Membrane Lipid Rafts Research Articles

Lipid rafts may affect the coalescence of milk fat globules through phase transition after thermal treatment

This study aimed to investigate changes of fat globules and their membranes, and further analyze evolution of lipid profile of lipid rafts in membranes during heat processing of cow milk. Size of fat globules increased from 3.16 μm to 3.70 μm and ζ-potential decreased from −0.53 mV to −0.38 mV after thermal treatment, suggesting that fat globule membrane was destroyed and fat globule occurred coalescence. Glycerophospholipids and cholesterol in fat globule membrane decreased while sphingomyelin increased after thermal treatment. Results of lipidomics show that total of 38 species of 5 lipids molecule showed ability to differentiate the samples. At high temperatures, highly unsaturated glycerophospholipids and sterol lipids were lost from rafts, meanwhile, sphingomyelin and ceramide increased in this region. Significant change of lipid profile in the raft region during thermal treatment suggested a potential relationship between lipid rafts and fat globule coalescence behavior.

Cholesterol Protects the Liquid-Ordered Phase of Raft Model Membranes from the Destructive Effect of Ionic Liquids.

Ionic liquids (ILs), although being a class of promising green solvents, have received many reports on the toxicity to living organisms. In this work, aiming at elucidating the disruptive effect of ILs to cell membrane lipid rafts, we investigated the effect of three 1-octylimidazolium-based ILs on the properties of the liquid ordered phase (Lo, a commonly used lipid raft model) of egg sphingomyelin (SM)-cholesterol model membrane. We found that, in the absence of cholesterol, a very low IL:SM molar ratio of 0.01:1 could disrupt the integrity of the bilayer structure. In sharp contrast, the presence of cholesterol in lipid bilayers helps the Lo phase resist the damaging effect of the ILs. For the role of the IL headgroup, we found that the mono- and trisubstituted species show a stronger destructive effect on the structures of the model rafts than the commonly used disubstituted counterpart.

Interactions of polycyclic aromatic hydrocarbons and their nitro derivatives with bilayer and monolayer models of fungal membranes

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous persistent organic pollutants forming as byproducts in combustion processes. These compounds can become nitrated in the environment forming even more toxic nitro derivatives. Both PAHs and nitro-PAHs accumulate in the soils which can lead to the pollution of vast areas of arable land. The best means of their elimination from the environment is bioremediation that is the degradation by soil microorganisms. Important key players with high prospects are non-ligninolytic fungi, which can incorporate PAHs and their derivatives into their plasma membranes, oxidize and metabolize to nontoxic products. The accumulation of PAHs in the membrane can, however, be toxic to the fungal cell, but the mechanisms of the PAH-membrane lipid interactions and how they influence the membrane phase state and properties are still poorly understood. To shed light on these questions we employed fungal plasma membrane models and studied their interactions with selected PAHs and nitro-PAHs. Two complementary model systems were investigated: giant unilamellar vesicles (GUV) and Langmuir monolayers. We find that the native PAHs induce phase separation and the formation of solid ordered phase in the GUVs, whereas the nitro-derivatives do not disturb the liquid crystalline state of the membranes. The Langmuir monolayer studies indicate that some PAHs preferably partition in the liquid-expanded phase while others in the liquid-condensed domains. The possibility of the incorporation of PAHs into the condensed domains implies that these compounds can influence the order in the lipid bilayer and affect the function of the lipid rafts in plasma membranes.

Simvastatin Inhibits Brucella abortus Invasion into RAW 264.7 Cells through Suppression of the Mevalonate Pathway and Promotes Host Immunity during Infection in a Mouse Model.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and has been found to have protective effects against several bacterial infections. In this study, we investigate the effects of simvastatin treatment on RAW 264.7 macrophage cells and ICR mice against Brucella (B.) abortus infections. The invasion assay revealed that simvastatin inhibited the Brucella invasion into macrophage cells by blocking the mevalonic pathway. The treatment of simvastatin enhanced the trafficking of Toll-like receptor 4 in membrane lipid raft microdomains, accompanied by the increased phosphorylation of its downstream signaling pathways, including JAK2 and MAPKs, upon =Brucella infection. Notably, the suppressive effect of simvastatin treatment on Brucella invasion was not dependent on the reduction of cholesterol synthesis but probably on the decline of farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthesis. In addition to a direct brucellacidal ability, simvastatin administration showed increased cytokine TNF-α and differentiation of CD8+ T cells, accompanied by reduced bacterial survival in spleens of ICR mice. These data suggested the involvement of the mevalonate pathway in the phagocytosis of B. abortus into RAW 264.7 macrophage cells and the regulation of simvastatin on the host immune system against Brucella infections. Therefore, simvastatin is a potential candidate for studying alternative therapy against animal brucellosis.

