migration via Rac1 and MEK-ERK signaling by altering lipid distribution in the plasma membrane.

Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma.

Hepatocellular carcinoma is the third leading cause of deaths from cancer worldwide. Infection with the hepatitis B virus is one of the leading risk factors for developing hepatocellular carcinoma, particularly in East Asia1. Although surgical treatment may be effective in the early stages, the five-year overall rate of survival after developing this cancer is only 50-70%2. Here, using proteomic and phospho-proteomic profiling, we characterize 110 paired tumour and non-tumour tissues of clinical early-stage hepatocellular carcinoma related to hepatitis B virus infection. Our quantitative proteomic data highlight heterogeneity in early-stage hepatocellular carcinoma: we used this to stratify the cohort into the subtypes S-I, S-II and S-III, each of which has a different clinical outcome. S-III, which is characterized by disrupted cholesterol homeostasis, is associated with the lowest overall rate of survival and the greatest risk of a poor prognosis after first-line surgery. The knockdown of sterol O-acyltransferase 1 (SOAT1)-high expression of which is a signature specific to the S-III subtype-alters the distribution of cellular cholesterol, and effectively suppresses the proliferation and migration of hepatocellular carcinoma. Finally, on the basis of a patient-derived tumour xenograft mouse model of hepatocellular carcinoma, we found that treatment with avasimibe, an inhibitor of SOAT1, markedly reduced the size of tumours that had high levels of SOAT1 expression. The proteomic stratification of early-stage hepatocellular carcinoma presented in this study provides insight into the tumour biology of this cancer, and suggests opportunities for personalized therapies that target it.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in HRAS Expression of MCOLN1, which encodes TRPML1, is significantly elevated in HRAS-positive tumors and inversely correlated with patient prognosis. Concordantly, MCOLN1 knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type, HRAS Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of HRAS-driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

Distribution of Cholesterol and HER2 in Patient Breast Cancer Cells using Quantitative Single Molecule Localization Microscopy

The nano-scale organization of cholesterol in the plasma membrane is important for cell signaling and function. Cholesterol content of the outer membrane has been assessed previously with domain 4 of the Perfringolysin O toxin (PFO-D4). Here, we used PFO-D4 (C459A)-Alexa Fluor 647 to image cholesterol with quantitative single molecule localization microscopy (qSMLM). We first assessed cholesterol in cultured breast cancer cells. SK-BR-3 cells exhibited cholesterol-enriched domains with an average domain size of 29 nm. We then assessed tumors from breast cancer patients using a combination of qSMLM and tissue touch preparation (touch prep-qSMLM). Patient tumor samples exhibited cholesterol-enriched domains; their sizes ranged from ∼20 to 110 nm. Additionally, the tumor samples were assessed for human epidermal growth factor receptor 2 (HER2) using fluorescently labeled trastuzumab. Importantly, HER2 copy numbers from traditional testing (e.g. fluorescence in situ hybridization) showed a significant positive correlation with detected densities from touch prep-qSMLM. In the future, these single molecule studies on HER2 and cholesterol may help clarify mechanisms of oncogenic signaling and, ultimately, resistance to therapy.

Cholesterol-Dependent Bending Energies in Both Leaves Play a Significant Role in Determining the Cholesterol Distribution in the Plasma Membrane

We consider a plasma membrane that contains a cholesterol mol fraction of 0.4, and ask how that cholesterol is distributed between the two leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical potentials in the two leaves. When we consider only the contributions of entropy and interactions to the cholesterol chemical potential in our model system we find, not surprisingly, that due to the strong attraction between cholesterol and sphingomyelin, which is predominantly in the outer leaf, the cholesterol is mostly in that leaf. We find 74% of the total cholesterol there. The explicit interaction energy does not include the bending energy, which we consider next. Simulations indicate that the bending energy of cholesterol-sphingomyelin mixtures varies non-linearly with composition. This energy is obtained from the product of bending modulus and spontaneous curvature that is available from simulation. We find that the addition of cholesterol to the outer leaf reduces the spontaneous curvature, which is initially positive. It passes through zero when the mol fraction of cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward the inner leaf by the sphingomyelin. This is resisted by the bending energy contribution to the inner leaf. We find, again by simulation, that the addition of cholesterol monotonically increases the magnitude of the spontaneous curvature of the inner leaf, which is negative. This increases its bending energy. We conclude that, as a result of these competing effects, the percent of total cholesterol that is in the outer leaf is reduced to about 62% ±4%.

