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NADPH oxidase 2 (Nox2), a superoxide-generating enzyme, is a source of reactive oxygen species (ROS) that regulate the intracellular redox state, self-renewal, and fate of hematopoietic stem/progenitor cells (HSPCs). Nox2 complex expressed on HSPCs associated with several activated cell membrane receptors increases the intracellular level of ROS. In addition, ROS are also released from mitochondria and, all together, are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates the trafficking, proliferation, and metabolism of HSPCs. In the current study, we noticed that Nox2-deficient mice, despite the increased number of HSPCs in the bone marrow (BM), show hematopoietic defects illustrated by delayed recovery of peripheral blood (PB) hematopoietic parameters after sublethal irradiation and mobilize fewer HSPCs after administration of G-CSF and AMD3100. Moreover, Nox2-deficient HSPCs engraft poorly after transplantation into normal syngeneic recipients. To explain these defects at the molecular level, we hypothesized that Nox2-KO decreased ROS level does not efficiently activate Nlrp3 inflammasome, which plays a crucial role in regulating the trafficking of HSPCs. Herein, we report Nox2-deficient HSPCs display i) defective migration to major chemoattractant, ii) impaired intracellular activation of Nlrp3 inflammasome, and iii) a defect in membrane lipid raft (MLRs) formation that is required for a proper chemotactic response to pro-migratory factors. We conclude that Nox2-derived ROS enhances in Nlrp3 inflammasome-dependent manner HSPCs trafficking by facilitating MLRs assemble on the outer cell membranes, and defect in Nox2 expression results in impaired activation of Nlrp3 inflammasome, which affects HSPCs migration.
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Introduction: The study was designed to explore how cinobufagin (CB) regulates the development of non-small cell lung cancer (NSCLC) cells through lipid rafts. Material and methods: The effects of CB at gradient concentrations (0, 0.5, 1 and 2 µM) on NSCLC cell viability, apoptosis, reactive oxygen species (ROS) level, phosphorylation of Akt, and apoptosis- and lipid raft-related protein expression were assessed by MTT assay, flow cytometry and Western blot. Cholesterol and sphingomyelin were labeled with BODIPY to evaluate the effect of CB (2 µM) on them. Sucrose density gradient centrifugation was used to extract lipid rafts. The effect of CB on the expression and distribution of caveolin-1 was determined by immunofluorescence, quantitative reverse transcription polymerase chain reaction and Western blot. After overexpression of caveolin-1, the above experiments were performed again to observe whether the regulatory effect of CB was reversed. Results: CB inhibited NSCLC cell viability while promoting apoptosis and ROS level. CB redistributed the lipid content on the membrane surface and reduced the content of caveolin-1 in the cell membrane. In addition, CB repressed the activation of AKT. However, caveolin-1 overexpression reversed the effects of CB on apoptosis, AKT activation and lipid raft. Conclusions: CB regulates the activity of Akt in lipid rafts by inhibiting caveolin-1 expression to promote NSCLC cell apoptosis.
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The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.
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Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.
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Autofagia , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Linhagem Celular Tumoral , Microdomínios da Membrana/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Tetraspanina 30/metabolismo , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.
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The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.
