RESUMO
Phosphatidylinositol (PI) lipids control critical biological processes, so aberrant biosynthesis often leads to disease. As a result, the capability to track the production and localization of these compounds in cells is vital for elucidating their complex roles. Herein, we report the design, synthesis, and application of clickable myo-inositol probe 1 a for bioorthogonal labeling of PI products. To validate this platform, we initially conducted PI synthase assays to show that 1 a inhibits PI production in vitro. Fluorescence microscopy experiments next showed probe-dependent imaging in T-24 human bladder cancer and Candida albicans cells. Growth studies in the latter showed that replacement of myo-inositol with probe 1 a led to an enhancement in cell growth. Finally, fluorescence-based TLC analysis and mass spectrometry experiments support the labeling of PI lipids. This approach provides a promising means for tracking the complex biosynthesis and trafficking of these lipids in cells.
Assuntos
Corantes Fluorescentes/química , Inositol/química , Engenharia Metabólica , Fosfatidilinositóis/química , Candida albicans/citologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Células Cultivadas , Química Click , Corantes Fluorescentes/síntese química , Humanos , Inositol/síntese química , Imagem ÓpticaRESUMO
Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Isquemia/patologia , Rim/patologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Biotinilação , Linhagem Celular , Citoplasma/metabolismo , Cães , Dinaminas/metabolismo , Endocitose , Células Epiteliais/citologia , Humanos , Rim/lesões , Nefropatias/metabolismo , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosforilação , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Traumatismo por Reperfusão , Transdução de SinaisRESUMO
Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Fenômenos Biofísicos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Desenho de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Lipídeos de Membrana/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/toxicidade , Ligação Proteica , Conformação Proteica , SolubilidadeRESUMO
The pH-low insertion peptide (pHLIP) targets acidic diseases such as cancer. The acidity of the environment causes key aspartic acids in pHLIP to become protonated, causing the peptide to insert into membranes. Here we investigate how the negative charge of the membrane influences how pHLIP enters and exits the lipid bilayer. We found that electrostatic repulsion affected differently the membrane entry and exit of pHLIP for negatively charged membranes. As a consequence, a large hysteresis was observed. We propose this is not a consequence of structural changes but results from local changes in the environment of aspartic acids, shifting their pK values.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Condutividade Elétrica , Elétrons , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Transporte ProteicoRESUMO
For drug delivery purposes, the ability to conveniently attach a targeting moiety that will deliver drugs to cells and then enable controlled release of the active molecule after localization is desirable. Toward this end, we designed and synthesized clickable and photocleavable lipid analogue 1 to maximize the efficiency of bioconjugation and triggered release. This compound contains a dibenzocyclooctyne group for bioorthogonal derivatization linked via a photocleavable 2-nitrobenzyl moiety at the headgroup of a synthetic lipid backbone for targeting to cell membranes. To assess delivery and release using this system, we report fluorescence-based assays for liposomal modification and photocleavage in solution as well as through surface immobilization to demonstrate successful liposome functionalization and photoinduced release. In addition, fluorophore delivery to and release from live cells was confirmed and characterized using fluorescence microscopy and flow cytometry analysis in which 1 was delivered to cells, derivatized, and photocleaved. Finally, drug delivery studies were performed using an azide-tagged analogue of camptothecin, a potent anticancer drug that is challenging to deliver due to poor solubility. In this case, the ester attachment of the azide tag acted as a caging group for release by intracellular esterases rather than through photocleavage. This resulted in a dose-dependent response in the presence of liposomes containing delivery agent 1, confirming the ability of this compound to stimulate delivery to the cytoplasm of cells.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Preparações de Ação Retardada/química , Lipídeos/química , Lipossomos/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Azidas/química , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Luz , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Imagem Óptica , Processos Fotoquímicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismoRESUMO
Phosphatidylserine (PS) is a key lipid that plays important roles in disease-related biological processes, and therefore, the means to track PS in live cells are invaluable. Herein, we describe the metabolic labeling of PS in Saccharomyces cerevisiae cells using analogues of serine, a PS precursor, derivatized with azide moieties at either the amino (N-l-SerN3) or carbonyl (C-l-SerN3) groups. The conservative click tag modification enabled these compounds to infiltrate normal lipid biosynthetic pathways, thereby producing tagged PS molecules as supported by mass spectrometry studies, thin-layer chromatography (TLC) analysis, and further derivatization with fluorescent reporters via click chemistry to enable imaging in yeast cells. This approach shows strong prospects for elucidating the complex biosynthetic and trafficking pathways involving PS.
