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The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides. [ABSTRACT FROM AUTHOR]

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner. [ABSTRACT FROM AUTHOR]

Kearns-Sayre syndrome (KSS) is a rare multisystem mitochondrial disorder. It is caused by mitochondrial DNA (mtDNA) rearrangements, mostly large-scale deletions of 1.1–10 kb. These deletions primarily affect energy supply through impaired oxidative phosphorylation and reduced ATP production. This impairment gives rise to dysfunction of several tissues, in particular those with high energy demand like brain and muscles. Over the past decades, changes in respiratory chain complexes and energy metabolism have been emphasized, whereas little attention has been paid to other reports on ROS overproduction, protein synthesis inhibition, myelin vacuolation, demyelination, autophagy, apoptosis, and involvement of lipid raft and oligodendrocytes in KSS. Therefore, this paper draws attention towards these relatively underemphasized findings that might further clarify the pathologic cascades following deletions in the mtDNA. [ABSTRACT FROM AUTHOR]

The tumor develops defense tactics, including conversing the mechanical characteristics of tumor cells and their surrounding environment. A recent study reported that cholesterol depletion stiffens tumor cells, which could enhance adaptive T-cell immunotherapy. However, it remains unclear whether reducing the cholesterol in tumor cells contributes to re-educating the stiff tumor matrix, which serves as a physical barrier against drug penetration. Herein, we found that depleting cholesterol from tumor cells can demolish the intratumor physical barrier by disrupting the mechanical signal transduction between tumor cells and the extracellular matrix through the destruction of lipid rafts. This disruption allows nanoparticles (H/S@hNP) to penetrate deeply, resulting in improved photodynamic treatment. Our research also indicates that cholesterol depletion can inhibit the epithelial-mesenchymal transition and repolarize tumor-associated macrophages from M2 to M1, demonstrating the essential role of cholesterol in tumor progression. Overall, this study reveals that a cholesterol-depleted, softened tumor matrix reduces the difficulty of drug penetration, leading to enhanced antitumor therapeutics. [Display omitted] • H/S@hNP could soften the stiff tumor matrix via cholesterol-depletion strategy. • H/S@hNP mediated cholesterol depletion enhanced nanoparticle penetration and photodynamic therapy antitumor efficacy. • H/S@hNP could hamper epithelial-mesenchymal transition and switch tumor-associated macrophages from M2 to M1. [ABSTRACT FROM AUTHOR]

Background: Lipids are a key nutrient source for the growth and reproduction of Mycobacterium tuberculosis (Mtb). Urine-derived extracellular vesicles (EVs), because of its non-invasive sampling, lipid enrichment, and specific sorting character, have been recognized as a promising research target for biomarker discovery and pathogenesis elucidation in tuberculosis (TB). We aim to profile lipidome of Mtb-infected individuals, offer novel lipid signatures for the development of urine-based TB testing, and provide new insights into the lipid metabolism after Mtb infection. Methods: Urine-derived extracellular vesicles from 41 participants (including healthy, pulmonary tuberculosis, latent tuberculosis patients, and other lung disease groups) were isolated and individually detected using targeted lipidomics and proteomics technology platforms. Biomarkers were screened by multivariate and univariate statistical analysis and evaluated by SPSS software. Correlation analyses were performed on lipids and proteins using the R Hmisc package. Results: Overall, we identified 226 lipids belonging to 14 classes. Of these, 7 potential lipid biomarkers for TB and 6 for latent TB infection (LTBI) were identified, all of which were classified into diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acid (FFA), and cholesteryl ester (CE). Among them, FFA (20:1) was the most promising biomarker target in diagnosing TB/LTBI from other compared groups and also have great diagnostic performance in distinguishing TB from LTBI with AUC of 0.952. In addition, enhanced lipolysis happened as early as individuals got latent Mtb infection, and ratio of raft lipids was gradually elevated along TB progression. Conclusion: This study demonstrated individualized lipid profile of urinary EVs in patients with Mtb infection, revealed novel potential lipid biomarkers for TB/LTBI diagnosis, and explored mechanisms by which EV lipid raft-dependent bio-processes might affect pathogenesis. It lays a solid foundation for the subsequent diagnosis and therapeutic intervention of TB. [ABSTRACT FROM AUTHOR]

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors. [ABSTRACT FROM AUTHOR]

Simple Summary: E. coli harms over 100 million patients worldwide annually. Its toxin component is lipopolysaccharide, which has various forms, referred to as smooth or rough, based on their structure. Macrophages' main function is to eat up foreign organisms through phagocytosis. This process requires membrane fluidity, which means the phospholipid in the macrophage plasma membrane needs to be regularly remodelled. A lipid-modifying enzyme known as LPCAT2 performs phospholipid remodelling and participates in inflammation. Here, we studied how this enzyme influences the gene expression of receptors that recognise and bind to lipopolysaccharides. These receptors are TLR4, CD14, and MD2. Our results show that LPCAT2 only influenced these receptors when macrophages were infected with smooth lipopolysaccharides. We conclude that LPCAT2 does not influence the gene expression of the receptors in macrophages infected with rough lipopolysaccharides because they do not require lipid-raft microdomains to mediate inflammation, but smooth lipopolysaccharides do. Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor's cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7—a commonly used experimental murine macrophage model—to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts. [ABSTRACT FROM AUTHOR]

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions. [ABSTRACT FROM AUTHOR]