Interaction modes between nanosized graphene flakes and liposomes: Adsorption, insertion and membrane fusion

BackgroundUnderstanding the effects of graphene-based nanomaterials on lipid membranes is critical to determine their environmental impact and their efficiency in the biomedical context. Graphene has been reported to favourably interact with biological and model lipid membranes. MethodsWe report on a systematic coarse-grained molecular dynamics study of the interaction modes of graphene nanometric flakes with POPC/cholesterol liposome membranes. We have simulated graphene layers with a variety of sizes and oxidation degrees, and we have analyzed the trajectories, the interaction modes, and the energetics of the observed phenomena. ResultsThree interaction modes are reported. Graphene can be transiently adsorbed onto the liposome membrane and/or inserted in its hydrophobic region. Inserted nanosheets prefer a perpendicular orientation, and tilt in order to maximize the contact with phospholipid tails while avoiding the contact with cholesterol molecules. When placed between two liposomes, graphene facilitates their fusion in a single vesicle. ConclusionsGraphene can be temporary adsorbed on the liposome before insertion. Bilayer curvature has an influence on the orientation of inserted graphene particles. Cholesterol molecules are depleted from the surrounding of graphene particles. Graphene layers may catalyse membrane fusion by bypassing the energy barrier required in stalk formation. General significanceNanometric graphene layers can be adsorbed/inserted in lipid-based membranes in different manners and affect the cholesterol distribution in the membrane, implying important consequences on the structure and functionality of biological cell membranes, and on the bioaccumulation of graphene in living organisms. The graphene-mediated mechanism opens new possibilities for vesicle fusion in the experimental context.

Lateral Heterogeneity of Cholesterol Distribution in Cell Plasma Membrane: Investigation by Microfluorimetry, Immunofluorescence, and TOF-SIMS

Lateral heterogeneity of cholesterol distribution in cell plasma membrane was revealed by a complex study using microfluorimetry, immunofluorescence microscopy, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Glioblastoma cells U87MG exhibit a high motility, and cell movements are accompanied by redistribution of proteins along the membrane surface. The formation of the protein caveolin-1 clusters at one of the plasma membrane edges was detected by confocal microscopy on cells labeled with antibodies against caveolin 1. Using two-photon excitation fluorescence of a membrane probe 4-dimethylaminochalcone, membrane areas of about 200 × 200 nm were examined on glioblastoma cells. Simultaneous detection of the decay kinetics and the fluorescence spectrum revealed the presence of regions with an increased cholesterol concentration in the membrane at the poles of the live migrating cell. TOF-SIMS provided direct data with high spatial resolution indicating colocalization of cholesterol and caveolin 1 and confirmed previously published data on the association of caveolin 1 complexes with cholesterol clusters. Thus, three independent methods of cell membrane analysis testify that localization of cholesterol-enriched membrane regions correlates with morphological features of moving glioblastoma cells.

Intracellular and Plasma Membrane Cholesterol Labeling and Quantification Using Filipin and GFP-D4.

Cholesterol, a major component of biological membranes, is rapidly trafficked and unevenly distributed between organelles. Anomalies of intracellular cholesterol distribution are the hallmark of a number of lysosomal lipid storage disorders. A major methodological obstacle for studying cholesterol trafficking is tracing this molecule in situ. The use of fluorescent probes that specifically bind cholesterol allows the visualization and imaging of cellular cholesterol. Here, we describe a series of assays optimized for quantifying free cholesterol in cell populations and at the single cell level, both at the plasma membrane and inside cells. These methods use two fluorescent probes: the D4 fragment of perfringolysin O fused to GFP (GFP-D4) and the polyene macrolide filipin. First, we report a robust method for quantifying plasma membrane cholesterol by flow cytometry using the GFP-D4 probe. Second, to optically distinguish and quantify intracellular cholesterol accumulation, we have adapted the classical filipin cholesterol staining protocol. Indeed, we observed that treatment of living cells with methyl-β-cyclodextrin, a chemical known to extract cholesterol from the plasma membrane, improves the visualization of the intracellular cholesterol pool with filipin. To complement these staining procedures, we developed an image analysis protocol based on image segmentation to quantify, in a robust manner, intracellular cholesterol stained with filipin. Thus, this chapter is a guideline for cellular cholesterol staining and signal quantification.

Mass Spectrometry Imaging of Cholesterol.

