Lipid Raft-associated Protein Research Articles

Radix Rehmanniae Praeparata extracts ameliorate hepatic ischemia-reperfusion injury by restoring lipid metabolism in hepatocytes

Ethnopharmacological relevanceHepatic ischemia/reperfusion injury (HIRI) is a common occurrence during or after liver surgery, representing a major cause for postoperative complications or increased morbidity and mortality in liver diseases. Rehmanniae Radix Praeparata (RRP) is a traditional Chinese medicine frequently used and has garnered extensive attention for its therapeutic potential treating cardiovascular and hepatic ailments. Recent studies have indicated the possibility of RRP in regulating lipid accumulation and apoptosis in hepatocytes. Aim of the studyThis study aimed to investigate the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of lipid metabolism. Materials and methodsHigh-performance liquid chromatography (HPLC) was used to identify the major components of RRP water extract. C57BL/6J mice were orally given RRP at doses of 2.5 g/kg, 5 g/kg, and 10 g/kg for a duration of 7 days before undergoing HIRI surgery. Furthermore, we established a lipid-loaded in vitro model by exposing hepatocytes to oleic acid and palmitic acid (OAPA). The anti-HIRI effect of RRP was determined through transcriptomics and various molecular biology experiments. ResultsAfter identifying active ingredients in RRP, we observed that RRP exerted lipid-lowering and hepatoprotective effects on HIRI mice and OAPA-treated hepatocytes. RRP activated AMP-activated protein kinase (AMPK) and inhibited mammalian target of rapamycin (mTOR), which further on the one hand, inhibited the cleavage and activation of sterol regulatory element binding protein 2 (SREBP2) by limiting the movement of SREBPs cleavage-activating protein (SCAP)-SREBP2 complex with the help of endoplasmic reticulum lipid raft-associated protein 1 (ERLIN1) and insulin-induced gene 1 (INSIG1), and on the other hand, promoted liver X receptor α (LXRα) nuclear transportation and subsequent cholesterol efflux. Meanwhile, the anti-lipotoxic effect of RRP can be partly reversed by an LXRα inhibitor but largely blocked by the application of compound C, an AMPK inhibitor. ConclusionOur study elucidated that RRP served as a potential AMPK activator to alleviate HIRI by blocking SREBP2 activation and cholesterol synthesis, while also activating LXRα to facilitate cholesterol efflux. These findings shed new light on the potential therapeutic use of RRP for improving HIRI.

In vivo characterization of a podocyte-expressed short podocin isoform

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes’ foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the β-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the β-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.

Flotillin-1 promotes EMT of gastric cancer via stabilizing Snail.

Gastric cancer is one of the most common malignancies worldwide and has been identified as the third leading cause of cancer-related mortality. Flotillin-1 is a lipid raft-associated scaffolding protein and plays an important role in the progression and development of several malignant carcinomas. Flotillin-1 is involved in epithelial-mesenchymal transition (EMT) process of several solid tumors to promote metastasis. However, the detailed characteristics and mechanisms of Flotillin-1 in gastric cancer have rarely been investigated. In this study, we found Flotillin-1 upregulated in gastric cancer, and the high expression of Flotillin-1 correlated with a worse prognosis. The migration and invasion ability of gastric cancer cells was upregulated by overexpressing Flotillin-1. Knockdown of Flotillin-1 inhibits gastric cancer cells metastasis. Flotillin-1 is a key regulator of EMT process and promotes gastric cancer cells metastasis through inducing EMT. Flotillin-1 may interact with a deubiquitinase to inhibit the ubiquitination of Snail in gastric cancer cells to promote EMT process. Our study provides a rationale and potential target for the treatment of gastric cancer.

Molecular characterization and function of the lipid raft protein Lvflotillin-1A from Litopenaeus vannamei