Amyloid β (Aβ) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aβ disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aβ oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aβ mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aβ enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aβ-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aβ-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 traffickingAmyloid β (Aβ) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aβ disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aβ oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aβ mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aβ enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aβ-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aβ-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aβ, providing new insights into the mechanism underlying AD-associated cognitive dysfunction. induced by Aβ, providing new insights into the mechanism underlying AD-associated cognitive dysfunction. [ABSTRACT FROM AUTHOR]

Receptology, the science of receptors, is a multidimensional field of research which can be dissected into biosynthesis, membrane sorting, ligand binding and signal transduction. Plasma membrane receptors connect the cells with their environment and transmit signals that are translated into biological information. The historical paradigm of ligand–receptor interactions is the lock-and-key model. This model presupposes that both partners have a precise 3D shape that perfectly fits together to form the ligand–receptor complex. However, this simple model suffers from severe limitations due to several levels of simplifications: (i) water molecules and membrane lipids are not considered; (ii) not all ligands have a stable 3D structure; (iii) the ligand-binding pocket of the receptor is often flexible and conformationally rearranged after the initial binding step (induced fit mechanism) and/or subjected to conformational selection by the ligand; (iv) there are signal transduction mechanisms which can be either purely mechanical (conformational change of the receptor induced after binding of the ligand), lipid-assisted (e.g., by raft lipids such as cholesterol or gangliosides), or in some instances of quantic nature (detection of odorant molecules). The aim of the present review is to challenge the old paradigms and present new concepts of membrane receptology that consider the impact of critical parameters such as water molecules, membrane lipids, electrostatic surface potential and quantum mechanisms. [ABSTRACT FROM AUTHOR]

Cholesterol is an essential component of cell membranes and serves as an important precursor of steroidal hormones and bile acids, but elevated levels of cholesterol and its oxidation products have been accepted as a risk factor for maintenance of health. The free and ester forms of cholesterol and fatty acids are the two major biological lipids. The aim of this hypothesis paper is to address the long-standing dogma that cholesterol is less susceptible to free radical peroxidation than polyunsaturated fatty acids (PUFAs). It has been observed that cholesterol is peroxidized much slower than PUFAs in plasma but that, contrary to expectations from chemical reactivity toward peroxyl radicals, cholesterol appears to be more readily autoxidized than linoleates in cell membranes. The levels of oxidation products of cholesterol and linoleates observed in humans support this notion. It is speculated that this discrepancy is ascribed to the fact that cholesterol and phospholipids bearing PUFAs are localized apart in raft and non-raft domains of cell membranes respectively and that the antioxidant vitamin E distributed predominantly in the non-raft domains cannot suppress the oxidation of cholesterol lying in raft domains which are relatively deficient in antioxidant. [Display omitted] • Cholesterol and phospholipid linoleates, the two major lipids, are oxidized by enzymatic and non-enzymatic mechanisms. • Cholesterol hydroperoxides are produced exclusively by free radical and singlet oxygen oxidation. • Different from plasma, cholesterol is more readily oxidized than linoleates in cell membranes. • Cholesterol lying in lipid rafts might not be well protected by a-tocopherol compared with PUFAs which lie in non-raft domains. [ABSTRACT FROM AUTHOR]

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes. [ABSTRACT FROM AUTHOR]

Background: Vibrio cholerae N-acetylglucosamine-binding protein (GbpA) is a four-domain, secretory colonization factor which is essential for chitin utilization in the environment, as well as in adherence to intestinal cells. GbpA is also involved in inducing intestinal inflammation by enhancing mucin and interleukin-8 secretion. The underlying cell signaling mechanism involved in the induction of the pro-inflammatory response and IL-8 secretion has yet to be deciphered in detail. Methods: Herein, the process through which GbpA triggers the induction of IL-8 in intestinal cells was investigated by examining the role of GbpA in intestinal cell line HT 29. Results: GbpA, specifically through the fourth domain, forms a binding connection with Toll-like receptor 2 (TLR2) and additionally, recruits TLR1 along with CD14 within a lipid raft micro-domain to initiate the signaling pathway. Notably, disruption of this micro-domain complex resulted in a reduction in IL-8 secretion. The lipid raft association served as the catalyst that invoked a downstream cellular inflammatory signaling pathway. This cascade involved the activation of various MAP kinases and NFκB and assembly of the AP-1 complex. This coordinated activation of signaling molecules eventually leads to enhanced IL-8 transcription via increased promoter activity. These findings suggested that GbpA is a crucial protein in V. cholerae, capable of inciting a pro-inflammatory response during infection by orchestrating the formation of the GbpA-TLR1/2-CD14 lipid raft complex. Activation of AP-1 and NFκB in the nucleus eventually enhanced IL-8 transcription through increased promoter activity. Conclusions: Collectively, these findings indicated that GbpA plays a pivotal role within V. cholerae by triggering a pro-inflammatory response during infection. This response is instrumented by the formation of the GbpA-TLR1/2-CD14 lipid raft complex.

AbstractContext Dihydromyricetin (DMY) is extracted from vine tea, a traditional Chinese herbal medicine with anti-cancer, liver protection, and cholesterol-lowering effects.Objective This study investigated the mechanism of DMY against hepatocellular carcinoma (HCC).Materials and methods Potential DMY, HCC, and cholesterol targets were collected from relevant databases. PPI networks were created by STRING. Then, the hub genes of co-targets, screened using CytoHubba. GO and KEGG pathway enrichment, were performed by Metascape. Based on the above results, a series of in vitro experiments were conducted by using 40–160 μM DMY for 24 h, including transwell migration/invasion assay, western blotting, and Bodipy stain assay.Results Network pharmacology identified 98 common targets and 10 hub genes of DMY, HCC, and cholesterol, and revealed that the anti-HCC effect of DMY may be related to the positive regulation of lipid rafts. Further experiments confirmed that DMY inhibits the proliferation, migration, and invasion of HCC cells and reduces their cholesterol levels in vitro. The IC50 is 894.4, 814.4, 467.8, 1,878.8, 151.8, and 156.9 μM for 97H, Hep3B, Sk-Hep1, SMMC-7721, HepG2, and Huh7 cells, respectively. In addition, DMY downregulates the expression of lipid raft markers (CAV1, FLOT1), as well as EGFR, PI3K, Akt, STAT3, and Erk.Discussion and conclusion The present study reveals that DMY suppresses EGFR and its downstream pathways by reducing cholesterol to disrupt lipid rafts, thereby inhibiting HCC, which provides a promising candidate drug with low toxicity for the treatment of HCC.

