RESUMEN
Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1ß and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1ß expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.
Asunto(s)
Transportador 1 de Casete de Unión a ATP , Euphorbia , Receptores X del Hígado , Microdominios de Membrana , Receptor Toll-Like 4 , Euphorbia/química , Receptor Toll-Like 4/metabolismo , Receptores X del Hígado/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Animales , Ratones , Transportador 1 de Casete de Unión a ATP/metabolismo , Inflamación/tratamiento farmacológico , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células RAW 264.7 , HumanosRESUMEN
Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study, we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. Lipopolysaccharide (LPS) also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response, indicating interference between these signaling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, inhibition of the EV-associated heat shock protein (HSP70) chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV-induced lipid raft formation and autophagy. Pre-treatment of microglia with a selective inhibitor of αvß3/αvß5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, αVß3/αVß5, and P2X4R signaling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used to develop new therapeutic strategies targeting disease-associated microglia.
Asunto(s)
Autofagia , Vesículas Extracelulares , Lipopolisacáridos , Microdominios de Membrana , Microglía , Receptor Toll-Like 4 , Humanos , Vesículas Extracelulares/metabolismo , Autofagia/efectos de los fármacos , Microglía/metabolismo , Microglía/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Lipopolisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Células CultivadasRESUMEN
AIMS: Despite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood. METHODS: The TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates. RESULTS: Acquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane. CONCLUSIONS: Our findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP , Neoplasias Encefálicas , Resistencia a Antineoplásicos , Glioma , Molécula 1 de Adhesión Intercelular , Temozolomida , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/patología , Glioma/genética , Glioma/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Humanos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Temozolomida/farmacología , Línea Celular Tumoral , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Proliferación Celular/efectos de los fármacos , Ratones , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacosRESUMEN
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.
Asunto(s)
Esfingomielina Fosfodiesterasa , Canales Catiónicos TRPM , beta-Ciclodextrinas , Animales , Humanos , Ratones , Analgésicos/farmacología , Analgésicos/uso terapéutico , beta-Ciclodextrinas/farmacología , Supervivencia Celular/efectos de los fármacos , Células CHO , Colesterol/metabolismo , Cricetulus , Modelos Animales de Enfermedad , Células HEK293 , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Dolor/inducido químicamente , Dolor/tratamiento farmacológico , Dolor/metabolismo , Pregnenolona/farmacología , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/farmacología , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/genética , Pirimidinonas/farmacologíaRESUMEN
BACKGROUND: Docosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown. OBJECTIVES: The primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA's anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model. METHODS: MH-S cells, an alveolar macrophage line, were treated with 50 µM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers. RESULTS: DHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice. CONCLUSIONS: The data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation.
Asunto(s)
Ácidos Docosahexaenoicos , Macrófagos Alveolares , Microdominios de Membrana , Animales , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/efectos de los fármacos , Ácidos Docosahexaenoicos/farmacología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Inflamación/metabolismo , Pulmón/metabolismo , Infecciones por Orthomyxoviridae , Ratones Endogámicos C57BL , Línea Celular , Colesterol/metabolismoRESUMEN
Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy.
Asunto(s)
Apoptosis , Microdominios de Membrana , Sapogeninas , Humanos , Apoptosis/efectos de los fármacos , Células Hep G2 , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Sapogeninas/farmacología , Sapogeninas/química , Proteína Ligando Fas/metabolismo , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , beta-Ciclodextrinas/farmacología , beta-Ciclodextrinas/química , Antineoplásicos/farmacología , Antineoplásicos/química , Receptor fas/metabolismoRESUMEN
ß2-Adrenoceptors (ß2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of ß2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that ß2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. ß2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of ß2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of Gi protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the ß2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on ß2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, ß2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of ß2-AR agonist on neurotransmission probably via switching to Gi protein-dependent signaling. This phenomenon may reflect a dependence of the ß2-AR signaling on lipid raft integrity in the neuromuscular junctions.
