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1.
Proc Natl Acad Sci U S A ; 117(44): 27141-27147, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33024017

RESUMO

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need to rapidly develop therapeutic strategies for such emerging viruses without effective vaccines or drugs. Here, we report a decoy nanoparticle against COVID-19 through a powerful two-step neutralization approach: virus neutralization in the first step followed by cytokine neutralization in the second step. The nanodecoy, made by fusing cellular membrane nanovesicles derived from human monocytes and genetically engineered cells stably expressing angiotensin converting enzyme II (ACE2) receptors, possesses an antigenic exterior the same as source cells. By competing with host cells for virus binding, these nanodecoys effectively protect host cells from the infection of pseudoviruses and authentic SARS-CoV-2. Moreover, relying on abundant cytokine receptors on the surface, the nanodecoys efficiently bind and neutralize inflammatory cytokines including interleukin 6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and significantly suppress immune disorder and lung injury in an acute pneumonia mouse model. Our work presents a simple, safe, and robust antiviral nanotechnology for ongoing COVID-19 and future potential epidemics.


Assuntos
Infecções por Coronavirus/terapia , Citocinas/antagonistas & inibidores , Nanopartículas/uso terapêutico , Pneumonia Viral/terapia , Internalização do Vírus/efeitos dos fármacos , Animais , Betacoronavirus , Membrana Celular/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Células HEK293 , Humanos , Interleucina-6/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos ICR , Monócitos , Nanopartículas/química , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores de Citocinas/metabolismo , Células THP-1
2.
Int J Nanomedicine ; 15: 6749-6760, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32982231

RESUMO

Background: The combination of radiotherapy (RT) and chemotherapy, as a standard treatment for breast cancer in the clinic, is unsatisfactory due to chemoradioresistance and severe side effects. Methods and Results: To address these issues, a cancer cell-erythrocyte hybrid membrane-coated doxorubicin (DOX)-loaded gold nanocage (CM-EM-GNCs@DOX) was constructed for near-infrared light (NIR)-activated photothermal/radio/chemotherapy of breast cancer. CM-EM-GNCs@DOX inherited an excellent homologous target ability from the cancer cell membrane and an immune evasion capability from the erythrocyte membrane, together resulting in highly efficient accumulation in the tumor site with decreased clearance. Following the highly efficient uptake of CM-EM-GNCs@DOX in cancer cells, the RT efficacy was remarkably amplified due to the radiosensitization effect of CM-EM-GNCs@DOX, which reduced the needed radiotherapeutic dose. Importantly, with NIR irradiation, CM-EM-GNCs@DOX exerted a high photothermal effect, which not only ruptured CM-EM-GNCs@DOX to release DOX for precise and controllable chemotherapy, but also potentiated chemo/radiotherapy by photothermal therapy. Conclusion: Therefore, a highly efficient and safe combined photothermal/radio/chemotherapy approach was achieved in vitro and in vivo by CM-EM-GNCs@DOX, which provided a promising strategy for treating breast cancer.


Assuntos
Neoplasias da Mama/terapia , Membrana Celular/química , Doxorrubicina/administração & dosagem , Nanoestruturas/química , Fototerapia/métodos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Membrana Eritrocítica/química , Feminino , Ouro/química , Humanos , Hipertermia Induzida/métodos , Raios Infravermelhos , Células MCF-7 , Fusão de Membrana , Camundongos , Camundongos Nus , Nanoestruturas/administração & dosagem , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacologia , Células RAW 264.7 , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Chemosphere ; 254: 126918, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32957302

RESUMO

The increasing application of various surfactants nowadays, may lead to the contamination of the natural environment and represent potential threat to terrestrial higher plants. In this article, the effect of 13 surfactants, with dodecyl alkyl chain and various aromatic (imidazolium, pyridinium, thiazolium) and aliphatic (guanidinium, ammonium, thiosemicarbazidium) polar heads, on germination, development and growth of wheat and cucumber was investigated. The study aimed to prove how changes in lipophilicity of surfactants and their various structural modifications (existence of the aliphatic or aromatic polar group, the introduction of oxygen and sulfur) influence toxicity towards investigated plants. The calculated lipophilic parameter (AlogP) is shown to be a useful parameter for predicting potential toxicity of the compound. The strategy of using surfactants with aliphatic polar heads instead of aromatic prove to be a promising strategy in reducing harmful effect, as well as the introduction of polar groups in the structure of cation. From all investigated compounds, surfactants with imidazolium polar head displayed the most harmful effect towards wheat and cucumber. The cucumber seeds were more sensitive to the addition of surfactants comparing to wheat. All obtained experimental results were additionally investigated using computational methods, simulating the transport of surfactants through a lipid bilayer. The influence of cation tendency to fit in lipid bilayer structure was correlated with toxicity. For the first time, it is concluded that cation ability to mimic the structure of bilayer have less harmful effect on plant development.