White spot syndrome virus (WSSV) can cause a contagious, high virulent and pandemic disease for crustaceans, especially shrimps. However, the molecular mechanism of WSSV pathogenesis remains unclear. Flotillins are lipid raft-associated proteins, which mainly include flotillin-1 and flotillin-2. They are involved in the formation of large heteromeric protein complexes engaged in diverse signalling pathways at the membrane-cytosol interface. They defined a clathrin-independent endocytic pathway in mammalian cells. Our previous studies suggested that shrimp flotillin-2 might mediate endocytosis involved in WSSV infection. To further explore the function of shrimp flotillin, a flotillin-1 homologous, Lvflotillin-1A was identified and characterized in Litopenaeus vanamei. The transcription of Lvflotillin-1A showed a significant decline at 12h post-infection, followed by complete recovery and a slight up-regulation after the WSSV challenge. Gene silencing revealed that inhibition of Lvflotillin-1A raised the virus infection, suggesting Lvflotillin-1A might play an important role in shrimp immunity. Furthermore, co-immunoprecipitation and immunofluorescence illustrated that Lvflotillin-1A and Lvflotillin-2 could form hetero-oligomers, and co-expression promoted the accumulation of intracellular vesicles. The study revealed that WSSV might up-regulate Lvflotillin-2 expression and alter the subcellular location of Lvflotillin-1 protein to facilitate virus infection. These results will provide information for understanding the interaction between WSSV and shrimp.

The differential role of the lipid raft-associated protein flotillin 2 for progression of myeloid leukemia

AbstractLipid raft-associated proteins play a vital role in membrane-mediated processes. The lipid microdomain-associated protein flotillin 2 (FLOT2), which has a scaffolding function, is involved in polarization, as well as in actin cytoskeletal organization of primitive and mature hematopoietic cells and has been associated with different malignancies. However, its involvement in myeloid leukemias is not well studied. Using murine transplantation models, we show here that the absence of FLOT2 from leukemia-initiating cells (LICs) altered the disease course of BCR-ABL1+ chronic myeloid leukemia (CML), but not of MLL–AF9-driven acute myeloid leukemia (AML). While FLOT2 was required for expression of the adhesion molecule CD44 on both CML- and AML-LIC, a defect in the cytoskeleton, cell polarity, and impaired homing ability of LIC was only observed in FLOT2-deficient BCR-ABL1+ compared with MLL-AF9+ cells. Downstream of CD44, BCR-ABL1 kinase-independent discrepancies were observed regarding expression, localization, and activity of cell division control protein 42 homolog (CDC42) between wild-type (WT) and FLOT2-deficient human CML and AML cells. Inhibition of CDC42 by ML141 impaired the homing of CML LIC and, thereby, CML progression. This suggested that alteration of both CD44 and CDC42 may be causative of impaired CML progression in the absence of FLOT2. In summary, our data suggest a FLOT2-CD44-CDC42 axis, which differentially regulates CML vs AML progression, with deficiency of FLOT2 impairing the development of CML.

APP deficiency and HTRA2 modulates PrPc proteostasis in human cancer cells

Cellular protein homeostasis (proteostasis) requires an accurate balance between protein biosynthesis, folding, and degradation, and its instability is causally related to human diseases and cancers. Here, we created numerous engineered cancer cell lines targeting APP (amyloid ß precursor protein) and/or PRNP (cellular prion) genes and we showed that APP knocking-down impaired PRNP mRNA level and vice versa, suggesting a link between their gene regulation. PRNPKD, APPKD and PRNPKD/APPKD HeLa cells encountered major difficulties to grow in a 3D tissue-like environment. Unexpectedly, we found a cytoplasmic accumulation of the PrPc protein without PRNP gene up regulation, in both APPKD and APPKO HeLa cells. Interestingly, APP and/or PRNP gene ablation enhanced the chaperone/serine protease HTRA2 gene expression, which is a protein processing quality factor involved in Alzheimer's disease. Importantly, HTRA2 gene silencing decreased PRNP mRNA level and lowered PrPc protein amounts, and conversely, HTRA2 overexpression increased PRNP gene regulation and enhanced membrane-anchored and cytoplasmic PrPc fractions. PrPc, APP and HTRA2 destabilized membrane-associated CD24 protein, suggesting changes in the lipid raft structure. Our data show for the first time that APP and the dual chaperone/serine protease HTRA2 protein could modulate PrPc proteostasis hampering cancer cell behavior.

Novel monoallelic variant in ERLIN2 causes spastic paraplegia converted to amyotrophic lateral sclerosis

Hereditary spastic paraplegias (HSPs) are a group of inherited neurologic disorders characterized by corticospinal tract and dorsal column degeneration. Endoplasmic reticulum lipid raft-associated protein 2 (ERLIN2) has been identified as a causative gene of the autosomal recessive HSP family [1,2]. However, only one report has shown that ERLIN2 also causes amyotrophic lateral sclerosis (ALS) [3]; thus, further investigations are needed for its confirmation. Herein we report a family of HSP converted to ALS caused by a novel monoallelic variant in ERLIN2.

Role of ERLINs in the Control of Cell Fate through Lipid Rafts.