The synapse is a piece of information transfer machinery replacing the electrical conduction of nerve impulses at the end of the neuron. Like many biological mechanisms, its functioning is heavily affected by time constraints. The solution selected by evolution is based on chemical communication that, in theory, cannot compete with the speed of nerve conduction. Nevertheless, biochemical and biophysical compensation mechanisms mitigate this intrinsic weakness: (i) through the high concentrations of neurotransmitters inside the synaptic vesicles; (ii) through the concentration of neurotransmitter receptors in lipid rafts, which are signaling platforms; indeed, the presence of raft lipids, such as gangliosides and cholesterol, allows a fine tuning of synaptic receptors by these lipids; (iii) through the negative electrical charges of the gangliosides, which generate an attractive (for cationic neurotransmitters, such as serotonin) or repulsive (for anionic neurotransmitters, such as glutamate) electric field. This electric field controls the flow of glutamate in the tripartite synapse involving pre- and post-synaptic neurons and the astrocyte. Changes in the expression of brain gangliosides can disrupt the functioning of the glutamatergic synapse, causing fatal diseases, such as Rett syndrome. In this review, we propose an in-depth analysis of the role of gangliosides in the glutamatergic synapse, highlighting the primordial and generally overlooked role played by the electric field of synaptic gangliosides.

Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here, we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components, such as GD3 ganglioside and scaffold protein caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of the transition zone (TZ). Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; that is, rapid elongation of primary cilia and redistribution of cilia proteins from TZ to the growing axoneme. Indeed, as cilia elongated, endogenous galectin 8, caveolin 1, and TZ component, TMEM231, also transited from the TZ to the growing axoneme. We also noted that the interaction between caveolin 1 and TMEM231 could be perturbed by exogenous galectin 8. Taken together, we proposed that galectin 8 promoted primary cilia elongation through impeding the barrier function of the TZ by interfering with the interaction between caveolin 1 and TMEM231.The transition zone (TZ) serves as a diffusion barrier for ciliary transport and constitutes a region on the ciliary membrane enriched in both ganglioside and lipid raft scaffold proteins such as caveolin 1 (Cav1). During ciliogenesis, extracellular galectin 8 (Gal8) accumulates at TZ presumably through its interaction with sialylated oligosaccharide such as GD3 and Cav1 to promote primary cilia elongation by perturbing the balanced interaction between Cav1 and TZ component transmembrane protein 231 (TMEM231). [ABSTRACT FROM AUTHOR]

Dihydromyricetin (DMY) is extracted from vine tea, a traditional Chinese herbal medicine with anti-cancer, liver protection, and cholesterol-lowering effects. This study investigated the mechanism of DMY against hepatocellular carcinoma (HCC). Potential DMY, HCC, and cholesterol targets were collected from relevant databases. PPI networks were created by STRING. Then, the hub genes of co-targets, screened using CytoHubba. GO and KEGG pathway enrichment, were performed by Metascape. Based on the above results, a series of in vitro experiments were conducted by using 40–160 μM DMY for 24 h, including transwell migration/invasion assay, western blotting, and Bodipy stain assay. Network pharmacology identified 98 common targets and 10 hub genes of DMY, HCC, and cholesterol, and revealed that the anti-HCC effect of DMY may be related to the positive regulation of lipid rafts. Further experiments confirmed that DMY inhibits the proliferation, migration, and invasion of HCC cells and reduces their cholesterol levels in vitro. The IC50 is 894.4, 814.4, 467.8, 1,878.8, 151.8, and 156.9 μM for 97H, Hep3B, Sk-Hep1, SMMC-7721, HepG2, and Huh7 cells, respectively. In addition, DMY downregulates the expression of lipid raft markers (CAV1, FLOT1), as well as EGFR, PI3K, Akt, STAT3, and Erk. The present study reveals that DMY suppresses EGFR and its downstream pathways by reducing cholesterol to disrupt lipid rafts, thereby inhibiting HCC, which provides a promising candidate drug with low toxicity for the treatment of HCC. [ABSTRACT FROM AUTHOR]

Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1) are nonselective cation channels expressed in primary sensory neurons and several other non-neuronal structures such as immune cells, keratinocytes, and vascular smooth muscle cells. They play important roles in nociception, pain processing and their chanellopathies are associated with the development of several pathological conditions. They are located in cholesterol- and sphingolipid-rich membrane lipid raft regions serving as platforms to modulate their activations. We demonstrated earlier that disruption of these lipid rafts leads to decreased TRP channel activation and exerts analgesic effects. Cyclodextrins are macrocyclic molecules able to form host-guest complexes with cholesterol and deplete it from the membrane lipid rafts. The aim of this study was to investigate 8 structurally different (methylated and non-methylated) CD derivatives on cell viability, mitochondrial membrane potential, membrane composition and activation abilities of the TRPV1 and TRPA1 channels. We showed that non-methylated derivatives have preferable safety profiles compared to methylated ones. Furthermore, methylated derivatives reduced mitochondrial membrane potential. However, all investigated derivatives influence the ordered cell membrane structure depleting membrane cholesterol and inhibit the TRPV1 agonist capsaicin- and the TRPA1 agonist allyl isothiocyanate-induced Ca2+−influx. This mechanism of action might provide novel perspectives for the development of peripherally acting analgesics via indirectly decreasing the generation and transmission of nociceptive signals.

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5-bisphosphate (PIP2) clusters in mammalian cells. First, mechanical force deforms the ordered lipids, which disrupts the interaction of PLD2 with the GM1 lipids and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response.

Gangliosides are functional components of membrane lipid rafts that control critical functions in cell communication. Many pathologies involve raft gangliosides, which therefore represent an approach of choice for developing innovative therapeutic strategies. Beginning with a discussion of what a disease is (and is not), this review lists the major human pathologies that involve gangliosides, which includes cancer, diabetes, and infectious and neurodegenerative diseases. In most cases, the problem is due to a protein whose binding to gangliosides either creates a pathological condition or impairs a physiological function. Then, I draw up an inventory of the different molecular mechanisms of protein‐ganglioside interactions. I propose to classify the ganglioside‐binding domains of proteins into four categories, which I name GBD‐1, GBD‐2, GBD‐3, and GBD‐4. This structural and functional classification could help to rationalize the design of innovative molecules capable of disrupting the binding of selected proteins to gangliosides without generating undesirable effects. The biochemical specificities of gangliosides expressed in the human brain must also be taken into account to improve the reliability of animal models (or any animal‐free alternative) of Alzheimer's and Parkinson's diseases.

In plasma, iron is normally bound to transferrin, the principal protein in blood responsible for binding and transporting iron throughout the body. However, in conditions of iron overload when the iron-binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) appears in plasma. NTBI is taken up by hepatocytes and other parenchymal cells via NTBI transporters and can cause cellular damage by promoting the generation of reactive oxygen species. However, how NTBI affects endothelial cells, the most proximal cell type exposed to circulating NTBI, has not been explored. We modeled in vitro the effects of systemic iron overload on endothelial cells by treating primary human umbilical vein endothelial cells (HUVECs) with NTBI (ferric ammonium citrate [FAC]). We showed by RNA-Seq that iron loading alters lipid homeostasis in HUVECs by inducing sterol regulatory element-binding protein 2-mediated cholesterol biosynthesis. We also determined that FAC increased the susceptibility of HUVECs to apoptosis induced by tumor necrosis factor-α (TNFα). Moreover, we showed that cholesterol biosynthesis contributes to iron-potentiated apoptosis. Treating HUVECs with a cholesterol chelator hydroxypropyl-β-cyclodextrin demonstrated that depletion of cholesterol was sufficient to rescue HUVECs from TNFα-induced apoptosis, even in the presence of FAC. Finally, we showed that FAC or cholesterol treatment modulated the TNFα pathway by inducing novel proteolytic processing of TNFR1 to a short isoform that localizes to lipid rafts. Our study raises the possibility that iron-mediated toxicity in human iron overload disorders is at least in part dependent on alterations in cholesterol metabolism in endothelial cells, increasing their susceptibility to apoptosis.

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor’s cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7—a commonly used experimental murine macrophage model—to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.

Abstract It is unclear whether membrane vitamin D receptor (mVDR) exists on the macrophage membrane or whether mVDR is associated with lipopolysaccharide (LPS) tolerance. Herein, we report that interfering with caveolae and caveolae-dependent lipid rafts inhibited the formation of LPS tolerance. VDR was detected as co-localized with membrane molecular markers. VDR was detected on the cell membrane and its level was higher in LPS-tolerant cells than that in only LPS treatment cells. Anti-VDR antibodies could abolish the effect of artesunate (AS) to reverse LPS tolerance, and the wild-type peptides (H397 and H305) of VDR, but not the mutant peptide (H397D and H305A), led to the loss of AS’s effect. AS decreased the mVDR level in LPS-tolerant cells. In vivo, AS significantly reduced VDR level in the lung tissue of LPS-tolerant mice. In summary, mVDR exists on the cell membrane of macrophages and is closely associated with the formation of LPS tolerance and the effects of AS. Video Abstract

Abstract The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut.

Cisplatin resistance poses a substantial hurdle in effectively treating head and neck squamous cell carcinoma (HNSCC). Utilizing multiple tumor models and examining an internal HNSCC cohort, squalene epoxidase (SQLE) is pinpointed as a key driver of chemoresistance and tumorigenesis, operating through a cholesterol‐dependent pathway. Comprehensive transcriptomic analysis reveals that SQLE is essential for maintaining c‐Myc transcriptional activity by stabilizing the c‐Myc protein and averting its ubiquitin‐mediated degradation. Mechanistic investigation demonstrates that SQLE inhibition diminishes Akt's binding affinity to lipid rafts via a cholesterol‐dependent process, subsequently deactivating lipid raft‐localized Akt, reducing GSK‐3β phosphorylation at S9, and increasing c‐Myc phosphorylation at T58, ultimately leading to c‐Myc destabilization. Importantly, employing an Sqle conditional knockout mouse model, SQLE's critical role in HNSCC initiation and progression is established. The preclinical findings demonstrate the potent synergistic effects of combining terbinafine and cisplatin in arresting tumor growth. These discoveries not only provide novel insights into the underlying mechanisms of SQLE‐mediated cisplatin resistance and tumorigenesis in HNSCC but also propose a promising therapeutic avenue for HNSCC patients unresponsive to conventional cisplatin‐based chemotherapy. [ABSTRACT FROM AUTHOR]

