Project description:Influenza A virus (IAV) is widely disseminated across different species and can cause recurrent epidemics and severe pandemics in humans. During infection, IAV attaches to receptors that are predominantly located in cell membrane regions known as lipid rafts, which are highly enriched in cholesterol and sphingolipids. Following IAV entry into the host cell, uncoating, transcription, and replication of the viral genome occur, after which newly synthesized viral proteins and genomes are delivered to lipid rafts for assembly prior to viral budding from the cell. Moreover, during budding, IAV acquires an envelope with embedded cholesterol from the host cell membrane, and it is known that decreased cholesterol levels on IAV virions reduce infectivity. Statins are commonly used to inhibit cholesterol synthesis for preventing cardiovascular diseases, and several studies have investigated whether such inhibition can block IAV infection and propagation, as well as modulate the host immune response to IAV. Taken together, current research suggests that there may be a role for statins in countering IAV infections and modulating the host immune response to prevent or mitigate cytokine storms, and further investigation into this is warranted.
Project description:Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.
Project description:Fragile X syndrome (FXS) is the most prevalent monogenic cause of intellectual disability and autism spectrum disorder (ASD). Affected individuals have a high prevalence of hypocholesterolemia, however, the underlying mechanisms and the clinical significance remains unknown. We hypothesized that decrease in the plasma cholesterol levels is associated with an alteration of cholesterol content within the lipid rafts (LRs) which ultimately affects the clinical profile of FXS individuals. The platelets LRs were isolated by ultracentrifugation on sucrose gradient from 27 FXS and 25 healthy controls, followed by measurements of proteins, cholesterol, and gangliosides content. Autistic and adaptive behaviour of affected individuals were respectively assessed by the Social Communication Questionnaire and Adaptive Behavior Assessment System. Our results suggest a decrease in the cholesterol content of LRs in FXS individuals as compared to controls. As opposed to controls, LR cholesterol was significantly associated with plasma total cholesterol (r = 0.47; p = 0.042) in the FXS group. Furthermore, the correlation between LRs cholesterol and the clinical profile showed a significant association with autistic traits (r = - 0.67; p < 0.001) and adaptative behavior (r = 0.70; p < 0.001). These results support the clinical significance of LR cholesterol alterations in FXS. Further studies are warranted to investigate the implication of LRs in FXS pathophysiology and ASD.
Project description:?-Opioid receptor (OPRM1) is mainly localized in lipid raft microdomains but internalizes through clathrin-dependent pathways. Our previous studies demonstrated that disruption of lipid rafts by cholesterol-depletion reagent blocked the agonist-induced internalization of OPRM1 and G protein-dependent signaling. The present study demonstrated that reduction of cholesterol level decreased and culturing cells in excess cholesterol increased the agonist-induced internalization and desensitization of OPRM1, respectively. Further analyses indicated that modulation of cellular cholesterol level did not affect agonist-induced receptor phosphorylation but did affect membrane translocation of ?-arrestins. The translocation of ?-arrestins was blocked by cholesterol reduction, and the effect could be reversed by incubating with cholesterol. OptiPrep gradient separation of lipid rafts revealed that excess cholesterol retained more receptors in lipid raft domains and facilitated the recruitment of ?-arrestins to these microdomains upon agonist activation. Moreover, excess cholesterol could evoke receptor internalization and protein kinase C-independent extracellular signal-regulated kinases activation upon morphine treatment. Therefore, these results suggest that cholesterol not only can influence OPRM1 localization in lipid rafts but also can effectively enhance the recruitment of ?-arrestins and thereby affect the agonist-induced trafficking and agonist-dependent signaling of OPRM1.
Project description:ObjectiveAIBP (apolipoprotein A-I binding protein) is an effective and selective regulator of lipid rafts modulating many metabolic pathways originating from the rafts, including inflammation. The mechanism of action was suggested to involve stimulation by AIBP of cholesterol efflux, depleting rafts of cholesterol, which is essential for lipid raft integrity. Here we describe a different mechanism contributing to the regulation of lipid rafts by AIBP. Approach and Results: We demonstrate that modulation of rafts by AIBP may not exclusively depend on the rate of cholesterol efflux or presence of the key regulator of the efflux, ABCA1 (ATP-binding cassette transporter A-I). AIBP interacted with phosphatidylinositol 3-phosphate, which was associated with increased abundance and activation of Cdc42 and rearrangement of the actin cytoskeleton. Cytoskeleton rearrangement was accompanied with reduction of the abundance of lipid rafts, without significant changes in the lipid composition of the rafts. The interaction of AIBP with phosphatidylinositol 3-phosphate was blocked by AIBP substrate, NADPH (nicotinamide adenine dinucleotide phosphate), and both NADPH and silencing of Cdc42 interfered with the ability of AIBP to regulate lipid rafts and cholesterol efflux.ConclusionsOur findings indicate that an underlying mechanism of regulation of lipid rafts by AIBP involves PIP-dependent rearrangement of the cytoskeleton.
Project description:Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/ cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
Project description:Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
Project description:We previously showed that macrophages from macrophage-specific ATP-binding cassette transporter A1 (ABCA1) knockout (Abca1(-M/-M)) mice had an enhanced proinflammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1(-M/-M) macrophages were also hyper-responsive to specific agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1(-M/-M) vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1(-M/-M) and WT macrophages, significantly more TLR4 was distributed in membrane lipid rafts in Abca1(-M/-M) macrophages. Abca1(-M/-M) macrophages also exhibited increased trafficking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specific agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens inflammation by reducing MyD88-dependent TLRs trafficking to lipid rafts by selective reduction of FC content in lipid rafts.
Project description:Cholesterol and sphingolipid enriched lipid raft micro-domains in the plasma membrane play an important role in the life-cycle of numerous enveloped viruses. Although human respiratory syncytial virus (RSV) proteins associate with the raft domains of infected cells and rafts are incorporated in RSV virion particles, the functional role of raft during RSV infection was unknown. In the current study we have identified rafts as an essential component of host cell that is required for RSV infection. Treatment of human lung epithelial cells with raft disrupting agent methyl-beta-cyclodextrin (MBCD) led to drastic loss of RSV infectivity due to diminished release of infectious progeny RSV virion particles from raft disrupted cells. RSV infection of raft deficient Niemann-Pick syndrome type C human fibroblasts and normal human embryonic lung fibroblasts revealed that during productive RSV infection, raft is required for release of infectious RSV particles.
Project description:The Cyprinus herpesvirus 3 (CyHV-3) is a member of the new Alloherpesviridae virus family in the Herpesvirales order. CyHV-3 has been implicated in a large number of disease outbreaks in carp populations causing up to 100% mortality. The aim of this study was to investigate the requirement of cholesterol-rich lipid rafts in CyHV-3 entry and replication in carp cells. Plasma membrane cholesterol was depleted from common carp brain (CCB) cells with methyl-?-cyclodextrin (M?CD). Treated and non-treated cells were infected with CyHV-3 and virus binding and infection parameters were assessed using RT-qPCR, immunocytochemistry and virus titration. The effect of cholesterol reduction severely stunted virus entry in vitro, however after cholesterol replenishment virus entry and subsequent replication rates were similar to the control infection. Furthermore, cholesterol depletion did not significantly influence virus binding and the subsequent post-entry replication stage, however had an impact on virus egress. Comparative analysis of the lipid compositions of CyHV-3 and CCB membrane fractions revealed strong similarities between the lipid composition of the CyHV-3 and CCB lipid rafts. The results presented here show that cholesterol-rich lipid rafts are important for the CyHV-3 replication cycle especially during entry and egress.