Isolated Lipid Rafts Research Articles

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck

BackgroundT cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation.MethodsWhole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells.ResultsTRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation.ConclusionsTRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

Evidence for alterations in lipid profiles and biophysical properties of lipid rafts from spinal cord in sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-dependent neurodegenerative disease affecting motor neurons in the spinal cord and brainstem whose etiopathogenesis remains unclear. Recent studies have linked major neurodegenerative diseases with altered function of multimolecular lipid-protein complexes named lipid rafts. In the present study, we have isolated lipid rafts from the anterior horn of the spinal cords of controls and ALS individuals and analysed their lipid composition. We found that ALS affects levels of different fatty acids, lipid classes and related ratios and indexes. The most significant changes affected the contents of n-9/n-7 monounsaturated fatty acids and arachidonic acid, the main n-6 long-chain polyunsaturated fatty acid (LCPUFA), which were higher in ALS lipid rafts. Paralleling these findings, ALS lipid rafts lower saturates-to-unsaturates ratio compared to controls. Further, levels of cholesteryl ester (SE) and anionic-to-zwitterionic phospholipids ratio were augmented in ALS lipid rafts, while sulfatide contents were reduced. Further, regression analyses revealed augmented SE esterification to (mono)unsaturated fatty acids in ALS, but to saturates in controls. Overall, these changes indicate that lipid rafts from ALS spinal cord undergo destabilization of the lipid structure, which might impact their biophysical properties, likely leading to more fluid membranes. Indeed, estimations of membrane microviscosity confirmed less viscous membranes in ALS, as well as more mobile yet smaller lipid rafts compared to surrounding membranes. Overall, these results demonstrate that the changes in ALS lipid rafts are unrelated to oxidative stress, but to anomalies in lipid metabolism and/or lipid raft membrane biogenesis in motor neurons.Key messagesThe lipid matrix of multimolecular membrane complexes named lipid rafts are altered in human spinal cord in sporadic amyotrophic lateral sclerosis (ALS).Lipid rafts from ALS spinal cord contain higher levels of n-6 LCPUFA (but not n-3 LCPUFA), n-7/n-9 monounsaturates and lower saturates-to-unsaturates ratio.ALS lipid rafts display increased contents of cholesteryl esters, anomalous anionic-to-zwitterionic phospholipids and phospholipid remodelling and reduced sulphated and total sphingolipid levels, compared to control lipid rafts.Destabilization of the lipid structure of lipid raft affects their biophysical properties and leads to more fluid, less viscous membrane microdomains.The changes in ALS lipid rafts are unlikely related to increased oxidative stress, but to anomalies in lipid metabolism and/or raft membrane biogenesis in motor neurons.

Abstract 6259: Novel inhibitors of StAR related lipid transfer protein 5 inhibitors target plasma membrane lipid raft growth signaling