Assuntos
Fosfatidilserinas , Saccharomyces cerevisiae , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Química ClickRESUMO
Candida albicans causes severe invasive candidiasis. C. albicans infection requires the virulence factor candidalysin (CL) which damages target cell membranes. However, the mechanism that CL uses to permeabilize membranes is unclear. We reveal that CL forms membrane pores using a unique mechanism. Unexpectedly, CL readily assembled into polymers in solution. We propose that the basic structural unit in polymer formation is a CL oligomer, which is sequentially added into a string configuration that can close into a loop. CL loops appear to spontaneously insert into the membrane to become pores. A CL mutation (G4W) inhibited the formation of polymers in solution and prevented pore formation in synthetic lipid systems. Epithelial cell studies showed that G4W CL failed to activate the danger response pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of CL pores by atomic force microscopy revealed co-existence of simple depressions and more complex pores, which are likely formed by CL assembled in an alternate oligomer orientation. We propose that this structural rearrangement represents a maturation mechanism that stabilizes pore formation to achieve more robust cellular damage. To summarize, CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.
The fungus Candida albicans is the most common cause of yeast infections in humans. Like many other disease-causing microbes, it releases several virulent proteins that invade and damage human cells. This includes the peptide candidalysin which has been shown to be crucial for infection. Human cells are surrounded by a protective membrane that separates their interior from their external environment. Previous work showed that candidalysin damages the cell membrane to promote infection. However, how candidalysin does this remained unclear. Similar peptides and proteins cause harm by inserting themselves into the membrane and then grouping together to form a ring. This creates a hole, or 'pore', that weakens the membrane and allows other molecules into the cell's interior. Here, Russell, Schaefer et al. show that candidalysin uses a unique pore forming mechanism to impair the membrane of human cells. A combination of biophysical and cell biology techniques revealed that the peptide groups together to form a chain. This chain of candidalysin proteins then closes in on itself to create a loop structure that can insert into the membrane to form a pore. Once embedded within the membrane, the proteins within the loops rearrange again to make the pores more stable so they can cause greater damage. This type of pore formation has not been observed before, and may open up new avenues of research. For instance, researchers could use this information to develop inhibitors that stop candidalysin from forming chains and harming the membranes of cells. This could help treat the infections caused by C. albicans.
Assuntos
Candida albicans , Fatores de Virulência , Candida albicans/genética , Células Epiteliais/metabolismo , Proteínas Fúngicas , Lipídeos , Polímeros/metabolismo , Fatores de Virulência/metabolismoRESUMO
Alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion. AMP-activated protein kinase (AMPK), a cellular energy sensor, is rapidly activated in response to renal ischemia, and we demonstrate that its activity is upregulated by energy depletion in Madin-Darby canine kidney (MDCK) cells. We hypothesized that AMPK activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion. MDCK cells were ATP depleted through a 1-h incubation with antimycin A and 2-deoxyglucose. Immunofluoresence localization demonstrated that this regimen induces mislocalization of the Na-K-ATPase from its normal residence at the basolateral plasma membrane to intracellular vesicular compartments. When cells were pretreated with the AMPK activator metformin before energy depletion, basolateral localization of Na-K-ATPase was preserved. In MDCK cells in which AMPK expression was stably knocked down with short hairpin RNA, preactivation of AMPK with metformin did not prevent Na-K-ATPase redistribution in response to energy depletion. In vivo studies demonstrate that metformin activated renal AMPK and that treatment with metformin before renal ischemia preserved cellular integrity, preserved Na-K-ATPase localization, and led to reduced levels of neutrophil gelatinase-associated lipocalin, a biomarker of tubular injury. Thus AMPK may play a role in preserving the functional integrity of epithelial plasma membrane domains in the face of energy depletion. Furthermore, pretreatment with an AMPK activator before ischemia may attenuate the severity of renal tubular injury in the context of acute kidney injury.
Assuntos
Proteínas Quinases Ativadas por AMP/biossíntese , Injúria Renal Aguda/enzimologia , Rim/irrigação sanguínea , Rim/enzimologia , Metformina/farmacologia , Traumatismo por Reperfusão/enzimologia , Injúria Renal Aguda/patologia , Animais , Antimetabólitos/farmacologia , Antimicina A/farmacologia , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Cães , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Traumatismo por Reperfusão/patologia , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.