ER lipid raft-associated protein 1 (ERLIN1) and 2 (ERLIN2) are 40 kDa transmembrane glycoproteins belonging to the family of prohibitins, containing a PHB domain. They are generally localized in the endoplasmic reticulum (ER), where ERLIN1 forms a heteroligomeric complex with its closely related ERLIN2. Well-defined functions of ERLINS are promotion of ER-associated protein degradation, mediation of inositol 1,4,5-trisphosphate (IP3) receptors, processing and regulation of lipid metabolism. Until now, ERLINs have been exclusively considered protein markers of ER lipid raft-like microdomains. However, under pathophysiological conditions, they have been described within mitochondria-associated endoplasmic reticulum membranes (MAMs), tethering sites between ER and mitochondria, characterized by the presence of specialized raft-like subdomains enriched in cholesterol and gangliosides, which play a key role in the membrane scrambling and function. In this context, it is emerging that ER lipid raft-like microdomains proteins, i.e., ERLINs, may drive mitochondria-ER crosstalk under both physiological and pathological conditions by association with MAMs, regulating the two main processes underlined, survival and death. In this review, we describe the role of ERLINs in determining cell fate by controlling the “interchange” between apoptosis and autophagy pathways, considering that their alteration has a significant impact on the pathogenesis of several human diseases.

Flotillin-1 Interacts With and Sustains the Surface Levels of TRPV2 Channel.

Transient receptor potential vanilloid subtype 2 (TRPV2) channel is a polymodal receptor regulating neuronal development, cardiac function, immunity and oncogenesis. The activity of TRPV2 is regulated by the molecular interactions in the subplasmalemmel signaling complex. Here by yeast two-hybrid screening of a cDNA library of mouse dorsal root ganglia (DRG) and patch clamp electrophysiology, we identified that flotillin-1, the lipid raft-associated protein, interacts with TRPV2 channel and regulates its function. The interaction between TRPV2 and flotillin-1 was validated through co-immuoprecipitation in situ using endogenous DRG neurons and the recombinant expression model in HEK 293T cells. Fluorescent imaging and bimolecular fluorescence complementation (BiFC) further revealed that flotillin-1 and TRPV2 formed a functional complex on the cell membrane. The presence of flotillin-1 enhanced the whole-cell current density of TRPV2 via increasing its surface expression levels. Using site-specific mapping, we also uncovered that the SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain of flotillin-1 interacted with TRPV2 N-termini and transmembrane domains 1–4, respectively. Our findings therefore demonstrate that flotillin-1 is a key element in TRPV2 signaling complex and modulates its cellular response.

E-cig vapor condensate alters proteome and lipid profiles of membrane rafts: impact on inflammatory responses in A549 cells.

Electronic cigarettes (e-cigs) are battery-operated heating devices that aerosolize e-liquid, typically containing nicotine and several other chemicals, which is then inhaled by a user. Over the past decade, e-cigs have gained immense popularity among both smokers and non-smokers. One reason for this is that they are advertised as a safe alternative to conventional cigarettes. However, the recent reports of e-cig use associated lung injury have ignited a considerable debate about the relative harm and benefits of e-cigs. The number of reports about e-cig-induced inflammation and pulmonary health is increasing as researchers seek to better understand the effects of vaping on human health. In line with this, we investigated the molecular events responsible for the e-cig vapor condensate (ECVC)-mediated inflammation in human lung adenocarcinoma type II epithelial cells (A549). In an attempt to limit the variables caused by longer ingredient lists of flavored e-cigs, tobacco-flavored ECVC (TF-ECVC±nicotine) was employed for this study. Interestingly, we observed significant upregulation of cytokines and chemokines (IL-6, IL-8, and MCP-1) in A549 cells following a 48 h TF-ECVC challenge. Furthermore, there was a significant increase in the expression of pattern recognition receptors TLR-4 and NOD-1, lipid raft-associated protein caveolin-1, and transcription factor NF-кB in TF-ECVC with and/or without nicotine-challenged lung epithelial cells. Our results further demonstrate the harboring of TLR-4 and NOD-1 in the caveolae of TF-ECVC-challenged A549 cells. Proteomic and lipidomic analyses of lipid raft fractions from control and challenged cells revealed a distinct protein and lipid profile in TF-ECVC (w/wo nicotine)-exposed A549 cells. Interestingly, the inflammatory effects of TF-ECVC (w/wo nicotine) were inhibited following the caveolin-1 knockdown, thus demonstrating a critical role of caveolae raft-mediated signaling in eliciting inflammatory responses upon TF-ECVC challenge. Graphical Abstract Graphical Abstract.

