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1.
Cell Physiol Biochem ; 54(1): 27-39, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31935048

RESUMO

BACKGROUND/AIMS: To test whether the physiological regulation of the cardiac Kv4 channels by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) is restricted to lipid rafts and whether the interactions observed in rat cardiomyocytes also occur in the human ventricle. METHODS: Ventricular myocytes were freshly isolated from Sprague-Dawley rats. Ito was recorded by the whole-cell Patch-Clamp technique. Membrane rafts were isolated by centrifugation in a discontinuous sucrose density gradient. The presence of the proteins of interest was analysed by western blot. Immunogold staining and electron microscopy of heart vibrosections was performed to localize Kv4.2/Kv4.3 and CaMKII proteins. Protein-protein interactions were determined by co-immunoprecipitation experiments in rat and human ventricular mycoytes. RESULTS: Patch-Clamp recordings in control conditions and after lipid raft or caveolae disruption show that the CaMKII-Kv4 channel complex must associate in non-caveolar lipid rafts to be functional. Separation in density gradients, co-immunoprecipitation and electron microscopy show that there are two Kv4 channel populations: one located in caveolae, that is CaMKII independent, and another one located in planar membrane rafts, which is bound to CaMKII. CONCLUSION: CaMKII regulates only the Kv4 channel population located in non-caveolar lipid rafts. Thus, the regulation of cardiac Kv4 channels in rat and human ventricle depends on their subcellular localization.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Microdomínios da Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio Shal/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/análise , Cavéolas/metabolismo , Células Cultivadas , Humanos , Transporte de Íons , Potássio/metabolismo , Mapas de Interação de Proteínas , Ratos Sprague-Dawley , Canais de Potássio Shal/análise
2.
Cell Mol Life Sci ; 77(2): 275-287, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31422442

RESUMO

Plasma membranes are heterogeneous and laterally compartmentalized into distinct microdomains. These membrane microdomains consist of special lipids and proteins and are thought to act as signaling platforms. In plants, membrane microdomains have been detected by super-resolution microscopy, and there is evidence that they play roles in several biological processes. Here, we review current knowledge about the lipid and protein components of membrane microdomains. Furthermore, we summarize the dynamics of membrane microdomains in response to different stimuli. We also explore the biological functions associated with membrane microdomains as signal integration hubs. Finally, we outline challenges and questions for further studies.


Assuntos
Membrana Celular/fisiologia , Microdomínios da Membrana/fisiologia , Células Vegetais/fisiologia , Animais , Membrana Celular/metabolismo , Humanos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Células Vegetais/metabolismo , Transdução de Sinais/fisiologia
3.
Int J Mol Sci ; 20(19)2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31574943

RESUMO

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.


Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Músculo Esquelético/metabolismo , Animais , Biomarcadores , Imunofluorescência , Microdomínios da Membrana/metabolismo , Junção Neuromuscular/metabolismo , Ratos , Esfingomielina Fosfodiesterase/metabolismo
4.
Int J Mol Sci ; 20(20)2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614433

RESUMO

Alterations on the immune system caused by omega-3 fatty acids have been described for 30 years. This family of polyunsaturated fatty acids exerts major alterations on the activation of cells from both the innate and the adaptive immune system, although the mechanisms for such regulation are diverse. First, as a constitutive part of the cellular membrane, omega-3 fatty acids can regulate cellular membrane properties, such as membrane fluidity or complex assembly in lipid rafts. In recent years, however, a new role for omega-3 fatty acids and their derivatives as signaling molecules has emerged. In this review, we describe the latest findings describing the effects of omega-3 fatty acids on different cells from the immune system and their possible molecular mechanisms.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Ácidos Graxos Ômega-3/efeitos adversos , Imunidade Inata/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Fluidez de Membrana/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo
5.
J Agric Food Chem ; 67(39): 10863-10870, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31507180