Assuntos
Melanoma/patologia , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Receptor EphA2/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Movimento Celular , Humanos , Ligantes , Melanoma/metabolismo , Proteínas de Membrana/química , Membranas/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Receptor EphA2/química , Homologia de Sequência , Células Tumorais CultivadasRESUMO
pH-responsive peptides are promising therapeutic molecules that can specifically target the plasma membrane in the acidified extracellular medium that bathes cells in tumors. We designed the acidity-triggered rational membrane (ATRAM) peptide to have a pH-responsive membrane interaction. At physiological pH, ATRAM binds to the membrane surface in a largely unstructured conformation, while in acidic conditions it inserts into lipid bilayers forming a transmembrane helix. However, the molecular mechanism ATRAM uses to target and insert into tumor cells remains poorly understood. Here, we determined that ATRAM inserts into cancer cells with a preferential membrane orientation, where the C-terminus of the peptide traverses the plasma membrane and explores the cytoplasm. Using biophysical techniques, we determined that the membrane interaction of ATRAM is contingent on the concentration of the peptide. Kinetic studies showed that membrane insertion occurs in at least three steps, where only the first step was affected by the membrane density of ATRAM. These observations, combined with membrane binding and leakage data, indicate that the interaction of ATRAM with lipid membranes is dependent on its oligomerization state. SPECT/CT imaging in mice revealed that ATRAM accumulates in the blood pool, where it has a prolonged circulation time (> 4â¯h). Since fast peptide clearance and degradation in circulation are major problems for clinical development, we studied the mechanism ATRAM uses to remain in the blood stream. Using binding and transfer assays, we determined that ATRAM binds reversibly to human serum albumin. We propose that ATRAM uses albumin as a carrier in the blood stream to evade clearance and proteolysis before interacting with the plasma membrane of cancer cells. We also show that ATRAM is able to be deliver liposomes to cells in a pH dependent way. Our data highlight the potential of ATRAM as a specific therapeutic agent for diseases that lead to acidic tissues, including cancer.
Assuntos
Membrana Celular/metabolismo , Peptídeos/metabolismo , Albumina Sérica Humana/metabolismo , Animais , Neoplasias da Mama/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/metabolismo , Lipossomos , Células MCF-7 , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Boronic acid liposomes enable triggered content release and cell delivery driven by carbohydrate binding. Dye release assays using hydrophilic and hydrophobic fluorophores validate dose-dependent release upon carbohydrate treatment. Microscopy results indicate dramatic enhancements in cell delivery, showcasing the prospects of boronic acid lipids for drug delivery.
Assuntos
Ácidos Borônicos/química , Diglicerídeos/química , Portadores de Fármacos/química , Heparina/química , Lipossomas Unilamelares/química , Ácidos Borônicos/síntese química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/toxicidade , Linhagem Celular Tumoral , Diglicerídeos/síntese química , Diglicerídeos/metabolismo , Diglicerídeos/toxicidade , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Humanos , Oxazinas/química , Pinocitose/efeitos dos fármacos , Rodaminas/química , Lipossomas Unilamelares/síntese química , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/toxicidadeRESUMO
Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism.