Gangliosides are functional components of membrane lipid rafts that control critical functions in cell communication. Many pathologies involve raft gangliosides, which therefore represent an approach of choice for developing innovative therapeutic strategies. Beginning with a discussion of what a disease is (and is not), this review lists the major human pathologies that involve gangliosides, which includes cancer, diabetes, and infectious and neurodegenerative diseases. In most cases, the problem is due to a protein whose binding to gangliosides either creates a pathological condition or impairs a physiological function. Then, I draw up an inventory of the different molecular mechanisms of protein‐ganglioside interactions. I propose to classify the ganglioside‐binding domains of proteins into four categories, which I name GBD‐1, GBD‐2, GBD‐3, and GBD‐4. This structural and functional classification could help to rationalize the design of innovative molecules capable of disrupting the binding of selected proteins to gangliosides without generating undesirable effects. The biochemical specificities of gangliosides expressed in the human brain must also be taken into account to improve the reliability of animal models (or any animal‐free alternative) of Alzheimer's and Parkinson's diseases. [ABSTRACT FROM AUTHOR]

α-Synuclein has a critical role in Parkinson's disease, but the mechanism of how extracellular α-synuclein aggregates lead to astrocytic degeneration remains unknown. Our recent study in astrocytes highlighted that α-synuclein aggregates undergo lower endocytosis than the monomeric-form, even while displaying a higher impact on glutathione-machinery and glutamate-metabolism under sublethal conditions. As optimal intracellular calcium levels are essential for these functions, we aimed to study the effect of extracellular α-synuclein aggregates on ER calcium entry. We assessed the association of extracellular aggregated-α-synuclein (WT and A30P/A53T double-mutant) with the astrocytic membrane (lipid rafts) and studied its effects on membrane fluidity, ER stress, and ER calcium refilling in three systems—purified rat primary midbrain astrocyte culture, human iPSC-derived astrocytes, and U87 cells. The corresponding timeline effect on mitochondrial membrane potential was also evaluated. Post-24 h exposure to extracellular WT and mutant α-synuclein aggregates, fluorescence-based studies showed a significant increase in astrocyte membrane rigidity over control, with membrane association being significantly higher for the double mutant aggregates. α-Synuclein aggregates also showed preferentially higher association with lipid rafts of astrocytic membrane. A simultaneous increase in ER stress markers (phosphorylated PERK and CHOP) with significantly higher SOCE was also observed in aggregate-treated astrocytes, with higher levels for double mutant variant. These observations correlate with increased expression of SOCE markers, especially Orai3, on plasma membrane. Alterations in mitochondrial membrane potential were only noted post-48 h of exposure to α-synuclein aggregates. We therefore suggest that in astrocytes, α-synuclein-aggregates preferentially associate with lipid rafts of membrane, altering membrane fluidity and consequently inducing ER stress mediated by interaction with membrane SOCE proteins, resulting in higher Ca2+ entry. A distinct cascade of events of sequential impairment of ER followed by mitochondrial alteration is observed. The study provides novel evidence elucidating relationships between extracellular α-synuclein aggregates and organellar stress in astrocytes and indicates the therapeutic potential in targeting the association of α-synuclein aggregates with astrocytic membrane. [ABSTRACT FROM AUTHOR]

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane. [ABSTRACT FROM AUTHOR]

Various peptide amphiphile (PA) molecules have been developed to promote bone regeneration. Previously we discovered that a peptide amphiphile with a palmitic acid tail (C 16) attenuates the signaling threshold of leucine-rich amelogenin peptide (LRAP)-mediated Wnt activation by increasing membrane lipid raft mobility. In the current study, we found that treatment of murine ST2 cells with an inhibitor (Nystatin) or Caveolin-1-specific siRNA abolishes the effect of C 16 PA, indicating that Caveolin-mediated endocytosis is required. To determine whether hydrophobicity of the PA tail plays a role in its signaling effect, we modified the length of the tail (C 12 , C 16 and C 22) or composition (cholesterol). While shortening the tail (C 12) decreased the signaling effect, lengthening the tail (C 22) had no prominent effect. On the other hand, the cholesterol PA displayed a similar function as the C 16 PA at the same concentration of 0.001% w/v. Interestingly, a higher concentration of C 16 PA (0.005%) is cytotoxic while cholesterol PA at the higher concentration (0.005%) is well-tolerated by cells. Use of the cholesterol PA at 0.005% enabled a further reduction of the signaling threshold of LRAP to 0.20 nM, compared to 0.25 nM at 0.001%. Caveolin-mediated endocytosis is also required for cholesterol PA, as evidenced by Caveolin-1 siRNA knockdown experiments. We further demonstrated that the noted effects of cholesterol PA are also observed in human bone marrow mesenchymal stem cells (BMMSCs). Taken together, these results indicate that the cholesterol PA modulates lipid raft/caveolar dynamics, thereby increasing receptor sensitivity for activation of canonical Wnt signaling. Cell signaling involves not only the binding of growth factors (or other cytokines) and cognate receptors, but also their clustering on the cell membrane. However, little or no work has been directed thus far toward investigating how biomaterials can serve to enhance growth factor or peptide signaling by increasing diffusion of cell surface receptors within membrane lipid rafts. Therefore, a better understanding of the cellular and molecular mechanism(s) operating at the material-cell membrane interface during cell signaling has the potential to change the paradigm in designing future biomaterials and regenerative medicine therapeutics. In this study, we designed a peptide amphiphile (PA) with a cholesterol tail to enhance canonical Wnt signaling by modulating lipid raft/caveolar dynamics. [Display omitted] [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) converts lysophosphatidylcholine and sphingosyl-phosphorylcholine into lysophosphatidic acid and sphingosine 1-phosphate, respectively. Despite the pivotal function of ATX in lipid metabolism, mechanisms by which ATX regulates immune and inflammatory disorders remain elusive. Here, using myeloid cell lineage-restricted Atx knockout mice, we show that Atx deficiency disrupts membrane microdomains and lipid rafts, resulting in the inhibition of Toll-like receptor 4 (TLR4) complex formation and the suppression of adaptor recruitment, thereby inhibiting TLR4-mediated responses in macrophages. Accordingly, TLR4-induced innate immune functions, including phagocytosis and iNOS expression, are attenuated in Atx-deficient macrophages. Consequently, Atx-/- mice exhibit a higher bacterial prevalence in the intestinal mucosa compared to controls. When combined with global Il10-/- mice, which show spontaneous colitis due to the translocation of luminal commensal microbes into the mucosa, myeloid cell lineage-restricted Atx knockout accelerates colitis development compared to control littermates. Collectively, our data reveal that Atx deficiency compromises innate immune responses, thereby promoting microbe-associated gut inflammation.