Assuntos
Cucumis sativus/efeitos dos fármacos , Imidazóis/toxicidade , Compostos de Piridínio/toxicidade , Tensoativos/toxicidade , Triticum/efeitos dos fármacos , Cátions , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Germinação/efeitos dos fármacos , Imidazóis/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tensoativos/química , Triticum/crescimento & desenvolvimento
4.
J Am Chem Soc ; 142(40): 17024-17038, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-32926779

RESUMO

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.


Assuntos
Antivirais/química , Antivirais/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Organofosfatos/farmacologia , Proteínas do Envelope Viral/efeitos dos fármacos , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Amiloide/antagonistas & inibidores , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Arginina/química , Betacoronavirus/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/química , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Lipídeos/química , Lisina/química , Espectroscopia de Ressonância Magnética , Organofosfatos/química , Proteínas Secretadas pela Vesícula Seminal/química , Proteínas Secretadas pela Vesícula Seminal/metabolismo , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo , Zika virus/efeitos dos fármacos
5.
Adv Exp Med Biol ; 1274: 5-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894505

RESUMO

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.


Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/antagonistas & inibidores , Aciltransferases/antagonistas & inibidores , Membrana Celular/metabolismo , Desenvolvimento de Medicamentos , Glicerofosfolipídeos/biossíntese , Glicerofosfolipídeos/química , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Animais , Membrana Celular/química , Membrana Celular/enzimologia , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Humanos
6.
Nat Commun ; 11(1): 4314, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887878

RESUMO

Previous studies on the phase behaviour of multicomponent lipid bilayers found an intricate interplay between membrane geometry and its composition, but a fundamental understanding of curvature-induced effects remains elusive. Thanks to a combination of experiments on lipid vesicles supported by colloidal scaffolds and theoretical work, we demonstrate that the local geometry and global chemical composition of the bilayer determine both the spatial arrangement and the amount of mixing of the lipids. In the mixed phase, a strong geometrical anisotropy can give rise to an antimixed state, where the lipids are mixed, but their relative concentration varies across the membrane. After phase separation, the bilayer organizes in multiple lipid domains, whose location is pinned in specific regions, depending on the substrate curvature and the bending rigidity of the lipid domains. Our results provide critical insights into the phase separation of cellular membranes and, more generally, two-dimensional fluids on curved substrates.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Microdomínios da Membrana , Lipossomos/química
7.
Exp Parasitol ; 218: 107964, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32822697

RESUMO

Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. infantum and L. amazonensis are etiologic agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts, but such an approach tends to underrepresent membrane-associated proteins due to their high hydrophobicity and low solubility. Considering the relevance of this category of proteins in virulence, invasiveness and the host-parasite interface, this study applied label-free proteomics to assess the plasma membrane sub-proteome of L. infantum and L. amazonensis. The number of proteins identified in L. infantum and L. amazonensis promastigotes was 1168 and 1455, respectively. After rigorous data processing and mining, 157 proteins were classified as putative plasma membrane-associated proteins, of which 56 proteins were detected in both species, six proteins were detected only in L. infantum and 39 proteins were exclusive to L. amazonensis. The quantitative analysis revealed that two proteins were more abundant in L. infantum, including the glucose transporter 2, and five proteins were more abundant in L. amazonensis. The identified proteins associated with distinct processes and functions. In this regard, proteins of L. infantum were linked to metabolic processes whereas L. amazonensis proteins were involved in signal transduction. Moreover, transmembrane transport was a significant process among the group of proteins detected in both species and members of the superfamily of ABC transporters were highly represented. Interestingly, some proteins of this family were solely detected in L. amazonensis, such as ABCA9. GP63, a well-known virulence factor, was the only GPI-anchored protein identified in the membrane preparations of both species. Finally, we found several proteins with uncharacterized functions, including differentially abundant ones, highlighting a gap in the study of Leishmania proteins. Proteins characterization could provide a better biological understanding of these parasites and deliver new possibilities regarding the discovery of therapeutic targets, drug resistance and vaccine candidates.