Assuntos
Efrina-A1/genética , Efrina-A2/genética , Neoplasias/genética , Peptídeos/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Efrina-A1/química , Efrina-A2/química , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neoplasias/tratamento farmacológico , Peptídeos/administração & dosagem , Peptídeos/farmacologia , Fosforilação , Domínios Proteicos/genética , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética , Receptor EphA2RESUMO
Commercial plant extracts containing anthraquinones are being increasingly used for cosmetics, food and pharmaceuticals due to their wide therapeutic and pharmacological properties. In this work, the interaction with model membranes of two representative 1,8-dihydroxyanthraquinones, barbaloin (Aloe) and emodin (Rheum, Polygonum), has been studied in order to explain their effects in biological membranes. Emodin showed a higher affinity for phospholipid membranes than barbaloin did, and was more effective in weakening hydrophobic interactions between hydrocarbon chains in phospholipid bilayers. Whereas emodin induced the formation of hexagonal-H(II) phase, barbaloin stabilized lamellar structures. Barbaloin promoted the formation of gel-fluid intermediate structures in phosphatidylglycerol membranes at physiological pH and ionic strength values. It is proposed that emodin's chromophore group is located at the upper half of the membrane, whereas barbaloin's one is in a deeper position but having its glucopyranosyl moiety near the phospholipid/water interface. Moreover, membrane disruption by emodin or barbaloin showed specificity for the two major phospholipids present in bacterial membranes, phosphatidylethanolamine and phosphatidylglycerol. In order to relate their strong effects on membranes to their biological activity, the capacity of these compounds to inhibit the infectivity of the viral haemorrhagic septicaemia rhabdovirus (VHSV), a negative RNA enveloped virus, or the growth of Escherichia coli was tested. Anthraquinone-loaded liposomes showed a strong antimicrobial activity whereas these compounds in their free form did not. Both anthraquinones showed antiviral activity but only emodin was a virucidal agent. In conclusion, a molecular mechanism based on the effect of these compounds on the structure of biological membranes is proposed to account for their multiple biological activities. Anthraquinone-loaded liposomes may suppose an alternative for antimicrobial, pharmaceutical or cosmetic applications.
Assuntos
Antracenos/administração & dosagem , Emodina/administração & dosagem , Antracenos/química , Antracenos/metabolismo , Antraquinonas/administração & dosagem , Antraquinonas/química , Antraquinonas/metabolismo , Sistemas de Liberação de Medicamentos , Emodina/química , Emodina/metabolismo , Lipossomos , Membranas/metabolismo , Fosfatidilgliceróis/química , Espectrometria de Fluorescência , TemperaturaRESUMO
We have previously demonstrated that the highly conserved R209, that flanks the M1 transmembrane segment of nicotinic acetylcholine (ACh) receptors, is required for the transport of assembled homomeric neuronal α7 nicotinic ACh receptors to the cell surface. In the present paper we show that basic residues at positions 208 and 210 are necessary for the assembly of α7 receptors. On the contrary, a basic residue at position 210 of α3 subunit decreases the assembly of heteromeric neuronal α3ß4 nicotinic ACh receptors. A basic residue at position 210 of the ß4 subunit slightly decreases α3ß4 receptor expression. We conclude that a pre-M1 RRR motif is necessary for the biogenesis of homomeric α-bungarotoxin-sensitive neuronal α7 nicotinic ACh receptors.
Assuntos
Motivos de Aminoácidos/genética , Mutação , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Bungarotoxinas/metabolismo , Bovinos , Espaço Extracelular/metabolismo , Feminino , Radioisótopos do Iodo , Oócitos/metabolismo , Ligação Proteica , Multimerização Proteica , Piridinas/metabolismo , Ensaio Radioligante , Receptores Nicotínicos/química , Trítio , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7RESUMO
The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Linhagem Celular , Chlorocebus aethiops , Cães , Relação Dose-Resposta a Droga , Endocitose , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Proteínas Ativadoras de GTPase/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Barbaloin is a bioactive glycosilated 1,8-dihydroxyanthraquinone present in several exudates from plants, such as Aloe vera, which are used for cosmetic or food purposes. It has been shown that barbaloin interacts with DMPG (dimyristoylphosphatidylglycerol) model membranes, altering the bilayer structure (Alves, D. S.; Pérez-Fons, L.; Estepa, A.; Micol, V. Biochem. Pharm. 2004, 68, 549). Considering that ESR (electron spin resonance) of spin labels is one of the best techniques to monitor structural properties at the molecular level, the alterations caused by the anthraquinone barbaloin on phospholipid bilayers will be discussed here via the ESR signal of phospholipid spin probes intercalated into the membranes. In DMPG at high ionic strength (10 mM Hepes pH 7.4 + 100 mM NaCl), a system that presents a gel-fluid transition around 23 degrees C, 20 mol % barbaloin turns the gel phase more rigid, does not alter much the fluid phase packing, but makes the lipid thermal transition less sharp. However, in a low-salt DMPG dispersion (10 mM Hepes pH 7.4 + 2 mM NaCl), which presents a rather complex gel-fluid thermal transition (Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19), barbaloin strongly affects bilayer structural properties, both in the gel and fluid phases, extending the transition region to much higher temperature values. The position of barbaloin in DMPG bilayers will be discussed on the basis of ESR results, in parallel with data from sample viscosity, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering).