Cholesterol-rich Domains Research Articles

Heparan sulfate proteoglycan deficiency up-regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

Caveolin-1: Functional Insights into Its Role in Muscarine- and Serotonin-Induced Smooth Muscle Constriction in Murine Airways.

An increased bronchoconstrictor response is a hallmark in the progression of obstructive airway diseases. Acetylcholine and 5-hydroxytryptamine (5-HT, serotonin) are the major bronchoconstrictors. There is evidence that both cholinergic and serotonergic signaling in airway smooth muscle (ASM) involve caveolae. We hypothesized that caveolin-1 (cav-1), a structural protein of caveolae, plays an important regulatory role in ASM contraction. We analyzed airway contraction in different tracheal segments and extra- and intrapulmonary bronchi in cav-1 deficient (cav-1−/−) and wild-type mice using organ bath recordings and videomorphometry of methyl-beta-cyclodextrin (MCD) treated and non-treated precision-cut lung slices (PCLS). The presence of caveolae was investigated by electron microscopy. Receptor subtypes driving 5-HT-responses were studied by RT-PCR and videomorphometry after pharmacological inhibition with ketanserin. Cav-1 was present in tracheal epithelium and ASM. Muscarine induced a dose dependent contraction in all airway segments. A significantly higher Emax was observed in the caudal trachea. Although, caveolae abundancy was largely reduced in cav-1−/− mice, muscarine-induced airway contraction was maintained, albeit at diminished potency in the middle trachea, in the caudal trachea and in the bronchus without changes in the maximum efficacy. MCD-treatment of PLCS from cav-1−/− mice reduced cholinergic constriction by about 50%, indicating that cholesterol-rich plasma domains account for a substantial portion of the muscarine-induced bronchoconstriction. Notably, cav-1-deficiency fully abrogated 5-HT-induced contraction of extrapulmonary airways. In contrast, 5-HT-induced bronchoconstriction was fully maintained in cav-1-deficient intrapulmonary bronchi, but desensitization upon repetitive stimulation was enhanced. RT-PCR analysis revealed 5-HT1B, 5-HT2A, 5-HT6, and 5-HT7 receptors as the most prevalent subtypes in the airways. The 5-HT-induced-constriction in PCLS could be antagonized by ketanserin, a 5-HT2A receptor inhibitor. In conclusion, the role of cav-1, caveolae, and cholesterol-rich plasma domains in regulation of airway tone are highly agonist-specific and dependent on airway level. Cav-1 is indispensable for serotonergic contraction of extrapulmonary airways and modulates cholinergic constriction of the trachea and main bronchus. Thus, cav-1/caveolae shall be considered in settings such as bronchial hyperreactivity in common airway diseases and might provide an opportunity for modulation of the constrictor response.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships between viral proteins in the plasma membrane. Some proteins, such as HA and M2, inherently cocluster within the membrane, although M2 is found mostly at the periphery of regions of HA, consistent with the proposed role of M2 in scission at the end of budding. The association between some pairs of influenza virus proteins, such as M2 and NP, appears to be brokered by additional influenza virus proteins, in this case M1. HA and NA, while raft associated, reside in distinct domains, reflecting their distributions in the viral membrane.

Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix–helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Glycosyl-Phosphatidyl-Inositol (GPI)-Anchors and Metalloproteases: Their Roles in the Regulation of Exosome Composition and NKG2D-Mediated Immune Recognition.

Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma membrane or in intracellular compartments to produce such factors, vesicle release and proteolytic cleavage. Release of soluble factors in exosomes, a subset of vesicles that originate from intracellular compartments, depends generally on biochemical and lipid environment features. This physical environment allows proteins to be recruited to membrane microdomains that will be later endocytosed and further released to the extracellular milieu. Cholesterol and sphingolipid rich domains (also known as lipid rafts or detergent-resistant membranes, DRMs) often contribute to exosomes and these membrane regions are rich in proteins modified with Glycosyl-Phosphatidyl-Inositol (GPI) and lipids. For this reason, many palmitoylated and GPI-anchored proteins are preferentially recruited to exosomes. In this review, we analyse the biochemical features involved in the release of NKG2D-ligands as an example of functionally related gene families encoding both transmembrane and GPI-anchored proteins that can be released either by proteolysis or in exosomes, and modulate the intensity of the immune response. The immune receptor NKG2D is present in all human Natural Killer and T cells and plays an important role in the first barrier of defense against tumor and infection. However, tumor cells can evade the immune system by releasing NKG2D-ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression and immune function, while others are more susceptible to metalloprotease cleavage and are shed as soluble molecules. Strikingly, metalloprotease inhibition is sufficient to drive the accumulation in exosomes of ligands otherwise released by metalloprotease cleavage. In consequence, NKG2D-ligands appear as different entities in different cells, depending on cellular metabolism and biochemical structure, which mediate different intensities of immune modulation. We discuss whether similar mechanisms, depending on an interplay between metalloprotease cleavage and exosome release, could be a more general feature regulating the composition of exosomes released from human cells.

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.

Cholesterol Rich Domains Identified in Unilamellar Supported Biomimetic Membranes via Nano-Viscosity Measurements

Understanding the distribution of cholesterol in phospholipid membranes is of key importance in membrane biophysics, primarily since cholesterol enriched regions, rafts, are known to play a special role in protein function. In this work, quartz crystal microbalance with dissipation (QCM)-based viscosity measurements were used to study cholesterol-induced domain formation in partially suspended single bilayer membranes. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and its mixtures with different amounts of cholesterol were studied. QCM temperature ramping experiments identified domains of different phase transition temperatures in the mixed membranes. The phase transition of DMPC shifted from 23.4 °C toward lower temperatures with increasing cholesterol content. A second, continuous but much broader, transition peak has been observed for the DMPC: cholesterol mixtures suggest that a separate cholesterol rich domain coexists with the DMPC rich domain. Importantly, the sharp DMC phase transition peak gradually diminished and eventually disappeared over 15% cholesterol content, suggesting that the cholesterol rich domain has a definite stoichiometry and once this cholesterol concentration is reached the DMPC-rich domain disappears. DSC control experiments do not show the second domain, suggesting that the phase separation only happens in nontensioned (flat) membranes.

Lipid Structure and Control of Membrane Ordered Domain Formation And Size by Lipid Composition and Asymmetry in Vitro and in Vivo

We have studied the properties and formation of ordered lipid domains (“lipid rafts”) in model membrane vesicles and cells. For example, recent FRET studies on vesicles with sphingomyelin/phospholipid/cholesterol mixtures found that mammalian lipids have structures and compositions favoring “ultrananodomain” formation, i.e. formation of very small nanodomains 5 nm or less in size. To study domain formation in more biologically-relevant vesicles, we developed methods to prepare vesicles with an asymmetric lipid distributions using cyclodextrin-catalzyed lipid exchange. Recent studies in asymmetric giant vesicles containing raft domains large enough to see with microscopy revealed interleaflet coupling to sphingomyelin- and cholesterol-rich outer leaflet domains induces ordered domains in the portion of the DOPC and cholesterol containing inner leaflets with which they are in contact. Analogous studies using smaller asymmetric vesicles and FRET-based detection may reveal whether nanodomains can form and exhibit coupling between inner and outer leaflets ordered domains. Methods to prepare a wider range of asymmetric membrane compositions and make more facile asymmetry measurements are being developed. In addition, cyclodextrin-catalyzed lipid exchange is being extended to studies of membrane domain structure and function in living cells. Substituting for cholesterol shows the subset of sterols having the ability to support the formation of lipid rafts are necessary and sufficient for them to support membrane domain formation in Borrelia burgdorferi, a bacteriumcontaining fatty acyl cholesterol glycosides.Interestingly,we find fatty acyl cholesterol glycosides are able to form ordered membrane domains without sphingolipids. Studies with cyclodextrin-catalyzed sterol exchange in mammalian cells show that sterols having an ability to promote the formation of ordered membrane domains are necessary and sufficient for them to support both clathrin-dependent and independent endocytosis provided they also contain a 3-beta OH. Efforts to carry out similar experiments with phospholipid and sphingolipid exchange are underway.

ABCA1-dependent sterol release: sterol molecule specificity and potential membrane domain for HDL biogenesis

Mammalian cells synthesize various sterol molecules, including the C30 sterol, lanosterol, as cholesterol precursors in the endoplasmic reticulum. The build-up of precursor sterols, including lanosterol, displays cellular toxicity. Precursor sterols are found in plasma HDL. How these structurally different sterols are released from cells is poorly understood. Here, we show that newly synthesized precursor sterols arriving at the plasma membrane (PM) are removed by extracellular apoA-I in a manner dependent on ABCA1, a key macromolecule for HDL biogenesis. Analysis of sterol molecules by GC-MS and tracing the fate of radiolabeled acetate-derived sterols in normal and mutant Niemann-Pick type C cells reveal that ABCA1 prefers newly synthesized sterols, especially lanosterol, as the substrates before they are internalized from the PM. We also show that ABCA1 resides in a cholesterol-rich membrane domain resistant to the mild detergent, Brij 98. Blocking ACAT activity increases the cholesterol contents of this domain. Newly synthesized C29/C30 sterols are transiently enriched within this domain, but rapidly disappear from this domain with a half-life of less than 1 h. Our work shows that substantial amounts of precursor sterols are transported to a certain PM domain and are removed by the ABCA1-dependent pathway.

Leukocyte adhesion and polarization: Role of glycosylphosphatidylinositol-anchored proteins.

ABSTRACTLeukocyte traffic out of the blood stream is crucial for an adequate immune response. Leukocyte extravasation is critically dependent on the binding of leukocyte integrins to their endothelial counterreceptors. This interaction enables the firm adhesion of leukocytes to the luminal side of the vascular wall and allows for leukocyte polarization, crawling and diapedesis. Leukocyte adhesion, polarization and migration requires the orchestrated regulation of integrin adhesion/de-adhesion dynamics and actin cytoskeleton rearrangements. Adhesion strength depends on conformational changes of integrin molecules (affinity) as well as the number of integrin molecules engaged at adhesion sites (valency). These two processes can be independently regulated and several molecules modulate either one or both processes. Cholesterol-rich membrane domains (lipid rafts) participate in integrin regulation and play an important role in leukocyte adhesion, polarization and motility. In particular, lipid raft-resident glycosyl-phosphatidyl-inositol-anchored proteins (GPI-APs) have been reported to regulate leukocyte adhesion, polarization and motility in both integrin-dependent and independent manners. Here, we present our recent discovery concerning the novel role of the GPI-AP prion protein (PrP) in the regulation of β1 integrin-mediated monocyte adhesion, migration and shape polarization in the context of existing literature on GPI-AP-dependent regulation of integrins.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains.

Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine-containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol-rich domains in a GPMV model, thus documenting the potential of our approach for membrane-partitioning studies.

Flow shear stress differentially regulates endothelial uptake of nanocarriers targeted to distinct epitopes of PECAM-1

Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1–8dyn/cm2, typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest.

HIV gp41-mediated membrane fusion occurs at edges of cholesterol-rich lipid domains.

Lipid rafts in plasma membranes have emerged as possible platforms for entry of HIV and other viruses into cells. However, how lipid phase heterogeneity contributes to viral entry is little known due to the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T-cell membranes and showed that raft-like (Lo phase) lipid domains are necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion was low in homogeneous Ld and Lo phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo/Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld phase vesicles proceeded only to hemifusion. Thus, we propose that the edges, but not the areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.

Specific cellular incorporation of a pyrene-labelled cholesterol: lipoprotein-mediated delivery toward ordered intracellular membranes.