Molecular and Immune Characteristics for Lung Adenocarcinoma Patients With ERLIN2 Overexpression.

BackgroundEndoplasmic reticulum lipid raft-associated protein 2 (ERLIN2) is protein contained in the membrane of the endoplasmic reticulum. In lung adenocarcinoma (LUAD), the molecular function of ERLIN2 and the correlation between ERLIN2 and tumor-infiltrating immune cells have been unclear. The aim of our study was to determine the role of ERLIN2 in LUAD development to provide a better understanding of the molecular pathogenesis of this disease and identify new therapeutic targets for its treatment.MethodsImmunohistochemistry, Western blotting, and real-time quantitative polymerase chain reaction were used to detect protein and mRNA levels of ERLIN2 in LUAD and adjacent normal tissues. Using the A549, H1299 cell line, ERLIN2-short hairpin RNA was applied to silence ERLIN2 to determine its role in LUAD cell proliferation and invasion. Based on mRNA expression of ERLIN2 from the Cancer Genome Atlas (TCGA) database, we identified ERLIN2-related protein-coding genes and analyzed the Kyoto Encyclopedia of Genes and Genomes pathway to explore its potential biological functions and determined the correlation between ERLIN2 and tumor-infiltrating immune cells.ResultsERLIN2 was abnormally expressed in a variety of tumor tissues and is highly expressed in LUAD. This overexpression was associated with histological grade (P = 0.044), TNM stage (P = 0.01), and lymph node metastasis (P = 0.038). Patient overall survival was poorer with ERLIN2 overexpression. Downregulation of ERLIN2 inhibited LUAD cell proliferation and invasion in vitro. Based on mRNA expression of ERLIN2 from the TCGA database, 13 ERLIN2-related genes and 10 pathways were identified and showed a correlation between ERLIN2 and naive B cells and neutrophils.ConclusionERLIN2 could serve as a potential diagnostic and prognostic biomarker for LUAD and has demonstrated to be correlated with immune infiltrates, which suggests that it may represent a new therapeutic target for LUAD.

Hereditary spastic paraplegia associated with a rare endoplasmic reticulum lipid raft-associated protein 2 mutation

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurological disorders that are characterized by progressive spasticity of the lower extremities. It can present as pure form or complex form. It can be present from infancy to adulthood, but majority in adult population. Childhood onset HSP must be differentiated from common conditions like cerebral palsy, neurodegenerative disorders and metabolic disorders. Many patients with pediatric HSP are mistakenly diagnosed with cerebral palsy. In children with spastic paraplegia in whom no acquired cause identified, HSP should be considered. Here we diagnosed a 6-year-old boy with HSP who presented with progressive spastic paraplegia, intellectual disability, seizures, joint contractures and cataract. His genetic study revealed exonic deletion of endoplasmic reticulum lipid raft-associated protein gene, which is associated with complicated Autosomal recessive HSP 18 (SPG18). HSP 18 was rarely described in literature.

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells.

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.

Abstract 359: Cardiac-specific Deletion of Prohibitin-1 Leads to Fetal Demise, Gross Mitochondrial Abnormalities and Heart Failure in Adolescent Mice

Prohibitins (PHB1, 2) are lipid raft-associated proteins which form a multimeric, alternating ring-like structure in cellular membranes, and are particularly abundant in mitochondria and plasma membranes, but have also been found in nuclei. These pleiotropic proteins are associated with numerous signaling pathways responsible for cell differentiation, growth, and metabolism. In mitochondria, PHBs are thought to act as scaffold-like chaperone proteins responsible for stabilizing enzymes responsible for electron transport and oxidative phosphorylation (OXPHOS), minimizing reactive oxygen species (ROS), and coordinating fission/fusion. However, the extent to which PHB1 regulates mitochondria and electromechanical function of the heart, is not known. We established a cardiac-specific PHB1-deficient mouse strain by crossing floxxed phb1 mice with mice expressing Cre recombinase under control of myosin heavy chain (MHC) promoter. The vast majority of cardiac-specific PHB1-deficient mice (cPHB1ko) died in utero during pregnancy (E12-E15). Uterine sonography of the expired cPHB1ko embryos showed significantly decreased crown-rump length and increased placenta thickness with large amount of fluids, restricted uterine artery flow and detachment between amnion and chorion compared with normal embryos (P < 0.001). The few that were born develop severe dilated cardiomyopathy, resulting in mortality by 8-9 weeks of age. Heart weight/body weight ratio is ~2-fold greater in the KO mice vs. WT. Echocardiography reveals severely enlarged right atria and ventricles in cPHB1ko mice, corresponding to significantly decreased ejection faction (EF), increased end diastolic volume (EDV) and end systolic volume (ESV) compared to wild-type littermates (P < 0.05). In mitochondria, cPHB1ko mice have derangements in OXPHOS as evidenced by decreased pyruvate and palmitoylcarnitine-supported respiration and ATP production. Moreover, both the rate of calcium uptake (P = 0.05) and the total calcium retention capacity (P < 0.01) were diminished in the KO mice. These preliminary findings implicate PHB1 as a critical regulator of myocardial development and function, due in part to its role in maintaining OXPHOS and controlling metabolism.