Mass spectrometry imaging (MSI) has evolved as a significant tool to map biomolecules in situ without tags. This chapter describes advancements in mass spectrometry imaging technology and applications focused on mapping cholesterol. Secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption/ionization (MALDI), and desorption electrospray (DESI) modes are described. These MSI technologies range in spatial resolution, and therefore different levels of mapping ranging from intracellular to whole tissue can be achieved. A variety of technical examples showing methods for cholesterol and cholesterol-derived molecular imaging are provided as well as imaging results from membrane dynamics, and genetic and drug treatment models. This emerging application of MSI for mapping cholesterol in biological specimens provides a new means to gain a deeper understanding of cholesterol distribution, and thereby insights into function.

ORP2 interacts with phosphoinositides and controls the subcellular distribution of cholesterol

ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation.Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L.ORP2 showed specific affinity for PI(4,5)P2, PI(3,4,5)P3 and PI(4)P, with suggestive Kd values in the μM range. Also binding of cholesterol by ORP2 was detectable, but a Kd could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions.Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.

The Affinity of Sterols for Different Phospholipid Classes and Its Impact on Lateral Segregation

Cholesterol is an essential molecule in the membranes of mammalian cells. It is known to be distributed heterogeneously within the cells, between the bilayer leaflets, as well as between lateral domains within the bilayer. However, we do not know exactly how cholesterol is distributed and what forces drive this sorting process because it extremely difficult to study using currently available methods. To further elucidate this distribution, we measured how cholesterol partitions between different phospholipid (PL) environments using different methods based on cholesterol, TopFluor-cholesterol, and cholesta-5,7,9(11)-triene-3-β-ol. Based on the obtained relative partition coefficients, we made predictions regarding how cholesterol would be distributed between lateral domains and between the inner and outer leaflets of the plasma membrane. In addition, using a trans-parinaric acid fluorescence-based method, we tested how cholesterol could influence lateral segregation through its interaction with unsaturated PLs with different headgroups. The results showed that the lower the affinity of cholesterol was for the different unsaturated PLs, the more cholesterol stimulated lateral segregation in a ternary bilayer of unsaturated PL/N-palmitoyl-D-erythro-sphingomyelin and cholesterol. Overall, the results indicate that both the distribution of cholesterol between different lipid environments and the impact of cholesterol on lateral segregation can be predicted relatively accurately from determined relative partition coefficients.

Paradox of hypercholesterolaemia in highly trained, keto-adapted athletes

ObjectiveA growing number of ultra-endurance athletes have switched to a very low-carbohydrate/high-fat eating pattern. We compared markers of cholesterol and the lipoprotein profile in a group of elite ultra-runners consuming...

Reinterpretation of the localization of the ATP binding cassette transporter ABCG1 in insulin-secreting cells and insights regarding its trafficking and function.

The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1’s trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.

Molecular Dynamics Investigation of the Ternary Bilayer Formed by Saturated Phosphotidylcholine, Sphingomyelin, and Cholesterol

Using the all-atom CHARMM36 force field, we have performed molecular dynamics simulations of the equimolar ternary dimyristoylphosphatidylcholine (DMPC)/ N-palmitoylsphingomyelin (PSM)/cholesterol bilayer. Analysis of its structural and kinetic properties has led us to the following conclusions. First, the DMPC/PSM/cholesterol bilayer features favorable interactions of cholesterol with DMPC and, particularly, PSM lipids, which are supported by hydrogen bonds. In contrast, the interactions between cholesterol molecules are strongly suppressed. Further analysis shows that about 60% of PSM molecules form hydrogen bonds with other PSM or cholesterol molecules and, to a lesser degree, with DMPC lipids. Second, local lipid packing around PSM molecules favors parallel or antiparallel alignments, resulting in the appearance of large anisotropic chain-like clusters formed by PSM lipids. In contrast, the distribution of DMPC lipids is closer to ideal, whereas the cholesterol distribution is sharply shifted toward small clusters or isolated molecules. Third, intermolecular interactions and mismatch in fatty acid tail lengths significantly slow down PSM rotational relaxation compared to that for DMPC, and PSM, but not DMPC lipids, tend to combine axial rotation with wobbling. Fourth, the analysis of cholesterol hydration indicates that lipid headgroups at most block about 30% of water molecules from reaching cholesterol, hinting at limited applicability of the umbrella model. Finally, we show that PSM and DMPC lipids predominantly exist in two equally probable conformational states, with and without a kink in their fatty acid tails. The appearance of these states is linked to the impact of cholesterol. Comparisons with previous studies allow us to delineate contributions of cholesterol, PSM, and DMPC to bilayer properties.