Cholesterol-dependent Manner Research Articles

Prostaglandin A1 Inhibits the Cognitive Decline of APP/PS1 Transgenic Mice via PPARγ/ABCA1-dependent Cholesterol Efflux Mechanisms.

Prostaglandins (PGs) are early and key contributors to chronic neurodegenerative diseases. As one important member of classical PGs, PGA1 has been reported to exert potential neuroprotective effects. However, the mechanisms remain unknown. To this end, we are prompted to investigate whether PGA1 is a useful neurological treatment for Alzheimer's disease (AD) or not. Using high-throughput sequencing, we found that PGA1 potentially regulates cholesterol metabolism and lipid transport. Interestingly, we further found that short-term administration of PGA1 decreased the levels of the monomeric and oligomeric β-amyloid protein (oAβ) in a cholesterol-dependent manner. In detail, PGA1 activated the peroxisome proliferator-activated receptor-gamma (PPARγ) and ATP-binding cassette subfamily A member 1 (ABCA1) signalling pathways, promoting the efflux of cholesterol and decreasing the intracellular cholesterol levels. Through PPARγ/ABCA1/cholesterol-dependent pathway, PGA1 decreased the expression of presenilin enhancer protein 2 (PEN-2), which is responsible for the production of Aβ. More importantly, long-term administration of PGA1 remarkably decreased the formation of Aβ monomers, oligomers, and fibrils. The actions of PGA1 on the production and deposition of Aβ ultimately improved the cognitive decline of the amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice.

Cholesterol Depletion by TASIN-1 Induces Apoptotic Cell Death through the ER Stress/ROS/JNK Signaling in Colon Cancer Cells.

Truncated APC selective inhibitor-1 (TASIN-1) is a recently identified small molecule that selectively kills colorectal cancer cells that express truncated adenomatous polyposis coli (APC) by reducing cellular cholesterol levels. However, the downstream mechanism responsible for its cytotoxicity is not well understood. In this study, we show that TASIN-1 leads to apoptotic cell death via inducing ER stress-dependent JNK activation in human truncated APC colon cancer cells, accompanied by production of reactive oxygen species (ROS). In addition, TASIN-1 inhibits AKT activity through a cholesterol-dependent manner. Human colon tumor xenografts in immunodeficient mice also show the same TASIN-1 induced molecular mechanisms of tumor cell death as observed in vitro Taken together, cholesterol depletion by TASIN-1 treatment induces apoptotic cell death through activating ER stress/ROS/JNK axis and inhibiting AKT pro-survival signaling in colon cancer cells with truncated APC both in vitro and in vivoMol Cancer Ther; 17(5); 943-51. ©2018 AACR.

Cerebral Vascular KIR2.x Channels are Distinctly Regulated by Membrane Lipids and Hemodynamic Forces.

This study examined membrane lipid (phosphatidylinositol‐bis‐phosphate (PIP2) and cholesterol) regulation of cerebral arterial KIR and whether these signaling molecules enable distinct channel pools to uniquely sense hemodynamic forces. Endothelial and smooth muscle cells were freshly isolated from rat cerebral arteries; patch‐clamp electrophysiology, Q‐PCR and immunohistochemistry delineated KIR channel activity and expression. Electrophysiology revealed a Ba2+‐sensitive KIR current in smooth muscle and endothelial cells, while Q‐PCR and immunohistochemistry confirmed KIR2.x mRNA and protein expression respectively. Each cellular pool of KIR channels was sensitive to particular membrane lipids and hemodynamic forces. Endothelial KIR responded dynamically to PIP2 manipulations, and laminar flow activated this channel pool in a PIP2 dependent manner. In contrast, smooth muscle KIR reacted to cholesterol perturbations, and pressure stimuli (e.g. hyposmotic challenge or negative pressure application) modulated this channel pool in a cholesterol dependent manner. The flow and pressure sensitivity of KIR channels was confirmed in intact cerebral arteries using vessel myography. In summary, while both vascular cell types express KIR2.x channels, each pool is distinctly regulated by membrane lipids and hemodynamic stimuli. This emerging picture of KIR regulation advances our mechanistic understanding of how hemodynamic forces interact to control arterial tone development.Support or Funding InformationThis research was supported by an operating grant from the Canadian Institute of Health Research. DG Welsh is Rorabeck Chair of Molecular Neuroscience and Vascular Biology at the University of Western Ontario.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Astemizole Inhibits mTOR Signaling and Angiogenesis by Blocking Cholesterol Trafficking.