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome.

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

Palmitoylation of Hepatitis C Virus NS2 Regulates Its Subcellular Localization and NS2-NS3 Autocleavage.

Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious-HCV replication. Blocking NS2 palmitoylation by introducing an NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of the NS2/C113S mutant was restored by inserting an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) between NS2 and NS3 to separate the two proteins independently of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting NS2-NS3 autoprocessing. The NS2/C113S mutation also impaired infectious-HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, i.e., NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine tune both HCV RNA replication and infectious-particle assembly.IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral-genome replication and infectious-virus assembly. However, the underlying mechanism that enables the protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious-particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, representing a new avenue for host-targeting strategies against HCV infection.

Novel Role of Raft-Associated Smoothened (SMO) in AKT Signal Regulation in Diffuse Large B Cell Lymphoma

Constitutive PI3K/AKT activation is relevant to multiple aspects of tumor growth and survival in numerous cancers including diffuse large B cell lymphoma (DLBCL). For example, PTEN loss is one of the mechanisms leading to constitutive PI3K/AKT activation in a subset of DLBCL. Smoothened (SMO) is a seven transmembrane spanning and Frizzled-class G-protein coupled receptor that functions as a Hedgehog (Hh) signal transducer. SMO is overexpressed in DLBCL cell lines and tumors. While canonical Hh signaling culminates in the activation of GLI transcription factors and is best understood in the context of cilia, "noncanonical" Hh signaling does not involve GLI transcriptional activity and remains less well characterized. Here, we found that SMO is not only an integral component of lipid rafts but also plays an unexpected central role in the organization of raft microdomains (specialized glycolipid-enriched microdomains known to serve as a highly dynamic signaling platform for cell surface receptors and signaling proteins) and in the sorting of lipid raft-associated proteins. To address whether SMO co-localizes to lipid rafts in the context of DLBCL, HBL1 cells were engineered to stably overexpress a C-terminal SMO-mCherry-fusion protein and were incubated with FITC-Cholera toxin. Within 15 min, a large fraction of SMO-mCherry was co-localized with FITC-Cholera toxin in lipid raft clusters. We also performed immunostaining of endogenous SMO and CD59 in HBL1 cells. CD59 is a glycol-phosphatidyl inositol-anchored lipid raft protein. Immunostaining of live HBL1 cells revealed the colocalization of SMO and CD59 on the extracellular surface of HBL1 cells. No colocalization was found between SMO and the transferrin receptor (TF-R), a plasma membrane protein not associated with lipid rafts. Finally, immunoblotting analysis of detergent-free fractionation further corroborated SMO as a bona fide component of lipid rafts. We then tried to explore the functional relevance of SMO association to the lipid compartment. First, we examined the effects of SMO stable knockdown in DLBCL. We observed a marked decrease in the expression of raft-associated receptors and signaling proteins (e.g. IGFR1, EGFR, IRS1) while caveolin and flotillin, two functional components of lipid rafts, remained unaltered in their levels and distribution. Although only a portion of the overall pool of AKT and pAKT were localized to lipid rafts, SMO loss significantly reduced raft-localized total AKT and pAKT (T308/S473). Consistent with the well-established role of AKT in cell survival, SMO silencing also resulted in reduced DLBCL cell viability. To evaluate whether SMO regulated IGF1R expression at the transcriptional level, we analyzed the mRNA levels of GLI1, the immediate transcriptional target of canonical SMO signaling, and IGF1R in SMO-/- MEFs. Even if GLI1 transcript levels were reduced, consistent with the established mode of GLI1 regulation by SMO through the GLI2 transcription factor, IGF1R mRNA levels remained unchanged. Using GLI1-/- MEFs, we could confirm that the effect of SMO on surface receptors was independent of canonical Hh signaling. We also determined that the rate of IGFR degradation was comparable in the presence and absence of SMO. Total IGF1R receptor levels at steady state represent the balance of total protein synthesis and the fraction of existing receptors that is directed towards lysosomal degradation. To test whether the absence of SMO results in increased degradation, we inhibited lysosomal function with chloroquine (CHLQ). CHLQ had a far more pronounced impact on restoring IGF1R protein levels in SMO deficient cells than it did in control cells. These last results suggested that in the absence of SMO, a larger fraction of this receptor is directed towards lysosomal degradation, and thus resulting in lower steady state levels. In summary, our data confirm that SMO is localized to raft microdomains in lymphoma cells and play a novel role in the sorting of surface proteins for degradation or recycling. In particular, SMO increases the levels of raft resident receptors and facilitates the assembly of an AKT activating machinery to enhance lymphoma cell survival. This novel role of SMO in signal regulation at the level of lipid rafts has broad implications for cancer biology. Disclosures Vega: National Cancer Institute, national Institutes of Health: Other: Grant Funding-R01CA222918.