In the aim of testing tools for tracing cell trafficking of exogenous cholesterol, two fluorescent derivatives of cholesterol, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), with distinctive chemico-physical characteristics, have been compared for their cell incorporation properties, using two cell models differently handling cholesterol, with two incorporation routes. In the Caco-2 cell model, the cholesterol probes were delivered in bile salt micelles, as a model of intestinal absorption. The two probes displayed contrasting behaviors for cell uptake characteristics, cell staining, and efflux kinetics. In particular, Pyr-met-Chol cell incorporation involved SR-BI, while that of NBD-Chol appeared purely passive. In the PC-3 cell model, which overexpresses lipoprotein receptors, the cholesterol probes were delivered via the serum components, as a model of systemic delivery. We showed that Pyr-met-Chol-labelled purified LDL or HDL were able to specifically deliver Pyr-met-Chol to the PC-3 cells, while NBD-Chol incorporation was independent of lipoproteins. Observations by fluorescence microscopy evidenced that, while NBD-Chol readily stained the cytosolic lipid droplets, Pyr-met-Chol labelling led to the intense staining of intracellular structures of membranous nature, in agreement with the absence of detectable esterification of Pyr-met-Chol. A 48 h incubation of PC-3 cells with either Pyr-met-Chol-labelled LDL or HDL gave same staining patterns, mainly colocalizing with Lamp1, caveolin-1 and CD63. These data indicated convergent trafficking downwards their respective receptors, LDL-R and SR-BI, toward the cholesterol-rich internal membrane compartments, late endosomes and multivesicular bodies. Interestingly, Pyr-met-Chol staining of these structures exhibited a high excimer fluorescence emission, revealing their ordered membrane environment, and indicating that Pyr-met-Chol behaves as a fair cholesterol tracer regarding its preferential incorporation into cholesterol-rich domains. We conclude that, while NBD-Chol is a valuable marker of cholesterol esterification, Pyr-met-Chol is a reliable new lipoprotein fluorescent marker which allows to probe specific intracellular trafficking of cholesterol-rich membranes.

Effect of an Apoptotic Membrane raft on the Conformational and Dynamical Changes of Calreticulin

Calreticulin (CRT) on the cell surface can mediate engulfment of apoptotic cells by interacting directly with LDL receptor-related protein (LRP1) (Cell. 123:321-34, 2005). Phosphatidyl serine (PS) lipids in the inner leaflet of the cell membrane are externalized and become exposed in cholesterol-rich domains during apoptosis and co-localized with cell surface surface CRT (Cell. 123:321-34, 2005). How the apoptotic cell membrane affects the structure and dynamics of CRT, further influencing CRT binding to LRP1 to signal apoptotic-cell clearance remain unknown. In this study, we investigated the interactions of a membrane raft in an apoptotic cell membrane with CRT and its effect on conformational and dynamical changes in CRT via atomically detailed molecular dynamics simulations. An apoptotic membrane raft membrane is modeled as a bilayer containing 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) lipids, cholesterol (CHOL) and palmitoyl-oleoyl phosphatidylserine (POPS) lipids (the ratio of the number of POPC lipid, POPS lipid and CHOL is: 5:1:4). The results are compared with the membrane raft without POPS lipids. POPS lipids in the membrane raft affected the microscopic and mesoscopic properties of the membrane raft. Apoptotic membrane raft resulted in the more direct interaction of the N-, partial C, and P-domains of CRT with membrane raft and more stabilized CRT conformation compared to that of CRT in a POPC-CHOL membrane raft, which could affect CRT recruitment of LRP1. Results from this study provided molecular insight into the effect of apoptotic membrane raft on CRT conformational and dynamical changes, further affecting CRT binding to LRP1 for signaling.Keywords: membrane raft, POPS lipids, TSP1-CRT complex, apoptotic cell clearance, molecular dynamics simulations

GPIbα Activation Triggers Platelet Tissue Factor/FVIIa-Dependent Procoagulant Activity, Which Is Dampened By Platelet Secreted TFPI and Protein S. Novel Platelet-Based Model of Hemostasis