Cholesterol plays a key role in membrane protein function and signaling in endothelial cells. Thus, disturbing cholesterol trafficking is an effective approach for inhibiting angiogenesis. We recently identified astemizole (AST), an antihistamine drug, as a cholesterol trafficking inhibitor from a phenotypic screen. In this study, we found that AST induced cholesterol accumulation in the lysosome by binding to the sterol-sensing domain of Niemann-Pick disease, type C1 (NPC1), a lysosomal surface protein responsible for cholesterol transport. Inhibition of cholesterol trafficking by AST led to the depletion of membrane cholesterol, causing SREBP1 nuclear localization. The depletion of membrane cholesterol resulted in dissociation of mammalian target of rapamycin (mTOR) from the lysosomal surface and inactivation of mTOR signaling. These effects were effectively rescued by addition of exogenous cholesterol. AST inhibited endothelial cell proliferation, migration and tube formation in a cholesterol-dependent manner. Furthermore, AST inhibited zebrafish angiogenesis in a cholesterol-dependent manner. Together, our data suggest that AST is a new class of NPC1 antagonist that inhibits cholesterol trafficking in endothelial cells and angiogenesis.

Unraveling of Lipid Raft Organization in Cell Plasma Membranes by Single-Molecule Imaging of Ganglioside Probes.

Ganglioside s are involved in a variety of physiological roles and particularly in the formation and function of lipid rafts in cell membranes. However, the dynamic behaviors of gangliosides have not been investigated in living cells owing to the lack of fluorescent probes that behave like their parental molecules. This has recently been resolved by developing new fluorescent ganglioside analogues that act similarly to their parental molecules, synthesized by only chemical methods. We performed single fluorescent-molecule imaging and revealed that ganglioside probes dynamically enter and exit rafts containing CD59, a glycosylphosphatidylinositol (GPI)-anchored protein, both before and after stimulation. The residency time of our ganglioside probes in CD59 oligomers was 48 ms after stimulation. The residency times in CD59 homodimer and monomer rafts were 40 and 12 ms, respectively. These results reveal the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they demonstrate that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner and at a much higher frequency than expected. In this chapter, we review methods for the development and single-molecule imaging of new fluorescent ganglioside analogues and discuss how raft domains are formed, both before and after receptor engagement.

Structure-Function Dissection of Pseudorabies Virus Glycoprotein B Fusion Loops.

Conserved across the family Herpesviridae, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomains of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the pseudorabies virus (PrV) gB ectodomain, revealing a typical class III postfusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct subregions of the lipid bilayer and their roles in membrane interactions. We tested 15 gB variants for the ability to bind to liposomes and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275, and Tyr276, which were essential for liposome binding and for fusion in cellular and viral contexts, form a continuous hydrophobic patch at the gB trimer surface. Together with results reported for other alphaherpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192, and Tyr276 form a rim that inserts into the more superficial interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gBs from beta- and gamma-herpesviruses suggests that this membrane interaction model is valid for gBs from all herpesviruses.IMPORTANCE Herpesviruses are common human and animal pathogens that infect cells by entering via fusion of viral and cellular membranes. Central to the membrane fusion event is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself in the target membrane via two polypeptide segments called fusion loops (FLs). The molecular details of how gB FLs insert into the lipid bilayer have not been described. Here, we provide structural and functional data regarding key FL residues of gB from pseudorabies virus, a porcine herpesvirus of veterinary concern, which allows us to propose, for the first time, a molecular model to understand how the initial interactions by gBs from all herpesviruses with target membranes are established.