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Colesterol , ATPases Transportadoras de Cálcio da Membrana Plasmática , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Colesterol/metabolismo , Humanos , Animais , Ratos , Simulação de Dinâmica Molecular , Motivos de Aminoácidos , Membrana Celular/metabolismoRESUMO
We have previously identified that a structural membrane protein Caveolin-1 (Cav1) is involved in the regulation of aberrant keratinocyte proliferation and differentiation. The aim of this study was to elucidate the role of Cav1, Caveolin-2 (Cav2), and Cavin-1 in the pathogenesis of psoriasis vulgaris and between psoriasis subtypes. We utilized human biopsies from validated cases of psoriasis vulgaris (n = 21) at the University of Miami Hospital and compared the expression of Cav1, Cav2, and Cavin-1 by immunohistochemistry staining with that in normal healthy age-/sex-/location-matched skin (n = 15) and chronic spongiotic dermatitis skin samples (as control inflammatory skin condition) and quantified using QuPath. Distinct subtypes of psoriasis included guttate, inverse, nail, plaque, palmoplantar, and pustular. All biopsy samples exhibited a trend toward downregulation of Cav1, with nail, plaque, and palmoplantar psoriasis exhibiting the most pronounced effects. Only nail and pustular psoriasis samples exhibited significant downregulation of Cav2 and Cavin-1, suggesting Cav1 to be the main caveolar contributor to the pathogenesis of psoriasis. Together, these data support caveolae as pathophysiological targets in nail and pustular psoriasis, whereas Cav1 seems to be a general biomarker of multiple subtypes of psoriasis.
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High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.
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Doenças Cardiovasculares , Hipercolesterolemia , Proteínas de Membrana , Humanos , LDL-Colesterol/metabolismo , Receptor A2A de Adenosina/metabolismo , Leucócitos Mononucleares/metabolismo , Adenosina , Fatores de Risco , Colesterol , Proteínas de Transporte , Fatores de Risco de Doenças Cardíacas , Microdomínios da Membrana/metabolismoRESUMO
This study presents a new approach to designing a lithocholic acid functionalized oligomer (OLithocholicAA-X) that can be used as a drug carrier with additional, beneficial activity. Namely, this novel oligomer can incorporate an anti-cancer drug due to the application of an effective backbone as its component (lithocholic acid) alone is known to have anticancer activity. The oligomer was synthesized and characterized in detail by nuclear magnetic resonance, attenuated total reflectance Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, thermal analysis, and mass spectrometry analysis. We selected lipid rafts as potential drug carrier-membrane binding sites. In this respect, we investigated the effects of OLithocholicAA-X on model lipid raft of normal and altered composition, containing an increased amount of cholesterol (Chol) or sphingomyelin (SM), using Langmuir monolayers and liposomes. The surface topography of the studied monolayers was additionally investigated by atomic force microscopy (AFM). The obtained results showed that the investigated oligomer has affinity for a system that mimics a normal lipid raft (SM:Chol 2:1). On the other hand, for systems with an excess of SM or Chol, thermodynamically unfavorable fluidization of the films occurs. Moreover, AFM topographies showed that the amount of SM determines the bioavailability of the oligomer, causing fragmentation of its lattice.
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Lipossomos , Ácido Litocólico , Ácido Litocólico/análise , Ácido Litocólico/metabolismo , Lipossomos/química , Sistemas de Liberação de Medicamentos , Espectroscopia de Ressonância Magnética , Microdomínios da Membrana/química , Esfingomielinas/química , Colesterol/químicaRESUMO
Chalcones are naturally produced by many plants, and constitute precursors for the synthesis of flavons and flavanons. They were shown to possess antibacterial, antifungal, anti-cancer, and anti- inflammatory properties. The goal of the study was to assess the suitability of three synthetic methoxychalcones as potential anticancer agents. In a panel of colon cancer cell lines they were demonstrated to be cytotoxic, proapoptotic, causing cell cycle arrest, and increasing intracellular level of reactive oxygen species. Anticancer activity of the compounds was not diminished in the presence of stool extract containing microbial enzymes that could change the structure of chalcones. Moreover, methoxychalcones interacted strongly with model phosphatidylcholine membranes as detected by differential scanning calorimetry. Metohoxychalcones particularly affected the properties of lipid domains in giant unilamellar liposomes formed from raft-mimicking lipid composition. This may be of importance since many molecular targets for therapy of metastatic colon cancer are raft-associated receptors (e.g., receptor tyrosine kinases). The importance of membrane perturbing potency of methoxychalcones for their biological activity was additionally corroborated by the results obtained by molecular modelling.