Abstract Background: Treatment of colorectal cancer (CRC) presents a major challenge due to chemotherapeutic resistance and recurrence, which are attributed to the presence of cancer stem cells (CSCs). Cholesterol metabolism is a critical regulator of CSCs. Lipid Rafts are cholesterol-rich microdomains in the plasma membrane (PM) that anchor various growth receptors tyrosine kinases (RTKs). STARD5 is a novel protein that helps transport cholesterol from the endoplasmic reticulum to the PM. We have identified specific small molecule inhibitors of the STARD5 which disrupt the lipid rafts, resulting in the inhibition of growth signaling pathways. Methods and Results: In-silico modeling studies using STARD5 crystal structure revealed 8 putative inhibitors (SD5i (A-H)). In vitro binding studies to determine secondary structural changes using circular dichroism spectroscopy revealed SD5iA and SD5iB as excellent STARD5 ligands. Both SD5iA and SD5iB caused a potent inhibition (IC50 0.3 -18 µM) of CSC-enriched 3D spheroids in 9 CRC cell lines representative of 3 consensus molecular subtypes. However, SD5iA but not SD5iB showed toxicity towards normal murine intestinal organoids at effective doses against CRC CSCs. Also, SD5iB enhanced cytotoxicity by oxaliplatin and panitumumab against CRC CSCs in HT-29 spheroids. Hence, SD5iB was advanced into in vivo studies. SD5iB caused no discernible toxicity in doses as high as 100 mg/kg in NCr nude mice at gross, serum chemistry, organ viability, or intestinal stem cell level (organoid formation). Subsequently, we generated s.c. xenograft using dual CSC marker sorted HT-29 cells and enriched them further with chemotherapy (5-fluorouracil and oxaliplatin weekly x 3) treatment followed by randomization to vehicle or SD5iB (100mg/kg i.p. 3/wk x 3 wks.). We observed xenograft growth inhibition and LGR5 expression in SD5iB-treated animals compared to vehicle controls. Mechanistically, we observed a decrease in free accessible PM cholesterol by SD5i measured by fluorescently tagged cholesterol-binding bacterial protein fALOD4 in HT-29 cells. Moreover, lipid raft staining (cholera toxin B staining) was significantly reduced in SD5iB-treated HT-29 cells compared to vehicle control using immunofluorescence. Further, lipid raft isolation was done by sucrose gradient ultracentrifugation to study the changes in the phosphorylation of RTKs SD5iB treatment in HT-29 spheroids resulted in decreased phosphorylation of EGFR, IGF1Rβ, and FGFR1 in isolated lipid rafts. Consequently, we observed a robust inhibition of ERK1/2 activation following SD5iB treatment compared to vehicle control. Conclusion: SD5i shows anti-CSC activity by targeting lipid-raft-driven growth factor signaling. Hence, STARD5 inhibitors represent a novel class of targeted therapy that will enhance the efficacy of FDA-approved therapies in CRC. Citation Format: Shoja Hajira, Chetna Sharon, Daniel RodriguezAgudo, Rio Boothelllo, Glenn Kellogg, Gregorio Gil, William Pandak, Bhaumik B. Patel. Novel inhibitors of StAR related lipid transfer protein 5 inhibitors target plasma membrane lipid raft growth signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6259.

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.

Fyn-kinase and caveolin-1 in the alveolar epithelial junctional adherence complex contribute to the early stages of pulmonary fibrosis

Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1β. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-β damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-β and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-β and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-β/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.

FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.

Atorvastatin suppressed proliferation and facilitated apoptosis of A549 cells through mediating recruitment of Fas and CD59 in lipid raft

Purpose: Lipid raft facilitated progression of NSCLC and atorvastatin could break cholesterol. The purpose of this study was to determine the potential mechanism of atorvastatin through lipid raft mediation in NSCLC.Methods: A549 cells were first grouped as NC, methyl-β-cyclodextrin (MβCD, lipid raft inhibitor, 5mM and 10mM), atorvastatin (0, 5mM, 10mM and 15mM) and MβCD (10mM) with atorvastatin (15mM). Later 10mM MβCD treated A549 cells were divided into three groups: NC, 3BDO (mTOR agonist) (60μM) and 3BDO (60μM) with atorvastatin (15mM) group. Thereafter, FLOT-2, SLP-2 expressions were assessed with RT-qPCR，mTOR proteins were measured by western blot and cell viability by CCK-8 method. Meanwhile, apoptosis was analyzed by flow cytometry. Moreover, lipid raft isolation was performed for acquiring Fas and CD59 and concentrations were detected by ELISA.Results: MβCD treatment significantly inhibited FLOT-2 and SLP-2 RNA expressions and cell viability of A549 cells but up-regulated apoptosis. Besides that, Fas protein level was promoted and CD59 was suppressed. Atorvastatin also repressed FLOT-2 and SLP-2 RNA levels. Meanwhile, atorvastatin downregulated cell viability and accelerated apoptosis. Moreover, Fas was increased and CD59 was inhibited by atorvastatin and MβCD enhanced functions of atorvastatin. MβCD inhibited mTOR RNA expression in A549 cells was increased by 3BDO but atorvastatin restored 3BDO caused up-regulation of mTOR. Furthermore, up regulated cell viability of A549 by 3BDO was declined with atorvastatin and decreased apoptosis by 3BDO was reversed through atorvastatin. Fas suppressed by 3BDO and promotion of CD59 were also resumed by atorvastatin.Conclusion: Atorvastatin suppressed FLOT-2 and SLP-2 and mediated recruitments of Fas and CD59 with suppressing cell viability and facilitating apoptosis of A549 cells via mTOR signaling pathway.

Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

BackgroundResistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known.MethodsA paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC–MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo.ResultsA higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression.ConclusionsThese findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

Concussion/Mild Traumatic Brain Injury (TBI) Induces Brain Insulin Resistance: A Positron Emission Tomography (PET) Scanning Study.

Brain injury/concussion is a growing epidemic throughout the world. Although evidence supports association between traumatic brain injury (TBI) and disturbance in brain glucose metabolism, the underlying molecular mechanisms are not well established. Previously, we reported the release of cellular prion protein (PrPc) from the brain to circulation following TBI. The PrPc level was also found to be decreased in insulin-resistant rat brains. In the present study, we investigated the molecular link between PrPc and brain insulin resistance in a single and repeated mild TBI-induced mouse model. Mild TBI was induced in mice by dropping a weight (~95 g at 1 m high) on the right side of the head. The procedure was performed once and thrice (once daily) for single (SI) and repeated induction (RI), respectively. Micro PET/CT imaging revealed that RI mice showed significant reduction in cortical, hippocampal and cerebellum glucose uptake compared to SI and control. Mice that received RI also showed significant motor and cognitive deficits. In co-immunoprecipitation, the interaction between PrPc, flotillin and Cbl-associated protein (CAP) observed in the control mice brains was disrupted by RI. Lipid raft isolation showed decreased levels of PrPc, flotillin and CAP in the RI mice brains. Based on observation, it is clear that PrPc has an interaction with CAP and the dislodgment of PrPc from cell membranes may lead to brain insulin resistance in a mild TBI mouse model. The present study generated a new insight into the pathogenesis of brain injury, which may result in the development of novel therapy.

Differential expression of two ATPases revealed by lipid raft isolation from gills of euryhaline teleosts with different salinity preferences.

In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H+-ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

Isolation of Lipid Rafts by the Detergent-Based and Non-detergent-Based Methods for Localization of GPCRs with Immunoblotting and Laser Scanning Confocal Microscopy.

The understanding of how biological membranes are organized and how they function has constantly been evolving over the past decades. Instead of just serving as a medium in which specific proteins are located, certain parts of the lipid bilayer contribute to platforms that assemble signaling complexes by providing a microenvironment that facilitates effective protein-protein interactions. G protein-coupled receptors (GPCRs) and relevant signaling molecules, including the heterotrimeric G proteins, key enzymes such as kinases and phosphatases, trafficking proteins, and secondary messengers, preferentially partition to these highly organized cell membrane microdomains, called lipid rafts. Lipid rafts are essential for the trafficking and signaling of GPCRs. The study of GPCR biology in the context of lipid rafts involves the localization of the GPCR of interest in lipid rafts, at the basal state and upon receptor agonism, and the evaluation of the biological functions of the GPCR in appropriate cell lines. The lack of standardized methodologies to study lipid rafts, in general, and of the workings of GPCRs in lipid rafts, in particular, and the inescapable drawbacks of current methods have hampered the complete understanding of the underlying molecular mechanisms. Newer methodologies that allow the study of GPCRs in their native form are needed. The use of complementary approaches that produce mutually supportive results appears to be the best way for drawing conclusions with regard to the distribution and activity of GPCRs in lipid rafts.

Lipid Raft Isolation by Sucrose Gradient Centrifugation and Visualization of Raft-Located Proteins by Fluorescence Microscopy: The Use of Combined Techniques to Assess Fas/CD95 Location in Rafts During Apoptosis Triggering.