Preferential selection of Arginine at the lipid-water-interface of TRPV1 during vertebrate evolution correlates with its snorkeling behaviour and cholesterol interaction

TRPV1 is a thermo-sensitive ion channel involved in neurosensory and other physiological functions. The trans-membrane helices of TRPV1 undergo quick and complex conformational changes governed by thermodynamic parameters and membrane components leading to channel opening. However, the molecular mechanisms underlying such events are poorly understood. Here we analysed the molecular evolution of TRPV1 at the lipid-water-interface region (LWI), typically defined as a layer of 6 Å thickness on each side of the membrane with less availability of free water. Amino acids demarcating the end of the trans-membrane helices are highly conserved. Residues present in the inner leaflet are more conserved and have been preferentially selected over others. Amino acids with snorkeling properties (Arginine and Tyrosine) undergo specific selection during the vertebrate evolution in a cholesterol-dependent and/or body temperature manner. Results suggest that H-bond formation between the OH- group of cholesterol and side chain of Arg557 or Arg575 at the inner leaflet is a critical parameter that can regulate channel functions. Different LWI mutants of TRPV1 have altered membrane localization and deficient colocalization with lipid raft markers. These findings may help to understand the lipid-protein interactions, and molecular basis of different neuronal functions. Such findings may have broad importance in the context of differential sensory responses, pathophysiologies, and application of pharmacological drugs such as anaesthetics acting on TRPVs.

Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR

The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid 31P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods.

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents.

Graphene quantum dots suppress proinflammatory T cell responses via autophagy-dependent induction of tolerogenic dendritic cells.

Graphene quantum dots (GQD) are atom-thick nanodimensional carbon sheets with excellent physico-chemical and biological properties, making them attractive for application in theranostics. However, their immunoregulatory properties are insufficiently investigated, especially in human primary immune cells. We found that non-toxic doses of GQD inhibit the production of proinflammatory and T helper (Th)1 cytokines, and augment the production of anti-inflammatory and Th2 cytokines by human peripheral blood mononuclear cells. While unable to affect T cells directly, GQD impaired the differentiation and functions of monocyte-derived dendritic cells (DC), lowering their capacity to stimulate T cell proliferation, development of Th1 and Th17cells, and T-cell mediated cytotoxicity. Additionally, GQD-treated DC potentiated Th2 polarization, and induced suppressive CD4+CD25highFoxp3+ regulatory T cells. After internalization in a dynamin-independent, cholesterol-dependent manner, GQD lowered the production of reactive oxygen species and nuclear translocation of NF-κB in DC. The activity of mammalian target of rapamycin (mTOR) was reduced by GQD, which correlated with the increase in transcription of autophagy genes and autophagic flux in DC. Genetic suppression of autophagy impaired the pro-tolerogenic effects of GQD on DC. Our results suggest that GQD-triggered autophagy promotes tolerogenic functions in monocyte-derived DC, which could be beneficial in inflammatory T-cell mediated pathologies, but also harmful in GQD-based anti-cancer therapy.

Line Activity of Ganglioside GM1 Regulates the Raft Size Distribution in a Cholesterol-Dependent Manner.

Liquid-ordered lipid domains, also called rafts, are assumed to be important players in different cellular processes, mainly signal transduction and membrane trafficking. They are thicker than the disordered part of the membrane and are thought to form to compensate for the hydrophobic mismatch between transmembrane proteins and the lipid environment. Despite the existence of such structures in vivo still being an open question, they are observed in model systems of multicomponent lipid bilayers. Moreover, the predictions obtained from model experiments allow the explanation of different physiological processes possibly involving rafts. Here we present the results of the study of the regulation of raft size distribution by ganglioside GM1. Combining atomic force microscopy with theoretical considerations based on the theory of membrane elasticity, we predict that this glycolipid should change the line tension of raft boundaries in two different ways, mainly depending on the cholesterol content. These results explain the shedding of gangliosides from the surface of tumor cells and the following ganglioside-induced apoptosis of T-lymphocytes in a raft-dependent manner. Moreover, the generality of the model allows the prediction of the line activity of different membrane components based on their molecular geometry.