RESUMO

Mastitis, inflammation of the mammary gland, occurs in both humans and animals. Staphylococcus aureus is the most common infectious bacterial pathogen associated with mastitis. We investigated the effects of allicin on S. aureus-induced mastitis in mice. Pathological histology revealed that allicin inhibited S. aureus-induced pathological damage and myeloperoxidase activity in mammary tissues. Enzyme-linked immunosorbent assays demonstrated that allicin reduced the production of IL-1ß and TNF-α as well as inhibited the NF-κB and mitogen-activated protein kinase pathway by reducing phosphorylation of p65, IκBα, p38, JNK, and ERK. Western blotting revealed that allicin reduced TLR2 and TLR6 expression in mammary tissues and cells but not in HEK293 cells. The lipid raft content was reduced by allicin, which inhibited signaling downstream of TLR2 and TLR6. Liver X receptor α (LXRα) luciferase reporter assays and LXRα interference experiments showed that allicin improved the LXRα activity and adenosine 5'-triphosphate-binding cassette G and A1 (ABCG and ABCA1) expression, thereby reducing the cholesterol level, lipid raft formation, and downstream TLR2 and TLR6 pathway activity. These results demonstrated that allicin exerted anti-inflammatory effects against S. aureus mastitis by improving the LXRα activity and reducing lipid raft formation.


Assuntos
Mastite/tratamento farmacológico , Microdomínios da Membrana/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Ácidos Sulfínicos/administração & dosagem , Animais , Feminino , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Mastite/genética , Mastite/metabolismo , Mastite/microbiologia , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , NF-kappa B/metabolismo , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo
6.
Lipids Health Dis ; 18(1): 168, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477154

RESUMO

BACKGROUND: The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. METHODS: RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. RESULTS: Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRß knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. CONCLUSION: Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Colestenonas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/genética , Microdomínios da Membrana/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Lipogênese/genética , Receptores X do Fígado/metabolismo , Células MCF-7 , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Células THP-1
7.
Nat Cell Biol ; 21(8): 991-1002, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31371828

RESUMO

Migrasomes are recently discovered cellular organelles that form as large vesicle-like structures on retraction fibres of migrating cells. While the process of migrasome formation has been described before, the molecular mechanism underlying migrasome biogenesis remains unclear. Here, we propose that the mechanism of migrasome formation consists of the assembly of tetraspanin- and cholesterol-enriched membrane microdomains into micron-scale macrodomains, which swell into migrasomes. The major finding underlying the mechanism is that tetraspanins and cholesterol are necessary and sufficient for migrasome formation. We demonstrate the necessity of tetraspanins and cholesterol via live-cell experiments, and their sufficiency by generating migrasome-like structures in reconstituted membrane systems. We substantiate the mechanism by a theoretical model proposing that the key factor driving migrasome formation is the elevated membrane stiffness of the tetraspanin- and cholesterol-enriched macrodomains. Finally, the theoretical model was quantitatively validated by experimental demonstration of the membrane-stiffening effect of tetraspanin 4 and cholesterol.


Assuntos
Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Tetraspaninas/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Organelas/metabolismo
8.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408934

RESUMO

Recent findings have revealed the role of membrane traffic in the signaling of transforming growth factor-ß (TGF-ß). These findings originate from the pivotal function of TGF-ß in development, cell proliferation, tumor metastasis, and many other processes essential in malignancy. Actin and unconventional myosin have crucial roles in subcellular trafficking of receptors; research has also revealed a growing number of unconventional myosins that have crucial roles in TGF-ß signaling. Unconventional myosins modulate the spatial organization of endocytic trafficking and tether membranes or transport them along the actin cytoskeletons. Current models do not fully explain how membrane traffic forms a bridge between TGF-ß and the downstream effectors that produce its functional responsiveness, such as cell migration. In this review, we present a brief overview of the current knowledge of the TGF-ß signaling pathway and the molecular components that comprise the core pathway as follows: ligands, receptors, and Smad mediators. Second, we highlight key role(s) of myosin motor-mediated protein trafficking and membrane domain segregation in the modulation of the TGF-ß signaling pathway. Finally, we review future challenges and provide future prospects in this field.