Lipid rafts are heterogeneous membrane domains enriched in cholesterol, sphingolipids, and gangliosides that serve as sorting platforms to compartmentalize and modulate signaling pathways. Death receptors and downstream signaling molecules have been reported to be recruited into these raft domains during the triggering of apoptosis. Here, we provide two protocols that support the presence of Fas/CD95 in lipid rafts during apoptosis, involving lipid raft isolation and confocal microscopy techniques. A detailed protocol is provided for the isolation of lipid rafts, by taking advantage of their resistance to Triton X-100 solubilization at 4°C, followed by subsequent sucrose gradient centrifugation and analysis of the protein composition of the different gradient fractions by Western blotting. In addition, we also provide a detailed protocol for the visualization of the coclustering of Fas/CD95 death receptor and lipid rafts, as assessed by using anti-Fas/CD95 antibodies and fluorescent dye-conjugated cholera toxin B subunit that binds to ganglioside GM1, a main component of lipid rafts, by immunofluorescence and confocal microscopy. These protocols can be extended to any protein of interest to be analyzed for its association to lipid rafts.

Isolation and Analysis of Lipid Rafts from Neural Cells and Tissues.

Lipid rafts are membrane areas characterized by the clustering of selected membrane lipids, as the result of their phase separation forming a liquid-ordered phase floating in the lipid-disordered bulk membrane. van Meer and Simons hypothesized the existence of lipid rafts to explain the differential composition of the apical and basolateral domains of polarized epithelial cells and proposed that association of given proteins with lipid rafts along the traffic route might represent an important mechanism for protein sorting. However, great attention was paid to the lipid raft theory after Simons and Ikonen highlighted the enrichment of several proteins involved in signal transduction in "detergent-insoluble, glycolipid-enriched complexes," and postulated that lipid rafts might serve as hubs in regulating intracellular signaling. Most notably, the feature of detergent-insolubility was incorporated in the definition of lipid rafts used in 1997 by these authors. "Lipid rafts" and "detergent-resistant membranes" became almost synonymous after the publication, in 1992, of the seminal paper by Brown and Rose, describing the separation of a low-density, Triton X-100-insoluble fraction from epithelial cells, enriched in GSL and apical GPI-anchored proteins and depleted of basolateral membrane marker proteins. This paper provided a working definition of lipid rafts and a putative biochemical method for their separation. More than 2000 papers have been published using "the Triton method." Evidences obtained by the use of alternative biochemical methods for the isolation of lipid rafts and of methods enabling to analyze the dynamics of lipid rafts in intact cells highlighted the several limitations of the Triton X-100 method. On the other hand, the main findings obtained by this method have not been confuted, and the method is still widely used.In this chapter, we will discuss the most relevant methodological aspects related to the preparation of detergent-resistant membrane fractions, with a special focus on neural cells and tissues.

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis.

Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and animal cells and comprehensively analyse their lipid composition.

PKCε-dependent H-Ras activation encompasses the recruitment of the RasGEF SOS1 and of the RasGAP neurofibromin in the lipid rafts of embryonic neurons

The spatial organization of plasma membrane proteins is a key factor in the generation of distinct signal outputs, especially for PKC/Ras/ERK signalling. Regulation of activation of the membrane-bound Ras, critical for neuronal differentiation and highly specialized functions, is controlled by exchanges in nucleotides catalyzed by nucleotide exchange factors (GEFs) for GTP loading and Ras activation, and by Ras GTPase Activated Proteins (RasGAPs) that lead to activation of the intrinsic GTPase activity of Ras and thus its inactivation. PKCs are potent Ras activators yet the mechanistic details of these interactions, or the involvement of specific PKC isoforms are now beginning to be addressed. Even less known is the topology where RasGAPs terminate Ras activation. Towards this aim, we isolated lipid rafts from chick embryo neural tissue and primary neuronal cultures when PKCε is the prominent isoform and in combination with in vitro kinase assays, we now show that, in response the PKCε-specific activating peptide ψεRACK, an activated PKCε is recruited to lipid rafts; similar mobility was established when PKCε was physiologically activated with the Cannabinoid receptor 1 (CB1) agonist methanandamide. Activation of H-Ras for both agents was then established for the first time using in vivo RasGAP activity assays, which showed similar temporal profiles of activation and lateral mobility. Moreover, we found that the GEF SOS1, and the major neuronal RasGAP neurofibromin, a specific PKCε substrate, were both transiently significantly enriched in the rafts. Finally, our in silico analysis revealed a highly probable, conserved palmitoylation site adjacent to a CARC motif on neurofibromin, both of which are included only in the RasGAP related domain type I (GRDI) with the known high H-RasGAP activity. Taken together, these results suggest that PKCε activation regulates the spatial plasma membrane enrichments of both SOS1 and neurofibromin, thus controlling the output of activated H-Ras available for downstream signalling in neurons.