Abstract 3020: Periodic fasting and cholesterol biosynthesis inhibitors achieve a synergistic antitumor activity in gastrointestinal cancers

Abstract Periodic fasting enhances the activity of several chemotherapeutics, molecularly-targeted drugs, endocrine agents and immune checkpoint inhibitors. Identifying drugs that are approved for non-oncological conditions, but acquire antitumor properties through fasting could pinpoint new cancer cell liabilities and define new treatment options. By screening over 800 approved drugs in PK9 pancreatic ductal adenocarcinoma (PDAC) cells, we identified several azoles (antifungal agents), including clotrimazole (CTZ), as agents whose cytotoxic activity against cancer cells is synergistically enhanced by starvation conditions. Since azoles inhibit 14alpha-demethylase, which is a key enzyme for cholesterol (CE) biosynthesis, we hypothesized that starvation and azoles would cooperate by blunting CE production in PDAC cells. Consistent with this notion, we found that simvastatin (an HMG-CoA reductase inhibitor) and terbinafine (TRB), which obstruct squalene epoxidase (SQLE), another key enzyme from the CE biosynthetic pathway, also had their antitumor effects strongly enhanced by starvation. Combined starvation and CTZ or starvation and TRB reduced intracellular CE in Capan-1 cells both in vitro and in vivo. Methyl-beta-cyclodextrin, which depletes intracellular CE, and starvation also showed a synergistic interaction in Capan-1 and in MiaPaCa2 cells (another PDAC cell line). Finally, culture media supplementation with a water-soluble CE formulation prevented the synergistic interaction between starvation and CE biosynthesis inhibitors. TRB potentiation through starvation was countered through supplementation with insulin, IGF1 and leptin, which are downregulated during fasting. Consistent with CE being an essential constituent of membrane lipid rafts, that harbor the growth- and survival-promoting PI3K/AKT signaling cascade, we found combined CTZ or TRB and starvation to markedly downregulate phosphorylated AKT in Capan-1 xenografts. Similar results were obtained in MiaPaCa2 cells. Capan-1 cell transduction with constitutively active, myristoylated AKT protected them from combined CTZ or TRB and starvation, indicating that AKT inhibition mediates the synergistic interaction between CE production inhibitors and fasting. Weekly 48h fasting enhanced CTZ and TRB antitumor effects in vivo in Capan-1 xenograft-bearing mice and, when combined with TRB, lowered circulating LDL (while increasing HDL) cholesterol as compared to TRB alone. Overall, these findings support the potential of fasting-based diets in combination with CE biosynthesis inhibitors against PDAC. Citation Format: Amr Khalifa, Ana Guijarro, Asmaa Namatalla, Moustafa Ghanem, Matteo Lambertini, Alessio Nencioni, Irene Caffa. Periodic fasting and cholesterol biosynthesis inhibitors achieve a synergistic antitumor activity in gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3020.

Biocompatible and optically stable hydrophobic fluorescent carbon dots for isolation and imaging of lipid rafts in model membrane.

Lateral heterogeneity in cell membranes features a variety of compositions that influence their inherent properties. One such biophysical variation is the formation of a membrane or lipid raft, which plays important roles in many cellular processes. The lipid rafts on the cell membrane are mostly identified by specific dyes and heavy metal quantum dots, which have their own drawbacks, such as cytotoxicity, photostability, and incompatibility. To this end, we synthesized special, hydrophobic, fluorescent, photostable, and non-cytotoxic carbon dots (CDs) by solvent-free thermal treatment using non-cytotoxic materials and incorporated into the lipid bilayers of giant unilamellar vesicles (GUVs) made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) lipids. A 2:2:1 mixture of DOPC, DPPC, and cholesterol (Chol) develops lipid rafts on the membrane by phase separation. The photophysical properties of the CDs get modulated on incorporation into the lipid rafts that identifies the membrane heterogeneity. The main attempt in this work is to develop a new, simple, cost-effective, and bio-friendly lipid raft marker, which can be used in biological applications, alongside other conventional raft markers, with more advantages.