Human platelets contain Tissue Factor (TF) demonstrated by western blotting (WB), IP, confocal IF microscopy and flow cytometry (FC) using an array of different α-TF antibodies. Moreover, TF is synthesized by platelets and has procoagulant activity (PCA) (Panes et al. Blood 2007). Platelets also contain the full-length α-isoform of TFPI that is exposed on the plasma membrane of activated, coated-platelets. A new role of Protein S (PS) is to function as TFPI co-factor for TF/FVIIa/FXa inactivation. In fact, TFPI function, enhanced by protein S (PS), dampens thrombin generation on the platelet surface (Wood, JP et al, 2014). In endothelial cells TFPI appears associated with the cell surface through glycosylphosphatidyl-inositol-mediated anchorage, suggesting some type of association with cholesterol-rich domains in cell membranes (lipid rafts, LR). Platelets also contain PS, but its functional association with platelet TFPI to inhibit platelet TF-dependent PCA remains still unknown. Aim: to disclose the physiologic role of TFPI and PS on the TF-dependent PCA of human platelets. Results: Stimulation of isolated, washed platelets with VWF-Ristocetin (VWF-R) resulted in 10-fold increase of TF-dependent FXa generation within 2-5min, compared with non-stimulated (N-S) platelets: [median 17(10-37) to 182(43-847) nM FXa/2x107 platelets, n=198]. VWF-R induced anionic phospholipid exposure, but no α-δ-granules release in washed platelets. FX activation could be triggered without external FVII, although this was required to sustain the reaction. FVII/FVIIa was demonstrated in washed platelets by WB, confocal IF microscopy and FC, and its membrane expression augmented after platelet activation. FXa induced by VWF-R was abolished by pre-incubation with TFPI or with several polyclonal or mAbs against to each TF, FVIIa or GPIbα. In contrast, TRAP stimulation (n=84) induced little or no FXa generation [Median 32(11-219) nM FXa/2x107platelets], but FX activation was increased by 54% (range 13-109%, p=0.0039) in platelets pre-incubated with α-TFPI. This increase was more pronounced with pre-incubation with α-PS (90%, range 31-227%, p<0.004). Combination of α-TFPI and α-PS did not enhance further FX activation. The releasate fraction of TRAP-stimulated platelets inhibited the FXa produced by VWF-R-stimulated platelets, supporting that TRAP-induced secretion of TFPI and PS explained this effect. Membrane exposure of platelet TFPI measured by FC was decreased after TRAP stimulation, a paradox explained by the high content of TFPI and PS in microparticles (MP) contained in the TRAP releasates. In contrast, the smaller number of MP released by VWF-R activation contained neither TFPI nor PS (WB assay). IP assays in platelet membrane fractions showed co-precipitation of TF, FVII/FVIIa and GPIbα. All these proteins co-precipitated also with TFPI in lipid raft fractions. Importantly, TFPI was notably augmented in LR fractions after TRAP stimulation, as compared with VWF-R stimulation. Moreover, TRAP stimulation resulted in co-precipitation of TFPI and PS in cytosolic, membrane and released platelet fractions. Conclusions: 1. TF-dependent PCA of washed human platelets is specifically and rapidly induced by GPIbα activation and it´s not accompanied with α-δ-granules release, including TFPI and PS. 2. Platelets contain enough membrane FVII to trigger the FX activation. 3. TRAP induces granule release, including TFPI and PS, which block TF-dependent PCA. 3. This TFPI is predominantly localized in LR fractions and co-precipitates with PS. 4. Secreted protein S likely localizes TFPI to the platelet membrane rich in anionic-rich phospholipids. 5. These results suggest that TF is translocated to LR for its inactivation. 6.These findings lead us to propose a novel model in which clotting is triggered by platelet TF during the first stage of platelet adhesion through GPIbα-VWF interaction; and TF/FVIIa/FXa would be dampened by the secreted TFPI and PS during subsequent platelet activation. Human platelets TF, FVII and the anionic phospholipid surface become central players to assemble and localize the whole clotting process into and around the platelet plug for both hemostasis and atherothrombosis; and platelet TFPI and PS would modulate its growth inactivating TF/FVIIa/FXa on the platelet surface. DisclosuresNo relevant conflicts of interest to declare.