Cellular prion protein (PrPC) is a widely expressed glycosylphosphatidylinositol-anchored membrane protein. Scrapie prion protein is a misfolded and aggregated form of PrPC responsible for prion-induced neurodegenerative diseases. Understanding the function of the nonpathogenic PrPC monomer is an important objective. PrPC may be shed from the cell surface to generate soluble derivatives. Herein, we studied a recombinant derivative of PrPC (soluble cellular prion protein, S-PrP) that corresponds closely in sequence to a soluble form of PrPC shed from the cell surface by proteases in the A Disintegrin And Metalloprotease (ADAM) family. S-PrP activated cell-signaling in PC12 and N2a cells. TrkA was transactivated by Src family kinases and extracellular signal-regulated kinase 1/2 was activated downstream of Trk receptors. These cell-signaling events were dependent on the N-methyl-d-aspartate receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1), which functioned as a cell-signaling receptor system in lipid rafts. Membrane-anchored PrPC and neural cell adhesion molecule were not required for S-PrP-initiated cell-signaling. S-PrP promoted PC12 cell neurite outgrowth. This response required the NMDA-R, LRP1, Src family kinases, and Trk receptors. In Schwann cells, S-PrP interacted with the LRP1/NMDA-R system to activate extracellular signal-regulated kinase 1/2 and promote cell migration. The effects of S-PrP on PC12 cell neurite outgrowth and Schwann cell migration were similar to those caused by other proteins that engage the LRP1/NMDA-R system, including activated α2-macroglobulin and tissue-type plasminogen activator. Collectively, these results demonstrate that shed forms of PrPC may exhibit important biological activities in the central nervous system and the peripheral nervous system by serving as ligands for the LRP1/NMDA-R system.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer’s and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer’s and diabetes.

Abstract Cisplatin resistance poses a substantial hurdle in effectively treating head and neck squamous cell carcinoma (HNSCC). Utilizing multiple tumor models and examining an internal HNSCC cohort, squalene epoxidase (SQLE) is pinpointed as a key driver of chemoresistance and tumorigenesis, operating through a cholesterol‐dependent pathway. Comprehensive transcriptomic analysis reveals that SQLE is essential for maintaining c‐Myc transcriptional activity by stabilizing the c‐Myc protein and averting its ubiquitin‐mediated degradation. Mechanistic investigation demonstrates that SQLE inhibition diminishes Akt's binding affinity to lipid rafts via a cholesterol‐dependent process, subsequently deactivating lipid raft‐localized Akt, reducing GSK‐3β phosphorylation at S9, and increasing c‐Myc phosphorylation at T58, ultimately leading to c‐Myc destabilization. Importantly, employing an Sqle conditional knockout mouse model, SQLE's critical role in HNSCC initiation and progression is established. The preclinical findings demonstrate the potent synergistic effects of combining terbinafine and cisplatin in arresting tumor growth. These discoveries not only provide novel insights into the underlying mechanisms of SQLE‐mediated cisplatin resistance and tumorigenesis in HNSCC but also propose a promising therapeutic avenue for HNSCC patients unresponsive to conventional cisplatin‐based chemotherapy.

ABSTRACT Many eukaryotic membrane-dependent functions are often spatially and temporally regulated by membrane microdomains (FMMs), also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and scaffolding proteins belonging to the stomatin, prohibitin, flotillin, and HflK/C (SPFH) protein superfamily that was also identified in Gram-positive bacteria. In contrast, little is still known about FMMs in Gram-negative bacteria. In Escherichia coli K-12, 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, were shown to localize in discrete polar or lateral inner membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner membrane FMMs and the regulation of cellular processes. Here, we studied the determinant of the localization of QmcA and HflC and showed that FMM-associated cardiolipin lipid biosynthesis is required for their native localization pattern. Using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress that is due to the absence of HflKC. Our study therefore provides further insights into the contribution of SPFH proteins to stress tolerance in E. coli. IMPORTANCE Eukaryotic cells often segregate physiological processes in cholesterol-rich functional membrane microdomains. These domains are also called lipid rafts and contain proteins of the stomatin, prohibitin, flotillin, and HflK/C (SPFH) superfamily, which are also present in prokaryotes but have been mostly studied in Gram-positive bacteria. Here, we showed that the cell localization of the SPFH proteins QmcA and HflKC in the Gram-negative bacterium E. coli is altered in the absence of cardiolipin lipid synthesis. This suggests that cardiolipins contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis, we also showed that HflKC contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli.