Galectin-3 facilitates cell-to-cell HIV-1 transmission by altering the composition of membrane lipid rafts in CD4 T cells.

Galectin-3 (GAL3) is a β-galactoside-binding lectin expressed in CD4 T cells infected with human immunodeficiency virus-1 (HIV-1). GAL3 promotes HIV-1 budding by associating with ALIX and Gag p6. GAL3 has been shown to localize in membrane lipid rafts in dendritic cells and positively regulate cell migration. HIV-1 spreads between T cells by forming supramolecular structures (virological synapses [VSs]), whose integrity depends on lipid rafts. Here, we addressed the potential role of GAL3 in cell-to-cell transmission of HIV-1 in CD4 T cells. GAL3 expressed in donor cells was more important for facilitating HIV-1 cell-to-cell transfer than GAL3 expressed in target cells. GAL3 was found to be co-transferred with Gag from HIV-1-positive donor to HIV-1-negative target T cells. HIV-1 infection induced translocation of GAL3 together with Gag to the cell-cell interfaces and colocalize with GM1, where GAL3 facilitated VS formation. GAL3 regulated the coordinated transfer of Gag and flotillin-1 into plasma membrane fractions. Finally, depletion of GAL3 reduced the cholesterol levels in membrane lipid rafts in CD4 T cells. These findings provide evidence that endogenous GAL3 stimulates lipid raft components and facilitates intercellular HIV-1 transfer among CD4 T cells, offering another pathway by which GAL3 regulates HIV-1 infection. These findings may inform the treatment of HIV-1 infection based on targeting GAL3 to modulate lipid rafts.

Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells.

Simple SummaryStem cell-like glioma-propagating cells (GPCs) are crucial for initiation, growth, and treatment resistance of glioblastoma multiforme. Due to their strong immunosuppressive activities, they essentially limit immunotherapeutic approaches. This study offers a new model of radio-selected patient-derived GPCs mimicking a clinical treatment regime of tumor irradiation which is especially useful for immunotherapeutic studies. We provide evidence that clinically relevant, sub-lethal fractions of γ radiation select for a more radio-resistant GPC phenotype with lower immunogenic potential, potentially hampering the success of adjuvant T-cell-based immunotherapies. The immune evasion in GPCs was characterized by quantitative proteomics. It revealed a marked downregulation of the antigen processing machinery in lipid rafts of these cells, leading to reduced MHC surface expression and weaker cytotoxic T lymphocyte (CTL) recognition.Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of γ radiation. GPCs were treated with 2.5 Gy of γ radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape.

0026 Targeted genome sequencing identifies multiple rare variants in Caveolin-1 associated with obstructive sleep apnea

Abstract Introduction Obstructive sleep apnea (OSA) is a common disorder associated with increased risk for cardiovascular disease, diabetes, and premature mortality. There is strong clinical and epidemiologic evidence supporting the importance of genetic factors influencing OSA, but limited data implicating specific genes. Methods Leveraging high depth genomic sequencing data from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program and imputed genotype data from multiple population-based studies, we performed linkage analysis in the Cleveland Family Study followed by multi-stage gene-based association analyses in independent cohorts to search for rare variants contributing to OSA severity as assessed by the apnea-hypopnea index (AHI) in a total of 7,708 European-Americans. Results We identified 21 non-coding rare variants in Caveolin-1 (CAV1) associated with lower AHI after accounting for multiple comparisons (P = 7.4×10-8). These non-coding variants together significantly contributed to the linkage evidence. Follow-up analysis revealed significant associations between increased CAV1 expression with lower AHI (P=0.024) and higher minimum overnight oxygen saturation (P=0.007). Conclusion Caveolin-1 is a membrane scaffolding protein that is essential in the formation of plasma membrane lipid rafts and mediates cholesterol trafficking; regulates several signaling molecules including transforming growth factor β (TGF-beta), Toll Like Receptor 4 (TLR4) and endothelial nitric oxide synthase (eNOS); with mutations implicated in disorders associated with OSA: pulmonary hypertension, diabetes, atherosclerosis, endothelial and cardiac dysfunction, and inflammation. Our results indicate that caveolin-1 also plays a significant role in OSA, with rare variants and higher CAV1 expression associated with lower AHI. Support (If Any) R35HL135818, R01HG011052, R01HL153814