Asunto(s)
Unión Neuromuscular , Receptores Adrenérgicos beta 2 , Transmisión Sináptica , Animales , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/metabolismo , Transmisión Sináptica/efectos de los fármacos , Receptores Adrenérgicos beta 2/metabolismo , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Masculino , Diafragma/efectos de los fármacos , Diafragma/inervación , Diafragma/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/efectos de los fármacos , Colesterol/metabolismo , Exocitosis/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin. METHODS: Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined. RESULTS: Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04). CONCLUSION: Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin. IMPACT: This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.
Asunto(s)
Bilirrubina , Cerebelo , Colina , Neuronas , Bilirrubina/farmacología , Bilirrubina/metabolismo , Colina/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Cerebelo/efectos de los fármacos , Cerebelo/citología , Animales , Fosforilación , Células Cultivadas , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Suplementos Dietéticos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Transducción de Señal/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Humanos , Neuritas/efectos de los fármacos , Neuritas/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) is currently a worldwide emergency caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). In observational clinical studies, statins have been identified as beneficial to hospitalized patients with COVID-19. However, experimental evidence of underlying statins protection against SARS-CoV-2 remains elusive. Here we reported for the first-time experimental evidence of the protective effects of simvastatin treatment both in vitro and in vivo. We found that treatment with simvastatin significantly reduced the viral replication and lung damage in vivo, delaying SARS-CoV-2-associated physiopathology and mortality in the K18-hACE2-transgenic mice model. Moreover, simvastatin also downregulated the inflammation triggered by SARS-CoV-2 infection in pulmonary tissue and in human neutrophils, peripheral blood monocytes, and lung epithelial Calu-3 cells in vitro, showing its potential to modulate the inflammatory response both at the site of infection and systemically. Additionally, we also observed that simvastatin affected the course of SARS-CoV-2 infection through displacing ACE2 on cell membrane lipid rafts. In conclusion, our results show that simvastatin exhibits early protective effects on SARS-CoV-2 infection by inhibiting virus cell entry and inflammatory cytokine production, through mechanisms at least in part dependent on lipid rafts disruption.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Regulación hacia Abajo/efectos de los fármacos , Inflamación/tratamiento farmacológico , Microdominios de Membrana/efectos de los fármacos , SARS-CoV-2/patogenicidad , Simvastatina/farmacología , Animales , COVID-19/virología , Modelos Animales de Enfermedad , Humanos , Inflamación/virología , Pulmón/virología , Ratones , Ratones Transgénicos , Replicación Viral/efectos de los fármacosRESUMEN
The increase in Aß1-42 is a neurotoxic effect induced by aluminum which can lead to impairment of learning and memory, but its mechanism has yet to be fully elucidated. Studies have shown that APP palmitoylation is appears to be involved in the production process of Aß1-42. Here, we investigated whether APP palmitoylation is related to the increase in Aß caused by aluminum and its specific mechanism of action. In this study, APP palmitoylation was studied in the setting of aluminum-induced increases in Aß1-42 from two perspectives: whole animal experiments and in vitro cell experiments. First, the learning and memory of rats were impaired and the number of rat cortical neurons was decreased after staining with aluminum. Second, the expression of palmitoyl APP, APP in lipid rafts and palmitoyl acyltransferase zDHHC7 both in rat cerebral cortex and PC12 cells increased with the production of Aß1-42 induced by aluminum in a dose-dependent manner. Finally, the intervention with the palmitoylation inhibitors 2-BP and siRNA zDHHC7 in PC12 cells reduced levels of palmitoyl APP, the expression of APP in lipid rafts and the content of Aß1-42 induced by aluminum to a certain extent. Our results indicate that increased APP palmitoylation levels may be related to the increase in Aß1-42 caused by aluminum, and the mechanism may involve APP palmitoylation promoting the accumulation of APP protein on lipid rafts and the cleavage of APP by BACE1 in amyloidogenic pathway. The increase in expression of zDHHC7 may be one of the reasons for the increase in levels of APP palmitoylation caused by aluminum.