RETRACTED ARTICLE: Receptor tyrosine kinase inhibitor Sunitinib and integrin antagonist peptide HM-3 show similar lipid raft dependent biphasic regulation of tumor angiogenesis and metastasis

BackgroundAnti-angiogenesis remains an attractive strategy for cancer therapy. Some anti-angiogenic reagents have bell-shape dose-response curves with higher than the effective doses yielding lower anti-angiogenic effects. In this study, two different types of anti-angiogenic reagents, a receptor tyrosine kinase inhibitor Sunitinib and an integrin antagonist peptide HM-3, were selected and their effects on tumor angiogenesis and metastasis were compared. The involved molecular mechanisms were investigated.MethodsThe effect of high dose Sunitinib and HM-3 on tumor angiogenesis and metastasis was investigated with two animal models: metastasis of B16F10 cells in syngeneic mice and metastasis of human MDA-MB-231 cells in nude mice. Furthermore, mechanistic studies were performed with cell migration and invasion assays and with biochemical pull-down assays of intracellular RhoGTPases. Distribution of integrin αvβ3, α5β1, VEGFR2 and the complex of integrin αvβ3 and VEGFR2 inside or outside of lipid rafts was detected with lipid raft isolation and Western-blot analysis.ResultsBoth Sunitinib and HM-3 showed a bell-shape dose-response curve on tumor angiogenesis and metastasis in both animal models. The effects of Sunitinib and HM-3 on endothelial cell and tumor cell proliferation and migration were characterized. Activation of intracellular RhoGTPases and actin stress fiber formation in endothelial and cancer cells following Sunitinib and HM-3 treatment correlated with cell migration analysis. Mechanistic studies confirmed that HM-3 and Sunitinib regulated distribution of integrin αvβ3, α5β1, VEGFR2 and αvβ3-VEGFR2 complexes, both inside and outside of the lipid raft regions to regulate endothelial cell migration and intracellular RhoGTPase activities.ConclusionsThese data confirmed that a general non-linear dose-effect relationship for these anti-angiogenic drugs exists and their mechanisms are correlative. It also suggests that the effective dose of an anti-angiogenic drug may have to be strictly defined to achieve its optimal clinical effects.

Shenmai injection maintains blood-brain barrier integrity following focal cerebral ischemia via modulating the expression and trafficking of occludin in lipid rafts

Ethnopharmacological relevanceShenmai injection (SMI), a traditional Chinese herbal medicine is widely used for the clinical treatment of cerebral infarction in China.Aim of the study: Tight junctions (TJs) are major components of the blood-brain barrier (BBB) that physically restrict the paracellular diffusion of blood-borne substances between endothelial cells into the CNS. TJ proteins are associated with cholesterol-enriched regions of plasma membrane known as lipid rafts, which are critical for the trafficking, positioning and function of TJ proteins. In this study, we investigated the effect of SMI on the expression and trafficking of the key TJ-associated protein, occludin, in lipid rafts. Materials and methodsUsing a neutral pH, rat cerebral microvessels were subjected to detergent-free density-gradient fractionation to isolate lipid rafts containing occludin. Transmission electron microscopy (TEM) was performed to study the effects of drug administration on ultrastructural changes to TJs. Western blotting (WB), immunofluorescence (IF), and co-immunoprecipitation (COIP) were used to observe the localization and function of TJ-associated proteins. ResultsWe successfully isolated cerebral microvessels and separated lipid rafts from plasma membranes. With SMI treatment, extravasation of FITC-albumin decreased around the cerebral vessels by IF, the tight junctions were found to still be intact and the basement membrane appeared to be of uniform thickness in TEM. Compared with the untreated group, the co-expression of flotillin-1 and occludin in microvascular endothelial cells was increased and distributed continuously in SMI treatment as shown in double label IF. SMI significantly increased the translocation of occludin to lipid raft fractions by WB and COIP. ConclusionsSMI helps maintain the proper assembly of the TJ multiprotein complex in lipid rafts, thereby helping to preserve BBB functional integrity during focal cerebral ischemic insult. Our findings enhance our understanding of the mechanisms underlying the neuroprotective effect of SMI in cerebral ischemia.

Abstract 3547: Properties of lipid rafts in two epigenetically distinct subtypes of the oncogenic cell-line SW13

Abstract The epithelial SW13 cancer cell line has two naturally occurring well-defined cell subtypes. The SW13(Vim-) has a phenotype that resembles typical epithelial tumors, displaying increased growth and proliferation with a low level of invasiveness. The SW13(Vim+) subtype has a mesenchymal-like phenotype, commonly associated with more invasive and metastatic states of epithelial cancers. SW13(Vim-) can be converted to a pseudo SW13(Vim+) phenotype when treated with histone deacetylase inhibitors (HDACi). This model is used to study the epigenetic phenotypic differences between tumor-like cancer cells and cells that display a more metastatic phenotype. Gene chip data obtained from the SW13 treated and untreated cells displays differential expression in many genes, specifically genes for cellular membrane proteins and genes involved in cholesterol, sphingolipid, and GPI-anchored protein metabolism; all of which are essential components of lipid rafts. Additionally, recent improved outcomes noted in patients using statin drugs further implicates that cholesterol plays a role in cancer progression, although the mechanism for this improvement is not well understood. This suggests lipid rafts are a candidate for a physical structure that manifests the differential effect of cholesterol perturbations in cancer phenotypes. We hypothesize that lipid raft protein composition is distinctly different between the two SW13 cell subtypes, and that the differences in the composition of lipid rafts may play a role in the unique oncogenic phenotypes of the two subtypes. Utilizing low-temperature detergent-resistant lipid raft isolation techniques, western blotting, and mass spectrometry we investigate protein composition differences between the two SW13 cell subtypes. Cholesterol assays are applied to assess the distribution and total cholesterol content of each SW13 subtype, this also provides translational confirmation of the gene chip data obtained from the subtypes. Cholesterol depletion techniques utilizing cyclodextrin for acute disruption and simvastatin for chronic disruption are being implemented to perturb lipid rafts and observe their role in the oncogenic phenotypes of the SW13 cell subtypes. Specifically, metastatic qualities are measured after lipid raft and cholesterol perturbations. Initial findings support differential cholesterol representation in the two SW13 cell subtypes. Potential findings may explain a mechanism in which cholesterol perturbations are capable of decreasing metastasis and improving the outcome of patients with epithelial cancers. Citation Format: Luis Espejo, Kathryn J. Leyva, Elizabeth E. Hull. Properties of lipid rafts in two epigenetically distinct subtypes of the oncogenic cell-line SW13 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3547.

Aggregation of lipid rafts activates c-met and c-Src in non-small cell lung cancer cells