Assuntos
Leishmania infantum/química , Leishmania mexicana/química , Proteínas de Membrana/análise , Proteômica/métodos , Proteínas de Protozoários/análise , Animais , Membrana Celular/química , Cromatografia Líquida , Biologia Computacional , Cricetinae , Transportador de Glucose Tipo 2/análise , Interações Hospedeiro-Parasita , Leishmania infantum/metabolismo , Leishmania infantum/patogenicidade , Leishmania infantum/ultraestrutura , Leishmania mexicana/ultraestrutura , Macrófagos Peritoneais/parasitologia , Espectrometria de Massas , Mesocricetus , Metaloendopeptidases/análise , Camundongos , Camundongos Endogâmicos BALB C , Transdução de Sinais , Espectrometria de Massas em Tandem , Virulência
8.
Proc Natl Acad Sci U S A ; 117(33): 19713-19719, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759217

RESUMO

Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.


Assuntos
Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Bicamadas Lipídicas/química , Membrana Celular/química , Microdomínios da Membrana/química , Microdomínios da Membrana/ultraestrutura
10.
Nature ; 584(7821): 479-483, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788728

RESUMO

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.


Assuntos
Membrana Celular/química , Escherichia coli/química , Escherichia coli/patogenicidade , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Amidoidrolases/química , Amidoidrolases/metabolismo , Motivos de Aminoácidos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Estabilidade Enzimática , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Genes Essenciais , Hidrolases/química , Hidrolases/metabolismo , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/biossíntese , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Periplasma/química , Periplasma/metabolismo , Ligação Proteica , Virulência
11.
Phys Rev Lett ; 125(6): 068101, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32845697

RESUMO

Shape, dynamics, and viscoelastic properties of eukaryotic cells are primarily governed by a thin, reversibly cross-linked actomyosin cortex located directly beneath the plasma membrane. We obtain time-dependent rheological responses of fibroblasts and MDCK II cells from deformation-relaxation curves using an atomic force microscope to access the dependence of cortex fluidity on prestress. We introduce a viscoelastic model that treats the cell as a composite shell and assumes that relaxation of the cortex follows a power law giving access to cortical prestress, area-compressibility modulus, and the power law exponent (fluidity). Cortex fluidity is modulated by interfering with myosin activity. We find that the power law exponent of the cell cortex decreases with increasing intrinsic prestress and area-compressibility modulus, in accordance with previous finding for isolated actin networks subject to external stress. Extrapolation to zero tension returns the theoretically predicted power law exponent for transiently cross-linked polymer networks. In contrast to the widely used Hertzian mechanics, our model provides viscoelastic parameters independent of indenter geometry and compression velocity.


Assuntos
Actinas/química , Fibroblastos/química , Fibroblastos/citologia , Modelos Biológicos , Actinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/química , Membrana Celular/fisiologia , Força Compressiva , Cães , Elasticidade , Microscopia de Força Atômica , Miosinas/química , Miosinas/fisiologia , Reologia/métodos , Viscosidade
12.
Phys Rev Lett ; 125(7): 078102, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32857533

RESUMO

Diffusion-mediated surface phenomena are crucial for human life and industry, with examples ranging from oxygen capture by lung alveolar surface to heterogeneous catalysis, gene regulation, membrane permeation, and filtration processes. Their current description via diffusion equations with mixed boundary conditions is limited to simple surface reactions with infinite or constant reactivity. In this Letter, we propose a probabilistic approach based on the concept of boundary local time to investigate the intricate dynamics of diffusing particles near a reactive surface. Reformulating surface-particle interactions in terms of stopping conditions, we obtain in a unified way major diffusion-reaction characteristics such as the propagator, the survival probability, the first-passage time distribution, and the reaction rate. This general formalism allows us to describe new surface reaction mechanisms such as for instance surface reactivity depending on the number of encounters with the diffusing particle that can model the effects of catalyst fooling or membrane degradation. The disentanglement of the geometric structure of the medium from surface reactivity opens far-reaching perspectives for modeling, optimization, and control of diffusion-mediated surface phenomena.