Lipid rafts as major platforms for signaling regulation in cancer

Cell signaling does not apparently occur randomly over the cell surface, but it seems to be integrated very often into cholesterol-rich membrane domains, termed lipid rafts. Membrane lipid rafts are highly ordered membrane domains that are enriched in cholesterol, sphingolipids and gangliosides, and behave as major modulators of membrane geometry, lateral movement of molecules, traffic and signal transduction. Because the lipid and protein composition of membrane rafts differs from that of the surrounding membrane, they provide an additional level of compartmentalization, serving as sorting platforms and hubs for signal transduction proteins. A wide number of signal transduction processes related to cell adhesion, migration, as well as to cell survival and proliferation, which play major roles in cancer development and progression, are dependent on lipid rafts. Despite lipid rafts harbor mainly critical survival signaling pathways, including insulin-like growth factor I (IGF-I)/phosphatidylinositol 3-kinase (PI3K)/Akt signaling, recent evidence suggests that these membrane domains can also house death receptor-mediated apoptotic signaling. Recruitment of this death receptor signaling pathway in membrane rafts can be pharmacologically modulated, thus opening up the possibility to regulate cell demise with a therapeutic use. The synthetic ether phospholipid edelfosine shows a high affinity for cholesterol and accumulates in lipid rafts in a number of malignant hematological cells, leading to an efficient invitro and invivo antitumor activity by inducing translocation of death receptors and downstream signaling molecules to these membrane domains. Additional antitumor drugs have also been shown to act, at least in part, by recruiting death receptors in lipid rafts. The partition of death receptors together with downstream apoptotic signaling molecules in membrane rafts has led us to postulate the concept of a special liquid-ordered membrane platform coined as "cluster of apoptotic signaling molecule-enriched rafts" (CASMER), referring to raft platforms enriched in apoptotic molecules. CASMERs act as scaffolds for apoptosis signaling compartmentalization, facilitating and stabilizing protein-protein interactions by local assembly of cross-interacting molecules, which leads to apoptosis amplification and a decrease in apoptotic signal threshold. Edelfosine also displaced survival PI3K/Akt signaling from lipid rafts, leading to Akt inhibition, in mantle cell lymphoma cells. Thus, membrane rafts could act as scaffold structures where segregation of pro- from anti-apoptotic molecules could take place. In this review, we summarize our view of how reorganization of the protein composition of lipid raft membrane domains regulates cell death and therefore it might be envisaged as a novel target in the treatment of cancer.

Metabolism, energetics, and lipid biology in the podocyte - cellular cholesterol-mediated glomerular injury.

Chronic kidney disease (CKD) is associated with a high risk of death. Dyslipidemia is commonly observed in patients with CKD and is accompanied by a decrease in plasma high-density lipoprotein, and an increase in plasma triglyceride-rich lipoproteins and oxidized lipids. The observation that statins may decrease albuminuria but do not stop the progression of CKD indicates that pathways other than the cholesterol synthesis contribute to cholesterol accumulation in the kidneys of patients with CKD. Recently, it has become clear that increased lipid influx and impaired reverse cholesterol transport can promote glomerulosclerosis, and tubulointerstitial damage. Lipid-rafts are cholesterol-rich membrane domains with important functions in regulating membrane fluidity, membrane protein trafficking, and in the assembly of signaling molecules. In podocytes, which are specialized cells of the glomerulus, they contribute to the spatial organization of the slit diaphragm (SD) under physiological and pathological conditions. The discovery that podocyte-specific proteins such as podocin can bind and recruit cholesterol contributing to the formation of the SD underlines the importance of cholesterol homeostasis in podocytes and suggests cholesterol as an important regulator in the development of proteinuric kidney disease. Cellular cholesterol accumulation due to increased synthesis, influx, or decreased efflux is an emerging concept in podocyte biology. This review will focus on the role of cellular cholesterol accumulation in the pathogenesis of kidney diseases with a focus on glomerular diseases.