A reengineered common chain cytokine augments CD8+ T cell-dependent immunotherapy.

Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3–mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell–based immunotherapy.

Clostridioides difficile surface layer protein triggered inflammasome activation is mediated by membrane lipid raft

Clostridioides difficile is the leading cause of nosocomial infectious diarrhea, with clinical symptoms ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon. Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of C. difficileinfection (CDI), clinical relapse rates remain high. Therefore, new alternative therapeutics to treat CDI are urgently required. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Here, we found SLPs as well as C. difficile induced inflammasome activation. In addition, the cholesterol‐rich microdomains, lipid rafts, on the cell membrane are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Furthermore, both binding ability and inflammasome activation induced by SLP were abrogated with membrane cholesterol depletion by methyl‐β‐cyclodextrin (MβCD). The coalescence of SLPs in the cholesterol‐rich microdomains was confirmed in C. difficile‐infected cells. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.

AAV9‐synapsin‐caveolin‐1 (AAV9‐SynCav1) gene delivery preserves motor neuron and neuromuscular junction morphology, motor function and body weight, and extends survival in hSOD1G93A mice

IntroductionAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from loss of upper and lower motor neurons in the brain and spinal cord. Due to the unknown etiology of sporadic ALS (SALS), interventions that are neuroprotective are urgently needed. Evidence shows that caveolin‐1 (Cav‐1), a membrane/lipid raft (MLR) scaffolding and neuroprotective protein, is decreased in degenerating neurons along with Cav‐1‐associated pro‐survival signaling proteins. We have previously shown that synapsin‐promoted Cav‐1 (SynCav1) augments neuroplasticity, preserves synaptic ultrastructure and hippocampal learning and memory in a mouse model of Alzheimer’s disease. The current study investigated whether spinal cord delivery of adeno‐associated virus serotype 9 (AAV9)‐SynCav1 could also be neuroprotective and extend survival in a mouse (hSOD1G93A) model of ALS.MethodsAAV9‐SynCav1 (10 ul; 1.9 x 1013 genomic copies (g.c.)/ml) subpial lumber spinal cord delivery was performed at 8 weeks (wk) of age. Weekly body weight (BW) measurements were recorded and survival was determined using Kaplan‐Meier curve. Running wheel (RW) tests were recorded at 8 (pre‐surgery baseline) and 16 wk (symptomatic age). Immunoblot (IB) and immunofluorescence microscopy (IF) assays for choline acetyltransferase (ChAT) and Cav‐1 were performed at 19 wk (before end‐stage age). To measure neuromuscular junctions (NMJs) in tibialis muscle, IF for bungarotoxin‐a (Btx, marker of post‐synaptic nicotinic acetylcholine receptors) and synaptoporin (Synphy, pre‐synaptic vesicle marker) was performed. Values were expressed as mean ± S.E.M while Kaplan‐Meier (Log‐rank (Mantel‐Cox) test) was expressed as median days (d) survival. Statistical significance was defined as p < 0.05.ResultsMale (Figure 1) and female (Figure 2) hSOD1G93A mice that received AAV9‐SynCav1 exhibited significantly longer survival compared to non‐treated hSOD1G93A mice (male: 176 d for SynCav1‐hSOD1G93A vs 158 d for non‐treated hSOD1G93A, p=0.004; female: 180 d for SynCav1‐hSOD1G93A vs 164 d for non‐treated hSOD1G93A, p<0.001). IF revealed that AAV9‐SynCav1 significantly increased motor neuron cell body area (Figure 3A, B), total ChAT‐positive motor neurons within the ventral horn (Figure 3C), increased Cav‐1 expression per motor neuron (Figure 3D), which resulted in a direct correlation between motor neuron area and Cav‐1 expression (R2 = 0.66) in hSOD1G93A mice at 19 wk. Further IF of anterior tibial muscle showed that preserved NMJ morphology (Btx) and NMJ occupancy of Synphy expression in AAV9‐SynCav1‐hSOD1G93A (Figure 4).ConclusionAAV9‐SynCav1 gene therapy significantly prolongs survival, preserves motor neurons and motor neuron morphology in the ventral horn of the spinal cord, and preservers NMJs in the tibialis muscle of hSOD1G93A mice. We are currently testing if alternative routes of AAV9‐SynCav1 delivery (e.g., intrathecal, intraventricular, systemic) can recapitulate the neuroprotective effects achieved using subpial delivery.

Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition and structure makes them less fluid than non‐raft membranes, and it is believed that lipid rafts regulate signaling of many proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl‐β‐cyclodextrin). Removing either the sphingolipids or cholesterol results in disruption of the lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p‐CREB) and p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p‐p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time‐dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p‐p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.

Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.

Abstract 426: Adipose Tissue Phosphatidylcholine Remodeling Influences Insulin Signaling

Lysophosphatidylcholine acyltransferase (LPCAT3) is the major isoform of phosphatidylcholine remodeling enzyme in adipose tissue. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice using the Cre-Loxp approach. We found LPCAT3 deficiency caused a significant reduction of LPCAT3 mRNA and activity by about 60%, and a significant reduction of polyunsaturated phosphatidylcholines in the plasma membrane. Further, the deficiency increased insulin sensitivity, reflected by glucose and insulin tolerance studies. Importantly, LPCAT3 deficiency promoted insulin-dependent insulin receptor kinase tyrosine phosphorylation and Akt phosphorylation through influencing membrane lipid rafts. Furthermore, the deficiency protected mice from high-fat, high-caloric diet-induced insulin resistance. Taken together, our findings identify LPCAT3 as one of the major players in maintaining cell membrane structure and illustrate the manipulation of cell membrane phospholipid saturation can be a new strategy to treat insulin resistance.

Chimeric Cytokine Targeting NKG2D and IL-2 Receptor Augments CD8+ T Cell-Metabolic Fitness and Improves Immunotherapy

Abstract T cell mediated immunotherapy has gained credible traction for cancer treatment. To date experimental and clinical strategies have relied on antibody-mediated co-stimulatory blockade, or the administration of high doses of naturally occurring or engineered cytokines to increase T cell activation. Cytokine therapy is by limited undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here we describe functionality of a unique chimeric cytokine by design, called OMCPmutIL-2, binds with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signals through the common γ-chain cytokine receptor, thus eliminating IL-2Ra mediated off-target side effects. In addition to precise activation of cytotoxic T cells due to redirected binding we describe that OMCPmutIL-2 results in superior activation of both human and murine CD8+ T cells by improving their survival, decreasing exhaustion, and improving memory cell generation. This functional improvement is the result altered signal transduction based on the reorganization of surface membrane lipid rafts resulting in JAK-mediated phosphorylation of the T cell receptor (TCR) rather than canonical STAT/AKT signaling intermediates activated by other common γ-chain cytokines. This altered signaling pathway increases CD8+ T cell response to low affinity antigens, activates Nuclear Factor of Activated T Cells (NFAT) transcription factors, and enhances mitochondrial biogenesis with improved T cell survival, cytotoxicity, and memory cell generation. OMCPmutIL-2 thus outperforms other common γ-chain cytokines as both a reagent for T cell expansion and in vivo CD8+ T cell-based immunotherapy. Supported by grant I01 BX002299

Nanoscale Structure and Dynamics of Model Membrane Lipid Raft Systems, Studied by Neutron Scattering Methods