Assuntos
Modelos Biológicos , Modelos Químicos , Membrana Celular/química , DNA/química , Difusão , Proteínas/química , Propriedades de Superfície , Termodinâmica
13.
Proc Natl Acad Sci U S A ; 117(36): 22378-22389, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839325

RESUMO

Hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (MRSI) is a noninvasive metabolic-imaging modality that probes carbon flux in tissues and infers the state of metabolic reprograming in tumors. Prevailing models attribute elevated hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in aggressive tumors to enhanced glycolytic flux and lactate dehydrogenase A (LDHA) activity (Warburg effect). By contrast, we find by cross-sectional analysis using genetic and pharmacological tools in mechanistic studies applied to well-defined genetically engineered cell lines and tumors that initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates as well as global conversion were highly dependent on and critically rate-limited by the transmembrane influx of [1-13C]pyruvate mediated predominately by monocarboxylate transporter-1 (MCT1). Specifically, in a cell-encapsulated alginate bead model, induced short hairpin (shRNA) knockdown or overexpression of MCT1 quantitatively inhibited or enhanced, respectively, unidirectional pyruvate influxes and [1-13C]pyruvate-to-[1-13C]lactate conversion rates, independent of glycolysis or LDHA activity. Similarly, in tumor models in vivo, hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion was highly dependent on and critically rate-limited by the induced transmembrane influx of [1-13C]pyruvate mediated by MCT1. Thus, hyperpolarized [1-13C]pyruvate MRSI measures primarily MCT1-mediated [1-13C]pyruvate transmembrane influx in vivo, not glycolytic flux or LDHA activity, driving a reinterpretation of this maturing new technology during clinical translation. Indeed, Kaplan-Meier survival analysis for patients with pancreatic, renal, lung, and cervical cancers showed that high-level expression of MCT1 correlated with poor overall survival, and only in selected tumors, coincident with LDHA expression. Thus, hyperpolarized [1-13C]pyruvate MRSI provides a noninvasive functional assessment primarily of MCT1 as a clinical biomarker in relevant patient populations.


Assuntos
Isótopos de Carbono/metabolismo , Membrana Celular/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/metabolismo , Simportadores/metabolismo , Animais , Isótopos de Carbono/análise , Isótopos de Carbono/química , Linhagem Celular Tumoral , Membrana Celular/química , Feminino , Humanos , Ácido Láctico/análise , Ácido Láctico/química , Imagem por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Ácido Pirúvico/análise , Ácido Pirúvico/química
14.
Proc Natl Acad Sci U S A ; 117(36): 21896-21905, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32843347

RESUMO

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (2H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.


Assuntos
Membrana Celular/química , Colesterol/metabolismo , Lipídeos de Membrana/química , Fenômenos Biomecânicos , Membrana Celular/metabolismo , Colesterol/química , Espectroscopia de Ressonância Magnética , Fluidez de Membrana , Lipídeos de Membrana/metabolismo , Simulação de Dinâmica Molecular
15.
PLoS One ; 15(7): e0236201, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687545

RESUMO

Interaction of phospholipase D2 (PLD2) with phosphatidylinositol (4,5)-bisphosphate (PIP2) is regarded as the critical step of numerous physiological processes. Here we build a full-length model of human PLD2 (hPLD2) combining template-based and ab initio modeling techniques and use microsecond all-atom molecular dynamics (MD) simulations of the protein in contact with a complex membrane to determine hPLD2-PIP2 interactions. MD simulations reveal that the intermolecular interactions preferentially occur between specific PIP2 phosphate groups and hPLD2 residues; the most strongly interacting residues are arginine at the pbox consensus sequence (PX) and pleckstrin homology (PH) domain. Interaction networks indicate formation of clusters at the protein-membrane interface consisting of amino acids, PIP2, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA); the largest cluster was in the PH domain.


Assuntos
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/química , Sequência Consenso , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfolipase D/química , Fosfolipase D/ultraestrutura , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos
16.
Proc Natl Acad Sci U S A ; 117(31): 18470-18476, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690682

RESUMO

Lipid membrane fusion is an essential process for a number of critical biological functions. The overall process is thermodynamically favorable but faces multiple kinetic barriers along the way. Inspired by nature's engineered proteins such as SNAP receptor [soluble N-ethylmale-imide-sensitive factor-attachment protein receptor (SNARE)] complexes or viral fusogenic proteins that actively promote the development of membrane proximity, nucleation of a stalk, and triggered expansion of the fusion pore, here we introduce a synthetic fusogen that can modulate membrane fusion and equivalently prime lipid membranes for calcium-triggered fusion. Our fusogen consists of a gold nanoparticle functionalized with an amphiphilic monolayer of alkanethiol ligands that had previously been shown to fuse with lipid bilayers. While previous efforts to develop synthetic fusogens have only replicated the initial steps of the fusion cascade, we use molecular simulations and complementary experimental techniques to demonstrate that these nanoparticles can induce the formation of a lipid stalk and also drive its expansion into a fusion pore upon the addition of excess calcium. These results have important implications in general understanding of stimuli-triggered fusion and the development of synthetic fusogens for biomedical applications.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Ouro/química , Bicamadas Lipídicas/metabolismo , Nanopartículas Metálicas/química , Cálcio/química , Membrana Celular/química , Ouro/metabolismo , Humanos , Bicamadas Lipídicas/química , Fusão de Membrana , Simulação de Dinâmica Molecular , Proteínas SNARE/metabolismo , Análise Serial de Tecidos
17.
Int J Nanomedicine ; 15: 4091-4104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606666

RESUMO

Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications. Methods and Materials: Here, AuNPs of three sizes, 5 nm, 10 nm, and 20 nm, were coated with a lipid bilayer composed of sodium oleate, hydrogenated phosphatidylcholine, and hexanethiol. To understand how the physical features of AuNPs influence uptake through cellular membranes, sum frequency generation (SFG) was utilized to assess the interactions of the AuNPs with a biomimetic lipid monolayer composed of a deuterated phospholipid 1.2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC). Results and Discussion: SFG measurements showed that 5 nm and 10 nm AuNPs are able to phase into the lipid monolayer with very little energetic cost, whereas, the 20 nm AuNPs warped the membrane conforming it to the curvature of hybrid lipid-coated AuNPs. Toxicity of the AuNPs were assessed in vivo to determine how AuNP curvature and uptake influence cell health. In contrast, in vivo toxicity tested in embryonic zebrafish showed rapid toxicity of the 5 nm AuNPs, with significant 24 hpf mortality occurring at concentrations ≥20 mg/L, whereas the 10 nm and 20 nm AuNPs showed no significant mortality throughout the five-day experiment. Conclusion: By combining information from membrane models using SFG spectroscopy with in vivo toxicity studies, a better mechanistic understanding of how nanoparticles (NPs) interact with membranes is developed to understand how the physiochemical features of AuNPs drive nanoparticle-membrane interactions, cellular uptake, and toxicity.


Assuntos
Membrana Celular/química , Ouro/toxicidade , Lipídeos/química , Membranas Artificiais , Nanopartículas Metálicas/toxicidade , Tamanho da Partícula , Testes de Toxicidade , Animais , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Humanos , Espectrofotometria Ultravioleta , Análise Espectral , Peixe-Zebra/embriologia
18.
Proc Natl Acad Sci U S A ; 117(28): 16690-16701, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601235

RESUMO

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of ß-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/µm2 to 0.54/µm2 Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.


Assuntos
Membrana Celular/metabolismo , Proteínas Desgrenhadas/metabolismo , Proteína Wnt3A/metabolismo , Membrana Celular/química , Membrana Celular/genética , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/genética , Células HEK293 , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ligação Proteica , Imagem Individual de Molécula , Via de Sinalização Wnt , Proteína Wnt3A/química , Proteína Wnt3A/genética
19.
Nat Protoc ; 15(8): 2443-2469, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32591769

RESUMO

Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Nanotecnologia/métodos , Membrana Celular/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Nanotubos/química
20.
Nat Commun ; 11(1): 2951, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528158

RESUMO

Biomembranes are two-dimensional assemblies of phospholipids that are only a few nanometres thick, but form micrometre-sized structures vital to cellular function. Explicit molecular modelling of biologically relevant membrane systems is computationally expensive due to the large number of solvent particles and slow membrane kinetics. Coarse-grained solvent-free membrane models offer efficient sampling but sacrifice realistic kinetics, thereby limiting the ability to predict pathways and mechanisms of membrane processes. Here, we present a framework for integrating coarse-grained membrane models with continuum-based hydrodynamics. This framework facilitates efficient simulation of large biomembrane systems with large timesteps, while achieving realistic equilibrium and non-equilibrium kinetics. It helps to bridge between the nanometer/nanosecond spatiotemporal resolutions of coarse-grained models and biologically relevant time- and lengthscales. As a demonstration, we investigate fluctuations of red blood cells, with varying cytoplasmic viscosities, in 150-milliseconds-long trajectories, and compare kinetic properties against single-cell experimental observations.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Simulação por Computador , Eritrócitos/metabolismo , Humanos , Hidrodinâmica , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfolipídeos/química
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