Inflammation can impair intestinal barrier, while increased epithelial permeability can lead to inflammation. In this study, we found that the expression of Tspan8, a tetraspanin expressed specifically in epithelial cells, is downregulated in mouse model of ulcerative disease (UC) but correlated with those of cell–cell junction components, such as claudins and E-cadherin, suggesting that Tspan8 supports intestinal epithelial barrier. Tspan8 removal increases intestinal epithelial permeability and upregulates IFN-γ-Stat1 signaling. We also demonstrated that Tspan8 coalesces with lipid rafts and facilitates IFNγ-R1 localization at or near lipid rafts. As IFN-γ induces its receptor undergoing clathrin- or lipid raft-dependent endocytosis and IFN-γR endocytosis plays an important role in Jak-Stat1 signaling, our analysis on IFN-γR endocytosis revealed that Tspan8 silencing impairs lipid raft-mediated but promotes clathrin-mediated endocytosis of IFN-γR1, leading to increased Stat1 signaling. These changes in IFN-γR1 endocytosis upon Tspan8 silencing correlates with fewer lipid raft component GM1 at the cell surface and more clathrin heavy chain in the cells. Our findings indicate that Tspan8 determines the IFN-γR1 endocytosis route, to restrain Stat1 signaling, stabilize intestine epithelium, and subsequently prevent intestine from inflammation. Our finding also implies that Tspan8 is needed for proper endocytosis through lipid rafts. [ABSTRACT FROM AUTHOR]

It is unclear whether membrane vitamin D receptor (mVDR) exists on the macrophage membrane or whether mVDR is associated with lipopolysaccharide (LPS) tolerance. Herein, we report that interfering with caveolae and caveolae-dependent lipid rafts inhibited the formation of LPS tolerance. VDR was detected as co-localized with membrane molecular markers. VDR was detected on the cell membrane and its level was higher in LPS-tolerant cells than that in only LPS treatment cells. Anti-VDR antibodies could abolish the effect of artesunate (AS) to reverse LPS tolerance, and the wild-type peptides (H397 and H305) of VDR, but not the mutant peptide (H397D and H305A), led to the loss of AS's effect. AS decreased the mVDR level in LPS-tolerant cells. In vivo, AS significantly reduced VDR level in the lung tissue of LPS-tolerant mice. In summary, mVDR exists on the cell membrane of macrophages and is closely associated with the formation of LPS tolerance and the effects of AS. 32cFMWcLnkNZ2YpWsjevit Video Abstract [ABSTRACT FROM AUTHOR]

The gel-phase domains in a binary supported lipid bilayer (SLB) comprising dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) were localized on graphene oxide (GO) deposited on a SiO2/Si substrate. We investigated the distribution of the gel-phase domains and the liquid crystalline (Lα) phase regions in DOPC+DPPC-SLB on thermally oxidized SiO2/Si substrates with GO flakes to understand the mechanism of the domain localization on GO. Fluorescence microscopy and atomic force microscopy revealed that the gel-phase domains preferably distributed on GO flakes, whereas the fraction of the Lα-phase increased on the bare SiO2 surface which was not covered with the GO flakes. The gel-phase domain was condensed on GO more effectively at the lower cooling rate. We propose that nucleation of the gel-phase domain preferentially occurred on GO, whose surface has amphiphilic property, during the gel-phase domain formation. The domains of the liquid ordered (Lo) phase were also condensed on GO in a ternary bilayer containing cholesterol that was phase-separated to the Lo phase and the liquid disordered phase. Rigid domains segregates on GO during their formation process, leaving fluid components to the surrounding region of GO. [ABSTRACT FROM AUTHOR]

Vaccination has drastically decreased mortality due to coronavirus disease 19 (COVID-19), but not the rate of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alternative strategies such as inhibition of virus entry by interference with angiotensin-I-converting enzyme 2 (ACE2) receptors could be warranted. Cyclodextrins (CDs) are cyclic oligosaccharides that are able to deplete cholesterol from membrane lipid rafts, causing ACE2 receptors to relocate to areas devoid of lipid rafts. To explore the possibility of reducing SARS-CoV-2 entry, we tested hydroxypropyl-β-cyclodextrin (HPβCD) in a HEK293T-ACE2hi cell line stably overexpressing human ACE2 and Spike-pseudotyped SARS-CoV-2 lentiviral particles. We showed that HPβCD is not toxic to the cells at concentrations up to 5 mM, and that this concentration had no significant effect on cell cycle parameters in any experimental condition tested. Exposure of HEK293T-ACEhi cells to concentrations of HPβCD starting from 2.5 mM to 10 mM showed a concentration-dependent reduction of approximately 50% of the membrane cholesterol content. In addition, incubation of HEK293T-ACEhi cells with HIV-S-CoV-2 pseudotyped particles in the presence of increasing concentrations of HPβCD (from 0.1 to 10 mM) displayed a concentration-dependent effect on SARS-CoV-2 entry efficiency. Significant effects were detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects. These data indicate that HPβCD is a candidate for use as a SARS-CoV-2 prophylactic agent. [ABSTRACT FROM AUTHOR]