Iron nanomedicines induce Toll-like receptor activation, cytokine production and complement activation.

Approximately a dozen of intravenous iron nanomedicines gained marketing authorization in the last two decades. These products are generally considered as safe, but have been associated with an increased risk for hypersensitivity-like reactions of which the underlying mechanisms are unknown. We hypothesized that iron nanomedicines can trigger the innate immune system. We hereto investigated the physico-chemical properties of ferric gluconate, iron sucrose, ferric carboxymaltose and iron isomaltoside 1000 and comparatively studied their interaction with Toll-like receptors, the complement system and peripheral blood mononuclear cells. Two out of four formulations appeared as aggregates by Scanning Transmission Electron Microscopy analysis and were actively taken up by HEK293T- and peripheral blood mononuclear cells in a cholesterol-dependent manner. These formulations triggered invitro activation of intracellular Toll-like receptors 3, -7 and -9 in a dose- and serum-dependent manner. In parallel experiments, we determined that these compounds activated the complement system. Finally, we found that uptake of aggregation-prone iron nanomedicines by peripheral blood mononuclear cells in whole blood induced production of the proinflammatory cytokine IL-1β, but not IL-6.

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations of asymmetric lipid membranes, whose composition is largely consistent with eukaryotic plasma membranes. We show that the membrane dipole potential changes in a cholesterol-dependent manner. Remarkably, moving cholesterol from the extracellular to the cytosolic leaflet increases the dipole potential on the cytosolic side, and vice versa. Biologically this implies that by altering the dipole potential, cholesterol can provide a driving force for cholesterol molecules to favor the cytosolic leaflet, in order to compensate for the intramembrane field that arises from the resting potential.

Cholesterol regulates DAF-16 nuclear localization and fasting-induced longevity in C. elegans

Cholesterol has attracted significant attention as a possible lifespan regulator. It has been reported that serum cholesterol levels have an impact on mortality due to age-related disorders such as cardiovascular disease. Diet is also known to be an important lifespan regulator. Dietary restriction retards the onset of age-related diseases and extends lifespan in various organisms. Although cholesterol and dietary restriction are known to be lifespan regulators, it remains to be established whether cholesterol is involved in dietary restriction-induced longevity. Here, we show that cholesterol deprivation suppresses longevity induced by intermittent fasting, which is one of the dietary restriction regimens that effectively extend lifespan. We also found that cholesterol is required for the fasting-induced upregulation of transcriptional target genes such as the insulin/IGF-1 pathway effector DAF-16 and that cholesterol deprivation suppresses the long lifespan of the insulin/IGF-1 receptor daf-2 mutant. Remarkably, we found that cholesterol plays an important role in the fasting-induced nuclear accumulation of DAF-16. Moreover, knockdown of the cholesterol-binding protein NSBP-1, which has been shown to bind to DAF-16 in a cholesterol-dependent manner and to regulate DAF-16 activity, suppresses both fasting-induced longevity and DAF-16 nuclear accumulation. Furthermore, this suppression was not additive to the cholesterol deprivation-induced suppression, which suggests that NSBP-1 mediates, at least in part, the action of cholesterol to promote fasting-induced longevity and DAF-16 nuclear accumulation. These findings identify a novel role for cholesterol in the regulation of lifespan.

LDL-Cholesterol Increases the Transcytosis of Molecules through Endothelial Monolayers.