Assuntos
Miosinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Humanos , Microdomínios da Membrana/metabolismo , Transporte Proteico
9.
Biosens Bioelectron ; 142: 111568, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31442945

RESUMO

A label-free, non-dispruptive, and real-time analytical device to monitor the dynamic features of biomolecules and their interactions with neighboring molecules is an essential prerequisite for biochip- and diagonostic assays. To explore one of the central questions on the lipid-lipid interactions in the course of the liquid-ordered (lo) domain formation, called rafts, we developed a method of reconstituting continuous but spatially heterogeneous lipid membrane platforms with molayer-bilayer juntions (MBJs) that enable to form the lo domains in a spatiotemporally controlled manner. This allows us to detect the time-lapse dynamics of the lipid-lipid interactions during raft formation and resultant membrane phase changes together with the raft-associated receptor-ligand binding through the surface plasmon resonance (SPR). For cross-validation, using epifluorescence microscopy, we demonstrated the underlying mechanisms for raft formations that the infiltration of cholesterols into the sphingolipid-enriched domains plays a crucial roles in the membrane phase-separation. Our membrane platform, being capable of monitoring dynamic interactions among lipids and performing the systematic optical analysis, will unveil physiological roles of cholesterols in a variety of biological events.


Assuntos
Colesterol/metabolismo , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Colesterol/análise , Desenho de Equipamento , Humanos , Cinética , Bicamadas Lipídicas/análise , Microdomínios da Membrana/química , Modelos Moleculares , Transição de Fase , Ligação Proteica , Ressonância de Plasmônio de Superfície/métodos
10.
Proc Jpn Acad Ser B Phys Biol Sci ; 95(7): 358-377, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406059

RESUMO

The neuronal growth cone is a highly motile, specialized structure for extending neuronal processes. This structure is essential for nerve growth, axon pathfinding, and accurate synaptogenesis. Growth cones are important not only during development but also for plasticity-dependent synaptogenesis and neuronal circuit rearrangement following neural injury in the mature brain. However, the molecular details of mammalian growth cone function are poorly understood. This review examines molecular findings on the function of the growth cone as a result of the introduction of novel methods such superresolution microscopy and (phospho)proteomics. These results increase the scope of our understating of the molecular mechanisms of growth cone behavior in the mammalian brain.


Assuntos
Cones de Crescimento/metabolismo , Mamíferos , Animais , Humanos , Microdomínios da Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Transdução de Sinais
11.
BMC Mol Cell Biol ; 20(1): 38, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455216

RESUMO

BACKGROUND: Flotillin-2 (Flot2) is a lipid raft scaffold protein that is thought to be related to neural differentiation. Flot2 is phosphorylated by Fyn, a Src kinase, and causes raft-dependent endocytosis; however, the exact role of Flot2 in neural differentiation remains unclear. To reveal the roles of lipid raft-associated proteins during neural differentiation, we tried to analyze the expression and localization. RESULTS: In this study, we found that the expression levels of the Flot2 and Fyn proteins increased in whole-cell lysates of P19C6 cells after neural differentiation. In addition, sucrose density fractionation and immunofluorescence experiments revealed an increase in the localization of Flot2 and Fyn to lipid rafts after neural differentiation. We also found that Fyn partially colocalized with Flot2 lipid rafts in neural cells. CONCLUSION: The observed distribution of Fyn and level of inactivated Fyn and/or c-Src in detergent-resistant membrane (DRM) fractions suggests that the amount of activated Fyn might increase in DRM fractions after neural differentiation. Overall these findings suggest that Flot2 lipid rafts are associated with Fyn, and that Fyn phosphorylates Flot2 during neural differentiation of P19C6 cells.


