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1.
Langmuir ; 40(13): 7038-7048, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38511880

RESUMO

The phospholipase A2 (PLA2) superfamily consists of lipolytic enzymes that hydrolyze specific cell membrane phospholipids and have long been considered a central hub of biosynthetic pathways, where their lipid metabolites exert a variety of physiological roles. A misregulated PLA2 activity is associated with mainly inflammatory-derived pathologies and thus has shown relevant therapeutic potential. Many natural and synthetic anti-inflammatory drugs (AIDs) have been proposed as direct modulators of PLA2 activity. However, despite the specific chemical properties that these drugs share in common, little is known about the indirect modulation able to finely tune membrane structural changes at the precise lipid-binding site. Here, we use a novel experimental strategy based on differential scanning calorimetry to systematically study the structural properties of lipid membrane systems during PLA2 cleavage and under the influence of several AIDs. For a better understanding of the AIDs-membrane interaction, we present a comprehensive and comparative set of molecular dynamics (MD) simulations. Our thermodynamic results clearly demonstrate that PLA2 cleavage is hindered by those AIDs that significantly reduce the lipid membrane cooperativity, while the rest of the AIDs oppositely tend to catalyze PLA2 activity to different extents. On the other hand, our MD simulations support experimental results by providing atomistic details on the binding, insertion, and dynamics of each AID on a pure lipid system; the drug efficacy to impact membrane cooperativity is related to the lipid order perturbation. This work suggests a membrane-based mechanism of action for diverse AIDs against PLA2 activity and provides relevant clues that must be considered in its modulation.


Assuntos
Simulação de Dinâmica Molecular , Fosfolipídeos , Fosfolipases A2/química , Fosfolipídeos/química , Membrana Celular/metabolismo , Fenômenos Biofísicos
2.
Nat Commun ; 14(1): 1167, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36859399

RESUMO

Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.


Assuntos
Síndrome de Angelman , Canais Iônicos , Ácido Linoleico , Animais , Feminino , Humanos , Masculino , Camundongos , Alelos , Síndrome de Angelman/tratamento farmacológico , Síndrome de Angelman/genética , Modelos Animais de Doenças , Deficiência Intelectual , Canais Iônicos/genética , Ácido Linoleico/farmacologia
3.
Biochim Biophys Acta Biomembr ; 1863(2): 183509, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33189718

RESUMO

Although sharing common properties with other divalent cations, calcium ions induce fine-tuned electrostatic effects essential in many biological processes. Not only related with protein structure or ion channels, calcium is also determinant for other biomolecules such as lipids or even drugs. Cellular membranes are the first interaction barriers for drugs. Depending on their hydrophilic, hydrophobic or amphipathic properties, they have to overcome such barriers to permeate and diffuse through inner lipid bilayers, cells or even tissues. In this context, the role of calcium in the permeation of cationic amphiphilic drugs (CADs) through lipid membranes is not well understood. We combine differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR) to investigate the effect of Ca2+ on the interlamellar diffusion kinetics of the local anesthetic tetracaine (TTC) in multilamellar artificial membrane systems. Our DSC results show the interesting phenomenon that TTC diffusion can be modified in two different ways in the presence of Ca2+. Furthermore, TTC diffusion exhibits a thermal-dependent membrane interaction in the presence of Ca2+. The FTIR results suggest the presence of ion-dipole interactions between Ca2+ and the carbonyl group of TTC, leading us to hypothesize that Ca2+ destabilizes the hydration shell of TTC, which in turn diffuses deeper into the multilamellar lipid structures. Our results demonstrate the relevance of the Ca2+ ion in the drug permeation and diffusion through lipid bilayers.


Assuntos
Anestésicos Locais/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Tetracaína/química , Varredura Diferencial de Calorimetria , Cinética , Espectroscopia de Infravermelho com Transformada de Fourier
4.
Nat Commun ; 10(1): 1200, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867417

RESUMO

Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell's response to mechanical cues.


Assuntos
Anemia Hemolítica Congênita/dietoterapia , Gorduras na Dieta/metabolismo , Hidropisia Fetal/dietoterapia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Anemia Hemolítica Congênita/genética , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Gorduras na Dieta/administração & dosagem , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/administração & dosagem , Ácidos Graxos Insaturados/metabolismo , Mutação com Ganho de Função , Células HEK293 , Humanos , Hidropisia Fetal/genética , Canais Iônicos/genética , Metabolismo dos Lipídeos/fisiologia , Camundongos , Microscopia de Força Atômica , Técnicas de Patch-Clamp
5.
J Vis Exp ; (137)2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-30035769

RESUMO

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.


Assuntos
Análise Espectral/métodos , Canais de Cátion TRPV/genética , Células HEK293 , Humanos , Canais de Cátion TRPV/metabolismo
6.
Sci Rep ; 7(1): 9861, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28852163

RESUMO

The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.


Assuntos
Expressão Gênica , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/isolamento & purificação , Sequência de Aminoácidos , Animais , Cisteína/química , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Conformação Proteica , Estabilidade Proteica , Prótons , Ratos , Proteínas Recombinantes , Análise Espectral , Canais de Cátion TRPV/química , Xenopus
7.
Cell Rep ; 21(1): 246-258, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978477

RESUMO

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoid's epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure.


Assuntos
Anti-Hipertensivos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Canais de Cátion TRPV/genética , Animais , Animais Geneticamente Modificados , Anti-Hipertensivos/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Expressão Gênica , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Metaboloma , Forbóis/farmacologia , Fosfolipídeos/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismo
8.
PLoS One ; 8(4): e59364, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23565149

RESUMO

We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine) and excitatory (produced by either caffeine or calcium) effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory) on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and dipalmitoyl phosphatidic acid (DPPA). Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Bicamadas Lipídicas , Lipídeos de Membrana/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Animais , Cafeína/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Lipossomos/química , Lipossomos/metabolismo , Masculino , Lipídeos de Membrana/química , Camundongos , Conformação Molecular , Simulação de Dinâmica Molecular , Motilidade dos Espermatozoides/fisiologia , Temperatura , Tetracaína/farmacologia
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