Cholesterol has been identified as a causative factor in numerous pathologies including atherosclerosis and cancer. One of the frequent effects of elevated cholesterol levels in humans is the compromise of endothelial function due to activation of pro-inflammatory signalling pathways. While the mechanisms involved in endothelial activation by cholesterol during an inflammatory response are well established, less is known about the mechanisms by which cholesterol may affect endothelial barrier function, which were the subject of the present study. Here we show that low density lipoprotein (LDL) increases the permeability of endothelial monolayers to high molecular weight dextrans in an LDL receptor and cholesterol-dependent manner. The increased permeability seen upon LDL treatment was not caused by disruption of cell-to-cell junctions as determined by a normal localization of VE-Cadherin and ZO-1 proteins, and no major alterations in transendothelial electrical resistance or permeability to fluorescein. We show instead that LDL increases the level of high molecular weight transcytosis and that this occurs in an LDL receptor, cholesterol and caveolae-dependent way. Our findings contribute to our understanding of the systemic pathological effects of elevated cholesterol and the transport of cargo through endothelial monolayers.

Data including GROMACS input files for atomistic molecular dynamics simulations of mixed, asymmetric bilayers including molecular topologies, equilibrated structures, and force field for lipids compatible with OPLS-AA parameters

In this Data in Brief article we provide a data package of GROMACS input files for atomistic molecular dynamics simulations of multicomponent, asymmetric lipid bilayers using the OPLS-AA force field. These data include 14 model bilayers composed of 8 different lipid molecules. The lipids present in these models are: cholesterol (CHOL), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidyl-ethanolamine (SOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (SOPS), N-palmitoyl-D-erythro-sphingosyl-phosphatidylcholine (SM16), and N-lignoceroyl-D-erythro-sphingosyl-phosphatidylcholine (SM24). The bilayers׳ compositions are based on lipidomic studies of PC-3 prostate cancer cells and exosomes discussed in Llorente et al. (2013) [1], showing an increase in the section of long-tail lipid species (SOPS, SOPE, and SM24) in the exosomes. Former knowledge about lipid asymmetry in cell membranes was accounted for in the models, meaning that the model of the inner leaflet is composed of a mixture of PC, PS, PE, and cholesterol, while the extracellular leaflet is composed of SM, PC and cholesterol discussed in Van Meer et al. (2008) [2]. The provided data include lipids׳ topologies, equilibrated structures of asymmetric bilayers, all force field parameters, and input files with parameters describing simulation conditions (md.mdp). The data is associated with the research article “Interdigitation of Long-Chain Sphingomyelin Induces Coupling of Membrane Leaflets in a Cholesterol Dependent Manner” (Róg et al., 2016) [3].

Treponema denticola invasion into human gingival epithelial cells

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [3H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-β-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.

Interdigitation of long-chain sphingomyelin induces coupling of membrane leaflets in a cholesterol dependent manner

It has been a long-standing question how the two leaflets in a lipid bilayer modulate each others' physical properties. In this paper, we discuss how this interaction may take place through interdigitation. We use atomistic molecular dynamics simulations to consider asymmetric lipid membrane models whose compositions are based on the lipidomics data determined for exosomes released by PC-3 prostate cancer cells. The simulations show interdigitation to be exceptionally strong for long-chain sphingomyelin (SM) molecules. In asymmetric membranes the amide-linked chain of SM is observed to extend deep into the opposing membrane leaflet. Interestingly, we find that the conformational order of the amide-linked SM chain increases the deeper it penetrates to the opposing leaflet. Analysis of this finding reveals that the amide-linked SM chain interacts favorably with the lipid chains in the opposite leaflet, and that cholesterol modulates the effect of SM interdigitation by influencing the conformational order of lipid hydrocarbon chains in the opposing (cytosolic) leaflet.

Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-dependent Manner

Our laboratory previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knock-out mice (Ldlr(-/-)xLcat(-/-) or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to protection from the diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr(-/-)xLcat(+/+) single-knock-out (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes, and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr(+/+)xLcat(-/-) (LCAT-KO) SC with DKO SC identified a role for LCAT deficiency in priming SC to express BAT genes. Additionally, we found that reduced cellular cholesterol is important for adipogenic differentiation, evidenced by increased induction of adipogenesis in cholesterol-depleted SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO mice is classical in origin, and its development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.