Quasi-elastic neutron scattering (QENS) and small angle neutron scattering (SANS), in combination with isotopic contrast variation, have been used to determine the structure and dynamics of three-component lipid membranes, in the form of vesicles, comprising an unsaturated [palmitoyl-oleoyl-phosphatidylcholine (POPC) or dioleoyl-phosphatidylcholine (DOPC)], a saturated phospholipid (dipalmitoyl-phosphatidylcholine (DPPC)), and cholesterol, as a function temperature and composition. SANS studies showed vesicle membranes composed of a 1:1:1 molar ratio of DPPC:DOPC:cholesterol and a 2:2:1 molar ratio of DPPC:POPC:cholesterol phase separated, forming lipid rafts of ∼18 and ∼7 nm diameter respectively, when decreasing temperature from 308 to 297 K. Phase separation was reversible upon increasing temperature. The larger rafts observed in systems containing DOPC are attributed to the greater mis-match in lipid alkyl chains between DOPC and DPPC, than for POPC and DPPC. QENS studies, over the temperature range 283–323K, showed that the resulting data were best modelled by two Lorentzian functions: a narrow component, describing the “in-plane” lipid diffusion, and a broader component, describing the lipid alkyl chain segmental relaxation. The overall “in-plane” diffusion was found to show a significant reduction upon increasing temperature due to the vesicle membranes transitioning from one containing rafts to one where the component lipids are homogeneously mixed. The use of different isotopic combinations allowed the measured overall reduction of in-plane diffusion to be understood in terms of an increase in diffusion of the saturated DPPC lipid and a corresponding decrease in diffusion of the unsaturated DOPC/POPC lipid. As the rafts are considered to be composed principally of saturated lipid and cholesterol, the breakdown of rafts decreases the exposure of the DPPC to cholesterol whilst increasing the exposure of cholesterol to unsaturated lipid. These results show the sensitivity of lipid diffusion to local cholesterol concentration, and the importance of considering the local, rather that the global composition of a membrane when understanding the diffusion processes of lipids within the membrane. The novel combination of SANS and QENS allows a non-intrusive approach to characterize the structure and dynamics occurring in phase-separated model membranes which are designed to mimic the lateral heterogeneity of lipids seen in cellular membranes–a heterogeneity that can have pathological consequences.

The Neuroprotective Lipocalin Apolipoprotein D Stably Interacts with Specific Subtypes of Detergent-Resistant Membrane Domains in a Basigin-Independent Manner

Accumulated evidence points to the lipocalin apolipoprotein D (ApoD), one of the few genes consistently upregulated upon brain ageing and neurodegeneration, as an endogenous controller of the redox state of cellular and extracellular lipid structures. This biochemical function has downstream consequences as apparently varied as control of glycocalyx and myelin compaction, cell viability upon oxidative stress or modulation of signalling pathways. In spite of this knowledge, it is still unclear if ApoD function requires canonical receptor-mediated transductions systems. This work aims to examine ApoD-cell membrane interaction and its dependence on a proposed ApoD receptor, Basigin. Whole and fractionated membrane preparations from the brain, primary astrocytes, glial and neuronal cell lines, reveal ApoD as a very specific component of particular subtypes of detergent-resistant microdomains (DRMs). ApoD interacts in vitro with neuronal membranes and is stably associated with astrocytic membranes. ApoD associates with DRMs with specific buoyancy properties that co-fractionate with plasma or late-endosome-lysosome markers. A mass spectrometry analysis reveals that these Triton X-114 DRMs contain both plasma membrane and endosomal-lysosomal compartment lipid raft proteins. ApoD-DRM association is maintained under metabolic and acute oxidative stress conditions. However, ApoD-membrane interaction, its internalization and its lipid-antioxidant function do not require the presence of Basigin. This work supports a stable association of ApoD with membranes, independent of Basigin, and provides the basis to fully understand ApoD antioxidant neuroprotective mechanism as a mechanism taking place in specific membrane subdomains.

Phosphatidylserine enhances anti-inflammatory effects of reconstituted HDL in macrophages via distinct intracellular pathways.

Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38mitogen-activated protein kinase (p38MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.

Anti-β2-GPI Antibodies Induce Endothelial Cell Expression of Tissue Factor by LRP6 Signal Transduction Pathway Involving Lipid Rafts.

In this study we analyzed whether anti-β2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-β2-GPI antibodies. Both LRP6 and β-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-β2-GPI antibodies induced LRP6 phosphorylation with consequent β-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-β-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-β2-GPI antibodies effect, indicating that the anti-β2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between β2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-β2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-β2-GPI binding triggers β-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).