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Antineoplásicos , Chalconas , Neoplasias do Colo , Humanos , Chalconas/farmacologia , Chalconas/química , Linhagem Celular , Fosfatidilcolinas , Antineoplásicos/química , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologiaRESUMO
Colorectal cancer (CRC) is characterized by a complex tumor inflammatory microenvironment, while angiogenesis and immunosuppression frequently occur concomitantly. However, the exact mechanism that controls angiogenesis and immunosuppression in CRC microenvironment remains unclear. Herein, we found that expression levels of lipid raft protein STOML2 were increased in CRC and were associated with advanced disease stage and poor survival outcomes. Intriguingly, we revealed that STOML2 is essential for CRC tumor inflammatory microenvironment, which induces angiogenesis and facilitates tumor immune escape simultaneously both in vitro and in vivo. Moreover, tumors with STOML2 overexpression showed effective response to anti-angiogenesis treatment and immunotherapy in vivo. Mechanistically, STOML2 regulates CRC proliferation, angiogenesis, and immune escape through activated NF-κB signaling pathway via binding to TRADD protein, resulting in upregulation of CCND1, VEGF, and PD-L1. Furthermore, treatment with NF-κB inhibitor dramatically reversed the ability of proliferation and angiogenesis. Clinically, we also observed a strong positive correlation between STOML2 expression and Ki67, CD31, VEGFC and PD-1 of CD8+T cell expression. Taken together, our results provided novel insights into the role of STOML2 in CRC inflammatory microenvironment, which may present a therapeutic opportunity for CRC.
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Neoplasias Colorretais , Proteínas de Membrana , NF-kappa B , Microambiente Tumoral , Humanos , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Regulação para Cima , Microdomínios da Membrana , Proteínas de Membrana/genéticaRESUMO
Tumor cells movement and migration are inseparable from the integrity of lipid rafts and the formation of lamellipodia, and lipid rafts are also a prerequisite for the formation of lamellipodia. Therefore, destroying the lipid rafts is an effective strategy to inhibit tumor metastasis. Herein, a multi-enzyme co-expressed nanomedicine: cholesterol oxidase (CHO) loaded CoâPN3 single-atom nanozyme (CoâPN3 SA/CHO) that can up-regulate cellular oxidative stress, disrupt the integrity of lipid rafts, and inhibit lamellipodia formation to induce anti-metastasis tumor therapy, is developed. In this process, CoâPN3 SA can catalyze oxygen (O2 ) and hydrogen peroxide (H2 O2 ) to generate reactive oxygen species (ROS) via oxidase-like and Fenton-like properties. The doping of P atoms optimizes the adsorption process of the intermediate at the active site and enhances the ROS generation properties of nanomedicine. Meantime, O2 produced by catalase-like catalysis can combine with excess cholesterol to generate more H2 O2 under CHO catalysis, achieving enhanced oxidative damage to tumor cells. Most importantly, cholesterol depletion in tumor cells also disrupts the integrity of lipid rafts and inhibits the formation of lamellipodia, greatly inhibiting the proliferation and metastasis of tumor cells. This strategy by up-regulating cellular oxidative stress and depleting cellular cholesterol constructs a new idea for anti-metastasis-oriented cancer therapy strategies.
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Nanomedicina , Neoplasias , Humanos , Espécies Reativas de Oxigênio , Estresse Oxidativo , Oxirredução , Colesterol , Linhagem Celular Tumoral , Peróxido de Hidrogênio/farmacologia , Microambiente TumoralRESUMO
Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide an evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs.