Asunto(s)
Aluminio/farmacología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Acetiltransferasas/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Aprendizaje/efectos de los fármacos , Lipoilación/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Memoria/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , RatasRESUMEN
OBJECTIVE: The aim of the study was to show the effect that two naturally occurring compounds, a cyclodextrin and hydroxytyrosol, can have on the entry of SARS-CoV-2 into human cells. MATERIALS AND METHODS: The PubMed database was searched to retrieve studies published from 2000 to 2020, satisfying the inclusion criteria. The search keywords were: SARS-CoV, SARS-CoV-2, coronavirus, lipid raft, endocytosis, hydroxytyrosol, cyclodextrin. Modeling of alpha-cyclodextrin and hydroxytyrosol were done using UCSF Chimera 1.14. RESULTS: The search results indicated that cyclodextrins can reduce the efficiency of viral endocytosis and that hydroxytyrosol has antiviral properties. Bioinformatic docking studies showed that alpha-cyclodextrin and hydroxytyrosol, alone or in combination, interact with the viral spike protein and its host cell receptor ACE2, thereby potentially influencing the endocytosis process. CONCLUSIONS: Hydroxytyrosol and alpha-cyclodextrin can be useful against the spread of SARS-CoV-2.
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Alcohol Feniletílico/análogos & derivados , SARS-CoV-2/fisiología , Internalización del Virus/efectos de los fármacos , alfa-Ciclodextrinas/farmacología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Sitios de Unión , COVID-19/patología , COVID-19/prevención & control , COVID-19/virología , Biología Computacional/métodos , Humanos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Simulación del Acoplamiento Molecular , Alcohol Feniletílico/química , Alcohol Feniletílico/metabolismo , Alcohol Feniletílico/farmacología , Alcohol Feniletílico/uso terapéutico , Unión Proteica , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , alfa-Ciclodextrinas/química , alfa-Ciclodextrinas/metabolismo , alfa-Ciclodextrinas/uso terapéuticoRESUMEN
The lipid raft-resident protein, MAL2, has been implicated as contributing to the pathogenesis of several malignancies, including breast cancer, but the underlying mechanism for its effects on tumorigenesis is unknown. Here, we show that MAL2-mediated lipid raft formation leads to HER2 plasma membrane retention and enhanced HER2 signaling in breast cancer cells. We demonstrate physical interactions between HER2 and MAL2 in lipid rafts using proximity ligation assays. Super-resolution structured illumination microscopy imaging displays the structural organization of the HER2/Ezrin/NHERF1/PMCA2 protein complex. Formation of this protein complex maintains low intracellular calcium concentrations in the vicinity of the plasma membrane. HER2/MAL2 protein interactions in lipid rafts are enhanced in trastuzumab-resistant breast cancer cells. Our findings suggest that MAL2 is crucial for lipid raft formation, HER2 signaling, and HER2 membrane stability in breast cancer cells, suggesting MAL2 as a potential therapeutic target.
Asunto(s)
Neoplasias de la Mama/patología , Proteínas del Citoesqueleto/metabolismo , Resistencia a Antineoplásicos , Microdominios de Membrana/metabolismo , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/metabolismo , Fosfoproteínas/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Receptor ErbB-2/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Antineoplásicos Inmunológicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/genética , Endocitosis , Femenino , Humanos , Microdominios de Membrana/efectos de los fármacos , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Fosfoproteínas/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Receptor ErbB-2/genética , Intercambiadores de Sodio-Hidrógeno/genética , Trastuzumab/farmacología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem-like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear. METHODS: This study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR-disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses. RESULTS: Miltefosine suppresses CSC populations and their self-renewal activities in CRC cells, a CSC-targeting effect leading to irreversible disruption of tumour-initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non-CSC counterparts by preventing cell death through CHEK1-mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death. CONCLUSION: Our findings underscore the therapeutic potential of LR-targeting APLs for CRC treatment that overcomes the therapy-resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.
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Neoplasias Colorrectales/tratamiento farmacológico , Microdominios de Membrana/efectos de los fármacos , Fosforilcolina/análogos & derivados , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/fisiopatología , Modelos Animales de Enfermedad , Ratones , Fosforilcolina/farmacología , Fosforilcolina/uso terapéuticoRESUMEN
The activity of local anesthetics (LAs) has been attributed to the inhibition of ion channels, causing anesthesia. However, there is a growing body of research showing that LAs act on a wide range of receptors and channel proteins far beyond simple analgesia. The current concept of ligand recognition may no longer explain the multitude of protein targets influenced by LAs. We hypothesize that LAs can cause anesthesia without directly binding to the receptor proteins just by changing the physical properties of the lipid bilayer surrounding these proteins and ion channels based on LAs' amphiphilicity. It is possible that LAs act in one of the following ways: They 1) dissolve raft-like membrane microdomains, 2) impede nerve impulse propagation by lowering the lipid phase transition temperature, or 3) modulate the lateral pressure profile of the lipid bilayer. This could also explain the numerous additional effects of LAs besides anesthesia. Furthermore, the concepts of membrane-mediated activity and binding to ion channels do not have to exclude each other. If we were to consider LA as the middle part of a continuum between unspecific membrane-mediated activity on one end and highly specific ligand binding on the other end, we could describe LA as the link between the unspecific action of general anesthetics and toxins with their highly specific receptor binding. This comprehensive membrane-mediated model offers a fresh perspective to clinical and pharmaceutical research and therapeutic applications of local anesthetics. SIGNIFICANCE STATEMENT: Local anesthetics, according to the World Health Organization, belong to the most important drugs available to mankind. Their rediscovery as therapeutics and not only anesthetics marks a milestone in global pain therapy. The membrane-mediated mechanism of action proposed in this review can explain their puzzling variety of target proteins and their thus far inexplicable therapeutic effects. The new concept presented here places LAs on a continuum of structures and molecular mechanisms in between small general anesthetics and the more complex molecular toxins.
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Potenciales de Acción/fisiología , Anestésicos Locales/metabolismo , Fenómenos Fisiológicos Celulares/fisiología , Microdominios de Membrana/metabolismo , Potenciales de Acción/efectos de los fármacos , Anestésicos Locales/administración & dosificación , Anestésicos Locales/química , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Fenómenos Fisiológicos Celulares/efectos de los fármacos , Humanos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.
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Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Venenos de Cnidarios/metabolismo , Venenos de Cnidarios/toxicidad , Microdominios de Membrana/metabolismo , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Esfingomielinas/metabolismo , Animales , Microdominios de Membrana/efectos de los fármacos , Microscopía de Fuerza Atómica , Microscopía ConfocalRESUMEN
Hereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.
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Axones/metabolismo , Gangliósidos/metabolismo , Neuropatías Hereditarias Sensoriales y Autónomas/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Neuroglía/metabolismo , Serina/farmacología , Envejecimiento/patología , Axones/efectos de los fármacos , Axones/ultraestructura , Secuencia de Bases , Caspasa 3/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Vaina de Mielina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuroglía/efectos de los fármacos , Proyección Neuronal/efectos de los fármacos , Proteína Nodal/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/ultraestructura , Transducción de Señal/efectos de los fármacos , Esfingolípidos/metabolismo , Transcriptoma/genéticaRESUMEN
Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.
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Caveolas/efectos de los fármacos , Colesterol/química , Células Endoteliales/efectos de los fármacos , Liposomas/química , Microdominios de Membrana/efectos de los fármacos , Transfección/métodos , Animales , Caveolas/química , Caveolas/metabolismo , Caveolina 1/antagonistas & inhibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular Transformada , Colesterol/metabolismo , Clatrina/metabolismo , ADN/química , ADN/metabolismo , Endocitosis/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/metabolismo , Filipina/química , Filipina/farmacología , Expresión Génica , Liposomas/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Nistatina/química , Nistatina/farmacología , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacología , Pinocitosis/efectos de los fármacos , Plásmidos/química , Plásmidos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , RatasRESUMEN
Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of Gα s signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of Gα s protein from lipid rafts to nonraft membrane regions. This results in augmented Gα s signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft-localized cAMP sensor, we show that Gα s signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in Gα s localization observed after antidepressant treatment. Furthermore, we show that suppression of Gα s signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. Gα s localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft Gα s signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane Gα s signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of Gα s out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT: This work explores, for the first time, the effects of antidepressants on Gα s signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment.
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Antidepresivos/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Microdominios de Membrana/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Microdominios de Membrana/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. METHODS: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-ß-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. RESULTS: MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. CONCLUSION: Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.
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Plaquetas/metabolismo , AMP Cíclico/sangre , Microdominios de Membrana/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/sangre , Agregación Plaquetaria , Sistemas de Mensajero Secundario , Alprostadil/farmacología , Animales , Plaquetas/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/genética , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Agregación Plaquetaria/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.