Assuntos
Diferenciação Celular , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Linhagem Celular Tumoral , Camundongos , Proteínas Proto-Oncogênicas c-fyn/metabolismo
12.
Int J Mol Sci ; 20(15)2019 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-31382686

RESUMO

Lipids in the brain are major components playing structural functions as well as physiological roles in nerve cells, such as neural communication, neurogenesis, synaptic transmission, signal transduction, membrane compartmentalization, and regulation of gene expression. Determination of brain lipid composition may provide not only essential information about normal brain functioning, but also about changes with aging and diseases. Indeed, deregulations of specific lipid classes and lipid homeostasis have been demonstrated in neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Furthermore, recent studies have shown that membrane microdomains, named lipid rafts, may change their composition in correlation with neuronal impairment. Lipid rafts are key factors for signaling processes for cellular responses. Lipid alteration in these signaling platforms may correlate with abnormal protein distribution and aggregation, toxic cell signaling, and other neuropathological events related with these diseases. This review highlights the manner lipid changes in lipid rafts may participate in the modulation of neuropathological events related to AD and PD. Understanding and characterizing these changes may contribute to the development of novel and specific diagnostic and prognostic biomarkers in routinely clinical practice.


Assuntos
Envelhecimento/metabolismo , Lipídeos/genética , Microdomínios da Membrana/metabolismo , Doenças Neurodegenerativas/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Microdomínios da Membrana/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Transdução de Sinais/genética
13.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261667

RESUMO

The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell-cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.


Assuntos
Microdomínios da Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Espermatozoides/metabolismo , Animais , Anexina A2/metabolismo , Catepsina D/metabolismo , Bovinos , Desmogleínas/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico/metabolismo , Hexoquinase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Placofilinas/metabolismo , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/genética
14.
PLoS Pathog ; 15(7): e1007907, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31344124

RESUMO

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities.


Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Efeito Espectador , Colesterol/metabolismo , Exossomos/metabolismo , Exossomos/virologia , Células HEK293 , HIV-1 , Humanos , Inflamação/metabolismo , Inflamação/virologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
15.
Biochim Biophys Acta Bioenerg ; 1860(12)2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31344362

RESUMO

Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 µm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.


Assuntos
Proteínas de Bactérias/metabolismo , Microdomínios da Membrana/metabolismo , Tilacoides/metabolismo , Imagem Tridimensional , Microscopia Confocal , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Synechocystis
16.
Cells ; 8(6)2019 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-31234505

RESUMO

Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells that can differentiate into myeloid or lymphoid cells. The mobilization and differentiation processes are affected by the external environment, such as extracellular matrix and soluble molecules in the niche, where the lipid rafts (LRs) of the HSCs act as the receptors and control platforms for these effectors. LRs are membrane microdomains that are enriched in cholesterol, sphingolipid, and proteins. They are involved in diverse cellular processes including morphogenesis, cytokinesis, signaling, endocytic events, and response to the environment. They are also involved in different types of diseases, such as cancer, Alzheimer's, and prion disease. LR clustering and disruption contribute directly to the differentiation, homing, hibernation, or mobilization of HSCs. Thus, characterization of LR integrity may provide a promising approach to controlling the fate of stem cells for clinical applications. In this review, we show the critical role of LR modification (clustering, disruption, protein incorporation, and signal responding) in deciding the fate of HSCs, under the effect of soluble cytokines such as stem cell factor (SCF), transforming growth factor- ß (TGF-ß), hematopoietic-specific phospholipase Cß2 (PLC-ß2), and granulocyte colony-stimulating factor (G-CSF).


Assuntos
Diferenciação Celular , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Humanos , Modelos Biológicos
17.
PLoS Comput Biol ; 15(5): e1007033, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31107861