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Melatonina , Glândula Pineal , Humanos , Animais , Camundongos , Inflamassomos/metabolismo , Melatonina/farmacologia , Melatonina/metabolismo , Glândula Pineal/metabolismo , Heme Oxigenase-1/metabolismo , Células-Tronco Hematopoéticas , Anti-Inflamatórios , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Triple-negative breast cancer stem cells (TCSCs) are considered as the origin of recurrence and relapse. It is difficult to kill not only for its resistance, but also the lacking of targetable molecules on membrane. Here, it is confirmed that ST6 ß-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal-1) is highly expressed in TCSCs that may be the key enzyme involved in glycoengineering via sialic acid (SA) metabolism. SA co-localizes with a microdomain on cell membrane termed as lipid rafts that enrich CSCs marker and necroptosis proteins mixed lineage kinase domain-like protein (MLKL), suggesting that TCSCs may be sensitive to necroptosis. Thus, the triacetylated N-azidoacetyl-d-mannosamine (Ac3ManNAz) is synthesized as the glycoengineering substrate and applied to introduce artificial azido receptors, dibenzocyclooctyne (DBCO)-modified liposome is used to deliver Compound 6i (C6), a receptor-interacting serine/threonine protein kinase 1(RIPL1)-RIP3K-mixed lineage kinase domain-like protein(MLKL) activator, to induce necroptosis. The pro-necroptosis effect is aggravated by nitric oxide (NO), which is released from NO-depot of cholesterol-NO integrated in DBCO-PEG-liposome@NO/C6 (DLip@NO/C6). Together with the immunogenicity of necroptosis that releases high mobility group box 1(HMGB1) of damage-associated molecular patterns, TCSCs are significantly killed in vitro and in vivo. The results suggest a promising strategy to improve the therapeutic effect on the non-targetable TCSCs with high expression of ST6Gal-1 via combination of glycoengineering and necroptosis induction.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/terapia , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Necroptose , Lipossomos , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , Células-Tronco/metabolismo , ApoptoseRESUMO
Polyphenols with different structures have shown distinct variations in their ability to inhibit the differentiation of 3T3-L1 preadipocytes. However, the underlying mechanisms for these differences remain unclear. In the present study, the surface binding of polyphenols to different membrane domains was explored using coarse-grained molecular dynamics simulation (CG-MDs). Subsequently, this surface binding was confirmed in the liposome system by microscale thermophoresis. Additionally, the interference of polyphenols on the membrane raft's structure was studied through atomic force microscopy and high-content screening fluorescence microscopy. The results indicated that polyphenols with a differentiation-inhibitory ability, such as epicatechin-3-gallate (ECG) and epicatechin-3-gallate-(4ß â 8, 2ß â O â 7)-epicatechin-3-gallate (A-type ECG dimer), exhibited strong binding to ordered domains enriched in sphingolipids and cholesterol. This binding led to the structural disruption of membrane rafts by altering their size and shape, with the binding constant of 3.8 µM for ECG and 0.3 µM for A-type ECG dimer, respectively. In contrast, epicatechin (EC) with little differentiation-inhibitory ability had no effects on membrane rafts, and its binding constant with the ordered domain was 380.6 µM. Overall, the surface binding of polyphenols to ordered domains and the resulting disruption of membrane rafts structure might be a fundamental mechanism by which polyphenols inhibited the differentiation of 3T3-L1 preadipocytes.
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Microdomínios da Membrana , Polifenóis , Animais , Camundongos , Polifenóis/farmacologia , Células 3T3-L1 , Diferenciação Celular , Simulação de Dinâmica MolecularRESUMO
The plethora of information about the expression of cancer cell-associated gangliosides, their role(s) in signal transduction, and their potential usefulness in the development of cancer treatments makes this an appropriate time to review these enigmatic glycosphingolipids. Evidence, reflecting the work of many, indicates that (1) expression of specific gangliosides, not generally found in high concentrations in most normal human cells, can be linked to certain types of cancer. (2) Gangliosides can affect the ability of cells to interact either directly or indirectly with growth factor receptors, thereby changing such things as a cell's mobility, rate of proliferation, and metastatic ability. (3) Anti-ganglioside antibodies have been tested, with some success, as potential treatments for certain cancers. (4) Cancer-associated gangliosides shed into the circulation can (a) affect immune cell responsiveness either positively or negatively, (b) be considered as diagnostic markers, and (c) be used to look for recurrence. (5) Cancer registries enable investigators to evaluate data from sufficient numbers of patients to obtain information about potential therapies. Despite advances that have been made, a discussion of possible approaches to identifying additional treatment strategies to inhibit metastasis, responsible for the majority of deaths of cancer patients, as well as for treating therapy-resistant tumors, is included.