The gut microbiome derived short chain fatty acids perform multitude of functions to maintain gut homeostasis. Here we studied how butyrate stymie enteric bacterial invasion in cell using a simplistic binary model. The surface of the mammalian cells is enriched with microdomains rich in cholesterol that are known as rafts and act as entry points for pathogens. We showed that sodium butyrate treated RAW264.7 cells displayed reduced membrane cholesterol and less cholera-toxin B binding coupled with increased membrane fluidity compared to untreated cells indicating that reduced membrane cholesterol caused disruption of lipid rafts. The implication of such cellular biophysical changes on the invasion of enteric pathogenic bacteria was assessed. Our study showed, in comparison to untreated cells, butyrate-treated cells significantly reduced the invasion of Shigella and Salmonella, and these effects were found to be reversed by liposomal cholesterol treatment, increasing the likelihood that the rafts' function against bacterial invasion. The credence of ex vivo studies found to be in concordance in butyrate fed mouse model as evident from the significant drift towards a protective phenotype against virulent enteric pathogen invasion as compared to untreated mice. To produce a cytokine balance towards anti-inflammation, butyrate-treated mice produced more of the gut tissue anti-inflammatory cytokine IL-10 and less of the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ. In histological studies of Shigella infected gut revealed a startling observation where number of neutrophils infiltration was noted which was correlated with the pathology and was essentially reversed by butyrate treatment. Our results ratchet up a new dimension of our understanding how butyrate imparts resistance to pathogen invasion in the gut. [ABSTRACT FROM AUTHOR]

Mast cells protect a host from invasion by infectious agents and environmental allergens through activation of innate and adaptive immune receptors, their excessive activation being tightly regulated by inhibitory receptors, such as leukocyte immunoglobulin-like receptor (LILR)B4 (gp49B in mice). However, the regulatory mechanism of LILRB4/gp49B expressed on mast cells remains to be clarified in relation to their recently identified ligand, fibronectin (FN), a direct activator of integrins and an indirect stimulator of highaffinity Fc receptor for IgE (Fcε RI). Confocal microscopic analysis suggested that gp49B is spatially close to integrin β 1 on non-adhered bone marrow-derived mast cells (BMMCs). Their spatial relatedness increases further at robust focal adhesion sites on cells adhering to immobilized FN. However, the confocal fluorescence signal of the α subunit of Fcε RI was found to be correlated to neither gp49B nor integrin β 1 on non-adherent and adherent BMMCs. Stimulation of Fcε RI with an immobilized antigen caused Fcε RIα signals to accumulate in an inside area surrounded by robust focal adhesion with a concomitant slight increase in the signal correlation of Fcε RIα and integrin β 1, accompanied by a less significant increase of the Fcε RIα and gp49 correlation. Thus, activating and inhibitory FN receptors integrin and gp49B, respectively, were co-localized via FN at robust focal adhesion sites on BMMCs, while Fcε RI was not close to gp49B spatially. [ABSTRACT FROM AUTHOR]

Ganglioside GD2 is associated with the proliferation and migration of breast cancer cells. However, the precise role of GD2 is unclear because its tendency to form dynamic and transient domains in cell plasma membranes (PMs), called lipid rafts, makes it difficult to observe. Previously, we developed fluorescent analogs of gangliosides (e.g., GM3 and GM1), which enabled the observation of lipid raft formation for the first time using single-molecule imaging. In this report, we describe the first chemical synthesis of a fluorescent ganglioside, GD2. A biophysical analysis of the synthesized analog revealed its raft-philic character, suggesting its potential to aid single-molecule imaging-based investigations into raft-associated interactions. [ABSTRACT FROM AUTHOR]

Onur Hur&scedil;ito&gbreve;lu,1 Ergul Belge Kurutas,2 Rebecca Strawbridge,3 Omer Faruk Uygur,4 Emrah Yildiz,5 Thomas J Reilly6 1Department of Psychiatry, Sular Academy Hospital, Kahramanmaras, Turkey; 2Department of Biochemistry, Faculty of Medicine, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkey; 3Centre for Affective Disorders, Department of Psychological Medicine, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UK; 4Department of Psychiatry, Faculty of Medicine, Ataturk University, Erzurum, Turkey; 5Private Clinic, Department of Psychiatry, Gaziantep, Turkey; 6Department of Psychosis Studies, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London, UKCorrespondence: Onur Hur&scedil;ito&gbreve;lu, Department of Psychiatry, Sular Academy Hospital, Kahramanmaras, 46100, Turkey, Tel +90 507 420 68 03, Email onurhursitoglu@gmail.comIntroduction: There is increasing evidence that oxidative stress (OS) and neuroinflammation play a role in the neuroprogression of schizophrenia (SCZ). Promising novel candidates which have been proposed in the search for biomarkers of psychotic illness include NADPH oxidase 1,2 (NOX1,2) and raftlin. NOX1 from the NOX family is the main source of physiological reactive oxygen species (ROS) and raftlin, the main lipid raft protein, is associated with inflammatory processes. The aim of the present study was to evaluate serum NOX1 and raftlin levels in chronic stable patients with SCZ.Methods: We measured serum NOX1 and raftlin levels from 45 clinically stable patients with SCZ and 45 healthy controls (HCs) matched for age, sex, and body-mass index. The Positive and Negative Syndrome Scale was applied to the patient group to evaluate the severity of psychotic symptoms.Results: NOX1 and raftlin levels in the patients were statistically significantly higher than the HCs (NOX1 p< 0.001, raftlin p< 0.001). Both parameters showed very good diagnostic performance (NOX1 AUC = 0.931, raftlin AUC = 0.915). We obtained positive and significant correlations between serum levels of both biomarkers and symptom severity.Discussion: This preliminary study indicating elevations in serum NOX1 and raftlin levels in patients with SCZ supports the importance of OS and inflammatory processes in the etiopathogenesis of the illness.Keywords: oxidative stress, oxidant, neuroinflammation, reactive oxygen species, lipid raft, diagnostic performance