RESUMO

G protein-coupled receptors (GPCRs) control cellular signaling and responses. Many of these GPCRs are modulated by cholesterol and polyunsaturated fatty acids (PUFAs) which have been shown to co-exist with saturated lipids in ordered membrane domains. However, the lipid compositions of such domains extracted from the brain cortex tissue of individuals suffering from GPCR-associated neurological disorders show drastically lowered levels of PUFAs. Here, using free energy techniques and multiscale simulations of numerous membrane proteins, we show that the presence of the PUFA DHA helps helical multi-pass proteins such as GPCRs partition into ordered membrane domains. The mechanism is based on hybrid lipids, whose PUFA chains coat the rough protein surface, while the saturated chains face the raft environment, thus minimizing perturbations therein. Our findings suggest that the reduction of GPCR partitioning to their native ordered environments due to PUFA depletion might affect the function of these receptors in numerous neurodegenerative diseases, where the membrane PUFA levels in the brain are decreased. We hope that this work inspires experimental studies on the connection between membrane PUFA levels and GPCR signaling.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células Receptoras Sensoriais/metabolismo , Encéfalo/metabolismo , Colesterol/metabolismo , Biologia Computacional , Simulação por Computador , Ácidos Docosa-Hexaenoicos/química , Ácidos Graxos Insaturados/metabolismo , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Modelos Neurológicos , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas-G/química , Células Receptoras Sensoriais/química , Transdução de Sinais , Termodinâmica
18.
Anesth Analg ; 129(4): 973-982, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31124840

RESUMO

BACKGROUND: Local anesthetics cause reversible block of pain and robustly inhibit TWIK-related K channel (TREK-1) currents. Before local anesthesia onset, injection of local anesthetics can cause unwanted transient pain. TREK-1 is an anesthetic-sensitive potassium channel that when inhibited produces pain. A disordered C-terminal loop of TREK-1 is thought to contribute to anesthetic sensitivity, but the molecular basis for TREK-1 inhibition by local anesthetics is unknown. Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA) required for TREK-1 activation and also binds to the channel's C terminus. METHODS: Here, we use biophysical and cellular techniques to characterize direct and indirect lipid-mediated mechanism for TREK-1 inhibition (respectively). We characterized direct binding of local anesthetic to TREK-1 by reconstituting the purified channel into artificial membranes and measuring ion flux. We characterized indirect PA-mediated inhibition of TREK-1 by monitoring lipid production in live whole cells using a fluorescent PLD2 product release assay and ion channel current using live whole-cell patch-clamp electrophysiology. We monitored anesthetic-induced nanoscale translocation of PLD2 to TREK-1 channels with super-resolution direct stochastic reconstruction microscopy (dSTORM). RESULTS: We find local anesthetics tetracaine, lidocaine, and bupivacaine directly bind to and inhibit PLD2 enzymatic activity. The lack of PLD2 activity indirectly inhibited TREK-1 currents. Select local anesthetics also partially blocked the open pore of TREK-1 through direct binding. The amount of pore block was variable with tetracaine greater than bupivacaine and lidocaine exhibiting a minor effect. Local anesthetics also disrupt lipid rafts, a mechanism that would normally activate PLD2 were it not for their direct inhibition of enzyme catalysis. CONCLUSIONS: We propose a mechanism of TREK-1 inhibition comprised of (1) primarily indirect PLD2-dependent inhibition of lipid catalysis and (2) limited direct inhibition for select local anesthetics through partial open pore block. The inhibition through PLD2 explains how the C terminus can regulate the channel despite being devoid of structure and putative binding sites for local anesthetics.


Assuntos
Anestésicos Locais/farmacologia , Bupivacaína/farmacologia , Lidocaína/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase D/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Tetracaína/farmacologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Domínios e Motivos de Interação entre Proteínas
19.
Dis Model Mech ; 12(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31036560

RESUMO

Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1-PI3K-Akt-mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions. This article has an associated First Person interview with the first author of the paper.


Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingolipidoses/genética , Animais , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Lisossomos/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Psicosina/farmacologia , Receptor IGF Tipo 1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esfingolipidoses/metabolismo
20.
J Pharmacol Exp Ther ; 370(1): 104-110, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31068382

RESUMO

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.


Assuntos
Adenilil Ciclases/metabolismo , Brônquios/citologia , AMP Cíclico/biossíntese , Miócitos de Músculo Liso/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/citologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Humanos , Isoproterenol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos
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