BackgroundActivation of c-Met, a receptor tyrosine kinase, induces radiation therapy resistance in non-small cell lung cancer (NSCLC). The activated residual of c-Met is located in lipid rafts (Duhon et al. Mol Carcinog 49:739-49, 2010). Therefore, we hypothesized that disturbing the integrity of lipid rafts would restrain the activation of the c-Met protein and reverse radiation resistance in NSCLC. In this study, a series of experiments was performed to test this hypothesis.MethodsNSCLC A549 and H1993 cells were incubated with methyl-β-cyclodextrin (MβCD), a lipid raft inhibitor, at different concentrations for 1 h before the cells were X-ray irradiated. The following methods were used: clonogenic (colony-forming) survival assays, flow cytometry (for cell cycle and apoptosis analyses), immunofluorescence microscopy (to show the distribution of proteins in lipid rafts), Western blotting, and biochemical lipid raft isolation (purifying lipid rafts to show the distribution of proteins in lipid rafts).ResultsOur results showed that X-ray irradiation induced the aggregation of lipid rafts in A549 cells, activated c-Met and c-Src, and induced c-Met and c-Src clustering to lipid rafts. More importantly, MβCD suppressed the proliferation of A549 and H1993 cells, and the combination of MβCD and radiation resulted in additive increases in A549 and H1993 cell apoptosis. Destroying the integrity of lipid rafts inhibited the aggregation of c-Met and c-Src to lipid rafts and reduced the expression of phosphorylated c-Met and phosphorylated c-Src in lipid rafts.ConclusionsX-ray irradiation induced the aggregation of lipid rafts and the clustering of c-Met and c-Src to lipid rafts through both lipid raft-dependent and lipid raft-independent mechanisms. The lipid raft-dependent activation of c-Met and its downstream pathways played an important role in the development of radiation resistance in NSCLC cells mediated by c-Met. Further studies are still required to explore the molecular mechanisms of the activation of c-Met and c-Src in lipid rafts induced by radiation.

A New Model for Understanding the Egg Cell Surface at Fertilization

The cell signaling pathway initiating fertilization has not been fully elucidated in any species. Several downstream components in the pathway leading to an internal Ca2+ release have been found; however, not much is known regarding the participation of proteins on the surface of the sperm and egg. The starfish Patiria miniata is an excellent model organism to study this process because they produce a significant quantity of large synchronized oocytes that are nearly transparent and rigid enough to hold their spherical shape despite the removal of extracellular layers. Recently, lipid rafts have been found on the surface of a variety of eggs, but their role at fertilization is unknown. Lipid rafts are small dynamic regions enriched with cholesterol and sphingolipids, which hold clusters of membrane bound proteins. Lipid rafts are found in a variety of cells and they are critical in certain signaling processes, such as the activation of T cells. This study develops the starfish egg as a model system for the study of lipid rafts and for the discovery of cell surface proteins necessary for fertilization. To visualize egg surface proteins in living cells, oocytes were labeled with a membrane impermeant form of biotin and imaged during fertilization by laser scanning confocal microscopy. Evidence of an increase in protein at the fertilization cone, which marks the site of sperm‐egg fusion, was seen. To further understand how these surface proteins were behaving at fertilization, samples prepared from the biotinylated eggs were subjected to gel electrophoresis followed by affinity interaction with streptavidin‐HRP to visualize the labeled proteins. This showed the presence of multiple egg surface proteins, ranging in size from 75 to 250 kDa in great quantity, as well as a few proteins ranging from 30 to 75kDa. Interestingly, biotinylated proteins were also recovered from the surrounding seawater following fertilization, suggesting the action of a protease that cleaves egg cell surface proteins. Experiments with protease inhibitors suggest that this protease activity is necessary for fertilization, leading to the idea that the protease may be involved in sperm‐egg fusion and/or signaling. To examine the idea that signaling may be initiated at the moment of sperm‐egg interaction, and to determine if lipid rafts could be involved, a detergent‐based lipid raft isolation assay was developed for the starfish egg. The eggs are separated into cytosolic, soluble membrane, and insoluble membrane (lipid rafts) fractions. The fractions were subjected to western blotting for tyrosine‐phosphorylated proteins, which were found almost exclusively in the insoluble fraction of the unfertilized egg. Therefore, the lipid rafts contain as of yet unidentified signaling molecules that could play a role in the initiation of fertilization. Understanding this mechanism at the molecular level in starfish can to eventually lead to understanding infertility issues as well as offer new possible non‐hormonal prophylactics in humans.Support or Funding InformationThis research was funded by the NSF and Florida Institute of TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.