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Human adenoviruses represent attractive candidates for the design of cancer gene therapy vectors. Modification of adenovirus tropism by incorporating a targeting ligand into the adenovirus capsid proteins allows retargeting of adenovirus towards the cells of interest. Human adenovirus type 5 (HAdV-C5) bearing NGR containing peptide (CNGRCVSGCAGRC) inserted into the fiber (AdFNGR) or the hexon (AdHNGR) protein demonstrated an increased transduction of endothelial cells showing expression of aminopeptidase N, also known as CD13, and αvß3 integrin both present on tumor vasculature, indicating that NGR-bearing adenoviruses could be used as tools for anti-angiogenic cancer therapy. Here we investigated how AdFNGR and AdHNGR infect cells lacking HAdV-C5 primary receptor, coxsackie and adenovirus receptor, and we showed that both AFNGR and AdHNGR enter cells by dynamin- and lipid raft-mediated endocytosis, while clathrin is not required for endocytosis of these viruses. We present evidence that productive infection of both AdFNGR and AdHNGR involves lipid rafts, with usage of flotillin-mediated cell entry for AdFNGR and limited role of caveolin in AdHNGR transduction efficiency. Lipid rafts play important role in angiogenesis and process of metastasis. Therefore, the ability of AdFNGR and AdHNGR to use lipid raft-dependent endocytosis, involving respectively flotillin- or caveolin-mediated pathway, could give them an advantage in targeting tumor cells lacking HAdV-C5 primary receptor.
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Adenovírus Humanos , Humanos , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismoRESUMO
BACKGROUND: Image-based screening improves the detection of early-stage lung adenocarcinoma (LUAD)but also highlights the issue of high false-positive diagnoses, which puts patients at a risk of unnecessary over-treatment. Therefore, more precise discrimination criteria are required to ensure that patients with early-stage LUAD receive appropriate treatments. METHODS: We integrated 158 early-stage LUAD cases from 2 independent cohorts, including 30 matched resected specimens with complete radiological and pathological information, and 128 retrospective pathological pair-samples with partial follow-up data. This integration allowed us to conduct a correlation analysis between clinical phenotype and transcriptome landscape. Immunohistochemistry was performed using tissue microarrays to examine the expression of phospholipid phosphatase 2 (PLPP2) and lipid-raft markers. Lipidomics analysis was used to determine the changes of lipid components in PLPP2-overexpressed cells. To assess the effects of PLPP2 on the malignant phenotypes of LUAD cells, we conducted mice tumor-bearing experiments and in vitro cellular experiments by knocking down PLPP2 and inhibiting lipid raft synthesis with MßCD, respectively. RESULTS: Bioinformatics analysis indicated that the co-occurrence of lipid raft formation and rapid cell proliferation might exhibit synergistic effects in driving oncogenesis from lung preneoplasia to adenocarcinoma. The enhanced activation of the cell cycle promoted the transition from non-invasive to invasive status in early-stage LUAD, which was related to an increase in lipid rafts within LUAD cells. PLPP2 participated in lipid raft formation by altering the component contents of lipid rafts, such as esters, sphingomyelin, and sphingosine. Furthermore, elevated PLPP2 levels were identified as an independent prognostic risk factor for LUAD patients. Further results from in vivo and in vitro experiments confirmed that PLPP2 could induce excessive cell proliferation by enhancing lipid raft formation in LUAD cells. CONCLUSIONS: Our study has revealed the characteristics of gene expression profiles in early-stage LUAD patients with the different radiological and pathological subtypes, as well as deciphered transcriptomic evolution trajectory from preneoplasia to invasive LUAD. Furthermore, it suggests that PLPP2-mediated lipid raft synthesis may be a significant biological event in the initiation of early-stage LUAD, offering a potential target for more precise diagnosis and therapy in clinical settings.
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Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/genética , Proliferação de Células , Neoplasias Pulmonares/genética , Prognóstico , Estudos Retrospectivos , TranscriptomaRESUMO
BACKGROUND: Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as neurological disorders, type 2 diabetes, and cancer metastasis. To better understand how PLLP interacts with raft components (gangliosides and cholesterol), we undertook a global study combining in silico simulations and physicochemical measurements of molecular interactions in various PLLP-ganglioside systems. METHODS: In silico studies consisted of molecular dynamics simulations in reconstructed membrane environments. PLLP-ganglioside interaction measurements were performed by microtensiometry at the water-air interface on ganglioside monolayers. RESULTS: We have elucidated the mode of interaction of PLLP with ganglioside GM1 and characterized this interaction at the molecular level. We showed that GM1 induces the structuring of the extracellular loops of PLLP and that this interaction propagates a conformational signal through the plasma membrane, involving a cholesterol molecule located between transmembrane domains. This conformational wave is finally transmitted to the intracellular domain of the protein, consistent with the role of PLLP in signal transduction. CONCLUSIONS: This study is a typical example of the epigenetic dimension of protein structure, a concept developed by our team to describe the chaperone effect of gangliosides on disordered protein motifs which associate with lipid rafts. From a physiological point of view, these data shed light on the role of gangliosides in myelin formation. From a pathological point of view, this study will help to design innovative therapeutic strategies focused on ganglioside-PLLP interactions in various PLLP-associated diseases.
Assuntos
Bainha de Mielina , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Humanos , Gangliosídeo G(M1) , Gangliosídeos , Microdomínios da Membrana , Proteolipídeos , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/químicaRESUMO
BACKGROUND & AIMS: Fatty acid translocase CD36 (CD36/FAT) is a widely expressed membrane protein with multiple immuno-metabolic functions. Genetic CD36 deficiency is associated with increased risk of metabolic dysfunction-associated fatty liver disease (MAFLD) in patients. Liver fibrosis severity mainly affects the prognosis in patients with MAFLD, but the role of hepatocyte CD36 in liver fibrosis of MAFLD remains unclear. METHODS: A high-fat high-cholesterol diet and a high-fat diet with high-fructose drinking water were used to induce nonalcoholic steatohepatitis (NASH) in hepatocyte-specific CD36 knockout (CD36LKO) and CD36flox/flox (LWT) mice. Human hepG2 cell line was used to investigate the role of CD36 in regulating Notch pathway in vitro. RESULTS: Compared to LWT mice, CD36LKO mice were susceptible to NASH diet-induced liver injury and fibrosis. The analysis of RNA-sequencing data revealed that Notch pathway was activated in CD36LKO mice. LY3039478, an inhibitor of γ-secretase, inhibited Notch1 protein S3 cleavage and Notch1 intracellular domain (N1ICD) production, alleviating liver injury and fibrosis in CD36LKO mice livers. Likewise, both LY3039478 and knockdown of Notch1 inhibited the CD36KO-induced increase of N1ICD production, causing the decrease of fibrogenic markers in CD36KO HepG2 cells. Mechanistically, CD36 formed a complex with Notch1 and γ-secretase in lipid rafts, and hence CD36 anchored Notch1 in lipid rafts domains and blocked Notch1/γ-secretase interaction, inhibiting γ-secretase-mediated cleavage of Notch1 and the production of N1ICD. CONCLUSIONS: Hepatocyte CD36 plays a key role in protecting mice from diet-induced liver injury and fibrosis, which may provide a potential therapeutic strategy for preventing liver fibrogenesis in MAFLD.