The Role of the Lipid Raft-Associated Protein Flotillin-2 during Development and Progression of Myeloid Leukaemia

Introduction The role of the bone marrow (BM) microenvironment (BMM) for the regulation of leukaemic stem cells (LSC) and their respective interplay is slowly being elucidated, but knowledge about how membrane structures or adhesion molecules on leukaemia cells may, specifically, interact with the BMM leading to regulation of leukemia cell maintenance and proliferation is limited. Flotillin (flot)-1 and -2 are highly conserved, ubiquitously expressed proteins localised in lipid microdomains in cellular membranes. These dynamic microdomains can serve as platforms for signal transduction, endocytosis and interactions with the actin cytoskeleton, as well as cell adhesion. Hypothesis As flotillins in the leukaemia cell membrane are associated with adhesion molecules known to be involved in the engraftment of leukaemia-initiating cells (LIC), we hypothesized that flotillins may play a role in LIC engraftment in the BMM. Results Using the murine retroviral transduction/transplantation model of BCR-ABL1-driven chronic myeloid leukaemia (CML) we observed a significant prolongation of survival of recipients of BCR-ABL1+, flot-2-deficient bone marrow compared to the respective control. The homing of BCR-ABL1+LIC to wild-type recipient bone marrow was reduced. Consistent with the known association of flotillins in the cell membrane with P-selectin glycoprotein ligand-1, a selectin ligand similar to CD44, both of which are known to be involved in the engraftment of CML LIC (Krause DS et al., Nat Med, 2006, and Krause DS et al., Blood, 2014), we demonstrated that flot-2 physically interacts and co-localizes with CD44. Flot-2-deficiency led to a significant reduction of the expression of CD44 and an impairment of the cytoskeleton in BCR-ABL1+ cells, as well as impaired migration. Further, Cdc42, a member of the Rho-GTPase family,was differentially distributed in wildtype versus flotillin-2-deficient BCR-ABL1+ LSC and may have compromised activity. Consistently, intrafemoral transplantation of flot-2-deficient CML-initiating cells or coexpression of CD44 in flot-2-deficient bone marrow ,rescued' or restored the CML-like disease. In contrast, in an MLL-AF9-driven model of acute myeloid leukemia (AML) a similar prolongation of survival or reduced homing of AML-initiating cells were not observed. However, expression of CD44, known to play a role for engraftment in this disease (Jin L et al., Nat Med, 2006), in AML-initiating cells was similarly reduced and migration, adhesion and the cytoskeleton was similarly compromised.We demonstrated that this might be due to compensatory upregulation of C-X-C chemokine receptor type 4 (CXCR-4), the receptor for stromal-derived factor (SDF)-1a. Conclusions In summary, these data suggest that lipid raft molecules, particularly flotillins, play a previously unknown and possibly leukaemia-specific role in CML progression via modification of the levels and function of CD44 and possible regulation of Cdc42. Disclosures Kumar: European Patent No. 16187926.7: Other: USE OF FIBRONECTIN OR ILK INHIBITORS FOR USE IN THE TREATMENT OF LEUKEMIA; Merck: Research Funding. Krause:Merck KGaA: Research Funding; Patent: Patents & Royalties: European Patents No. 16187926.7-1401, EP18184430.9-.