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1.
Nat Commun ; 11(1): 5661, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168829

RESUMO

Sarcopenia is characterized by decreased skeletal muscle mass and function with age. Aged muscles have altered lipid compositions; however, the role and regulation of lipids are unknown. Here we report that FABP3 is upregulated in aged skeletal muscles, disrupting homeostasis via lipid remodeling. Lipidomic analyses reveal that FABP3 overexpression in young muscles alters the membrane lipid composition to that of aged muscle by decreasing polyunsaturated phospholipid acyl chains, while increasing sphingomyelin and lysophosphatidylcholine. FABP3-dependent membrane lipid remodeling causes ER stress via the PERK-eIF2α pathway and inhibits protein synthesis, limiting muscle recovery after immobilization. FABP3 knockdown induces a young-like lipid composition in aged muscles, reduces ER stress, and improves protein synthesis and muscle recovery. Further, FABP3 reduces membrane fluidity and knockdown increases fluidity in vitro, potentially causing ER stress. Therefore, FABP3 drives membrane lipid composition-mediated ER stress to regulate muscle homeostasis during aging and is a valuable target for sarcopenia.


Assuntos
Envelhecimento/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Proteína 3 Ligante de Ácido Graxo/metabolismo , Lipídeos de Membrana/metabolismo , Músculo Esquelético/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteína 3 Ligante de Ácido Graxo/genética , Feminino , Técnicas de Silenciamento de Genes , Lipidômica , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/patologia , Mioblastos/patologia , Mioblastos/fisiologia , Fosfolipídeos/metabolismo , Proteínas Serina-Treonina Quinases , Sarcopenia , Regulação para Cima
2.
Ecotoxicol Environ Saf ; 203: 110999, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888604

RESUMO

Aluminium (Al) is a key element that plays a major role in inhibiting plant growth and productivity under acidic soils. While lipids may be involved in plant tolerance/sensitivity to Al, the role of monogalactosyldiacylglycerol (MGDG) in Al response remains unknown. In this study, Arabidopsis MGDG synthase (AtMGD) mutants (mgd1, mgd2 and mgd3) and wild-type (Col-0) plants were treated with AlCl3; the effect of aluminium on root growth, aluminium distribution, plasma membrane integrity, lipid peroxidation, hydrogen peroxide content and membrane lipid compositions were analysed. Under Al stress, mgd mutants exhibited a more severe root growth inhibition, plasma membrane integrity damage and lipid peroxidation compared to Col-0. Al accumulation in root tips showed no difference between Col-0 and mutants under Al stress. Lipid analysis demonstrated that under Al treatment the MGDG content in all plants and MGDG/DGDG (digalactosyldiacylglycerol) remarkably reduced, especially in mutants impairing the stability and permeability of the plasma membrane. These results indicate that the Arabidopsis mgd mutants are hypersensitive to Al stress due to the reduction in MGDG content, and this is of great significance in the discovery of effective measures for plants to inhibit aluminium toxicity.


Assuntos
Alumínio/toxicidade , Arabidopsis/efeitos dos fármacos , Galactolipídeos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poluentes do Solo/toxicidade , Alumínio/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Galactolipídeos/genética , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Mutação , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Poluentes do Solo/metabolismo
3.
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
4.
Proc Natl Acad Sci U S A ; 117(35): 21014-21021, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817429

RESUMO

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Imagem por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Permeabilidade , Estrutura Secundária de Proteína
5.
Biomed Pharmacother ; 130: 110582, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32763818

RESUMO

Given the speed of viral infection spread, repurposing of existing drugs has been given the highest priority in combating the ongoing COVID-19 pandemic. Only drugs that are already registered or close to registration, and therefore have passed lengthy safety assessments, have a chance to be tested in clinical trials and reach patients quickly enough to help in the current disease outbreak. Here, we have reviewed available evidence and possible ways forward to identify already existing pharmaceuticals displaying modest broad-spectrum antiviral activity which is likely linked to their high accumulation in cells. Several well studied examples indicate that these drugs accumulate in lysosomes, endosomes and biological membranes in general, and thereby interfere with endosomal pathway and intracellular membrane trafficking crucial for viral infection. With the aim to identify other lysosomotropic drugs with possible inherent antiviral activity, we have applied a set of clear physicochemical, pharmacokinetic and molecular criteria on 530 existing drugs. In addition to publicly available data, we have also used our in silico model for the prediction of accumulation in lysosomes and endosomes. By this approach we have identified 36 compounds with possible antiviral effects, also against coronaviruses. For 14 of them evidence of broad-spectrum antiviral activity has already been reported, adding support to the value of this approach. Presented pros and cons, knowledge gaps and methods to identify lysosomotropic antivirals, can help in the evaluation of many drugs currently in clinical trials considered for repurposing to target COVID-19, as well as open doors to finding more potent and safer alternatives.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Lisossomos/efeitos dos fármacos , Pandemias , Pneumonia Viral/tratamento farmacológico , Anti-Inflamatórios/farmacocinética , Antivirais/efeitos adversos , Antivirais/farmacocinética , Arritmias Cardíacas/induzido quimicamente , Azitromicina/farmacocinética , Azitromicina/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cloroquina/farmacocinética , Cloroquina/uso terapêutico , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Endossomos/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapêutico , Membranas Intracelulares/fisiologia , Lisossomos/química , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Fosfolipídeos/metabolismo , Tensoativos/farmacocinética , Internalização do Vírus
6.
J Membr Biol ; 253(5): 399-423, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32833058

RESUMO

Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified. Lipids act as critical determinants of bacterial and viral pathogenesis by altering the host cell membrane structure and functions.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Pneumonia Viral/epidemiologia , Esfingolipídeos/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Humanos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Transdução de Sinais
7.
Nat Commun ; 11(1): 4259, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32848156

RESUMO

The plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available to manipulate membrane protein location at the single-molecule level. Here, we use fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to control their movement. FMNPs allow single-particle tracking (SPT) at 10 nm and 5 ms spatiotemporal resolution, and using a magnetic needle, we pull membrane components laterally with femtonewton-range forces. In this way, we drag membrane proteins over the surface of living cells. Doing so, we detect barriers which we could localize to the submembrane actin cytoskeleton by super-resolution microscopy. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.


Assuntos
Nanopartículas de Magnetita , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Proteínas de Fluorescência Verde/metabolismo , Campos Magnéticos , Lipídeos de Membrana/metabolismo , Microscopia de Fluorescência , Nanotecnologia , Imagem Individual de Molécula/métodos
8.
Gene ; 759: 144987, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32712065

RESUMO

BACKGROUND: The immune response is influenced by the administration of omega-3 polyunsaturated fatty acids (PUFA). Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are affected by PUFA. The combination of evening primrose/hemp seed oil (EPO/HSO) has essential fatty acids (EFAs) for human optimal health due to the favorable ratio of omega-6/omega-3 and antioxidantal properties. The study was conducted to evaluate the effects of EPO/HSO on improving the membrane fatty acids composition of spleen and blood cells and immunologic factors in compared to rapamycin (RAPA) in the EAE model. METHODS AND MATERIALS: Chronic-EAE was induced by induction of MOG in C57BL/6J mice (female, age: 6-8 weeks, weight 18-21). Mice were assigned to 5 groups (6/group) to evaluate the therapeutic effects of EPO/HSO supplement in comparison with rapamycin: A group; EPO/HSO + RAPA, B group; RAPA, C group; EPO/HSO. Results were compared to two control groups (EAE and naive). The fatty acid profile of the spleen and blood cell membrane was evaluated. Real-time-polymerase chain reaction was used for the evaluate the genes expression levels of interleukin (IL) -4, IL-5, and IL-13 in lymphocytes. Also, IL-4 of serum was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our findings indicated that EPO/HSO therapy significantly increased the percentage of essential fatty acids in cell membrane of the spleen and blood. The relative expression of IL-4, IL-5, and IL-13 genes in lymphocytes and serum level of IL-4 was significantly increased in the HSO/EPO treated group versus other groups. CONCLUSION: These results point to potential therapeutic effects on the repair of the structure of cell membranes and suppression of inflammation by EPO/HSO in EAE.


Assuntos
Antioxidantes/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ácidos Graxos Essenciais/metabolismo , Fatores Imunológicos/uso terapêutico , Interleucinas/metabolismo , Óleos Vegetais/uso terapêutico , Sirolimo/uso terapêutico , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Cannabis/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Suplementos Nutricionais , Combinação de Medicamentos , Feminino , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óleos Vegetais/administração & dosagem , Primula/química , Sirolimo/administração & dosagem
9.
Proc Natl Acad Sci U S A ; 117(32): 18977-18983, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719116

RESUMO

Parkinson's disease is associated with α-synuclein (α-syn), a cytosolic protein enriched in presynaptic terminals. The biological function of α-syn remains elusive; however, increasing evidence suggests that the protein is involved in the regulation of synaptic vesicle fusion, signifying the importance of α-syn-lipid interactions. We show that α-syn preferentially binds to GM1-rich, liquid-ordered lipid domains on cytoplasmic membranes by using unroofed cells, which encapsulates lipid complexity and cellular topology. Moreover, proteins (Rab3a, syntaxin-1A, and VAMP2) involved in exocytosis also localize with α-syn, supporting its proposed functional role in exocytosis. To investigate how these lipid/protein interactions influence α-syn at the residue level, α-syn was derivatized with an environmentally sensitive fluorophore (7-nitrobenz-2-oxa-1,3-diazol-4-yl [NBD]) at different N- and C-terminal sites. Measurements of NBD fluorescence lifetime distributions reveal that α-syn adopts a multitude of membrane-bound conformations, which were not recapitulated in simple micelle or vesicle models, indicating an exquisite sensitivity of the protein to the complex lipid environment. Interestingly, these data also suggest the participation of the C terminus in membrane localization, which is generally overlooked and thus emphasize the need to use cellularly derived and biologically relevant membranes for biophysical characterization. Collectively, our results demonstrate that α-syn is more conformationally dynamic at the membrane interface than previously appreciated, which may be important for both its physiological and pathological functions.


Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , alfa-Sinucleína/química , Gangliosídeo G(M1)/metabolismo , Humanos , Cinética , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Microdomínios da Membrana/genética , Ligação Proteica , Transporte Proteico , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
10.
Nature ; 584(7819): 125-129, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32528175

RESUMO

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Receptores de Dopamina D2/química , Receptores de Dopamina D2/ultraestrutura , Bromocriptina/química , Bromocriptina/metabolismo , Dopamina/química , Dopamina/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Lipídeos de Membrana/química , Modelos Moleculares , Conformação Proteica , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transdução de Sinais
11.
Proc Natl Acad Sci U S A ; 117(25): 14178-14186, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513706

RESUMO

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).


Assuntos
Lipídeos de Membrana/metabolismo , Membranas/metabolismo , alfa-Sinucleína/metabolismo , Fluoresceínas , Humanos , Cinética , Bicamadas Lipídicas/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Fosfatidilgliceróis/química , Ligação Proteica , alfa-Sinucleína/genética
12.
FASEB J ; 34(6): 7253-7264, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367579

RESUMO

Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID-19. By describing the life cycle of the newly emergent coronavirus, SARS-CoV-2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID-19. Moreover, we propose that Niemann-Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS-CoV-2, by highlighting key established features of the disorder that together result in an "unfavorable" host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID-19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC-like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID-19.


Assuntos
Antivirais/farmacocinética , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Endossomos/virologia , Hidroxicloroquina/farmacologia , Lisossomos/virologia , Doença de Niemann-Pick Tipo C/patologia , Pneumonia Viral/tratamento farmacológico , Proteína ADAM17/fisiologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Transporte Biológico , Catepsina L/fisiologia , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/fisiologia , Glicopeptídeos/farmacologia , Glicopeptídeos/uso terapêutico , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Doença de Niemann-Pick Tipo C/metabolismo , Oxisteróis/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Serina Endopeptidases/fisiologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Internalização do Vírus/efeitos dos fármacos
13.
Phys Rev Lett ; 124(15): 158102, 2020 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-32357050

RESUMO

We analyze the nonequilibrium shape fluctuations of giant unilamellar vesicles encapsulating motile bacteria. Owing to bacteria-membrane collisions, we experimentally observe a significant increase in the magnitude of membrane fluctuations at low wave numbers, compared to the well-known thermal fluctuation spectrum. We interrogate these results by numerically simulating membrane height fluctuations via a modified Langevin equation, which includes bacteria-membrane contact forces. Taking advantage of the lengthscale and timescale separation of these contact forces and thermal noise, we further corroborate our results with an approximate theoretical solution to the dynamical membrane equations. Our theory and simulations demonstrate excellent agreement with nonequilibrium fluctuations observed in experiments. Moreover, our theory reveals that the fluctuation-dissipation theorem is not broken by the bacteria; rather, membrane fluctuations can be decomposed into thermal and active components.


Assuntos
Vesículas Citoplasmáticas/química , Lipídeos de Membrana/química , Modelos Biológicos , Modelos Químicos , Bacillus subtilis/química , Bacillus subtilis/citologia , Bacillus subtilis/metabolismo , Vesículas Citoplasmáticas/metabolismo , Locomoção , Lipídeos de Membrana/metabolismo , Fosfatidiletanolaminas/química , Rodaminas/química , Termodinâmica , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
14.
Food Chem ; 320: 126684, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32229394

RESUMO

Refrigeration is used to retard senescence and extend the storage life of 'Nanguo' pears, but fruits subjected to long-term refrigeration are prone to pericarp browning during subsequent shelf life. To uncover the potential effects of membrane lipid changes during fruit pericarp browning, changes in fruit appearance and cell ultrastructure were observed after different storage durations. Membrane lipid content as well as the activity and gene expression of enzymes involved in membrane lipid metabolism and membrane stability were analyzed. Results showed that long-term refrigeration increased the activity and expression of PLD, LOX, lipase, and membrane stability-related genes that promoted membrane lipid degradation and peroxidation, reduced membrane lipid unsaturation, and led to severe browning. Overall, membrane instability induced by disordered membrane lipid metabolism under low temperature stress may account for pericarp browning of cold stored 'Nanguo' pears.


Assuntos
Membrana Celular/metabolismo , Frutas/química , Frutas/metabolismo , Pyrus/química , Pyrus/metabolismo , Armazenamento de Alimentos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Refrigeração
15.
Nat Commun ; 11(1): 1531, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210233

RESUMO

Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.


Assuntos
Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Linhagem Celular Tumoral , Colesterol/metabolismo , Células HEK293 , Humanos , Microscopia Intravital , Metabolismo dos Lipídeos , Lipidômica , Espectroscopia de Ressonância Magnética , Fusão de Membrana , Domínios Proteicos/genética , Multimerização Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise Espacial , Proteína 2 Associada à Membrana da Vesícula/genética
16.
Phys Rev Lett ; 124(10): 108102, 2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32216409

RESUMO

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.


Assuntos
Lipídeos de Membrana/administração & dosagem , Microdomínios da Membrana/química , Colesterol/química , Colesterol/metabolismo , Diglicerídeos/química , Diglicerídeos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Microscopia Confocal/métodos , Modelos Químicos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Espectrometria de Fluorescência/métodos
17.
Chemistry ; 26(28): 6247-6256, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32166806

RESUMO

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.


Assuntos
Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Lipopolissacarídeos/química , Lipídeos de Membrana/química , Pseudomonas aeruginosa/química , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Hemólise , Lipopolissacarídeos/metabolismo , Lipídeos de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína
18.
Nat Commun ; 11(1): 1455, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32193379

RESUMO

The lipopeptide daptomycin is used as an antibiotic to treat severe infections with gram-positive pathogens, such as methicillin resistant Staphylococcus aureus (MRSA) and drug-resistant enterococci. Its precise mechanism of action is incompletely understood, and a specific molecular target has not been identified. Here we show that Ca2+-daptomycin specifically interacts with undecaprenyl-coupled cell envelope precursors in the presence of the anionic phospholipid phosphatidylglycerol, forming a tripartite complex. We use microbiological and biochemical assays, in combination with fluorescence and optical sectioning microscopy of intact staphylococcal cells and model membrane systems. Binding primarily occurs at the staphylococcal septum and interrupts cell wall biosynthesis. This is followed by delocalisation of components of the peptidoglycan biosynthesis machinery and massive membrane rearrangements, which may account for the pleiotropic cellular events previously reported. The identification of carrier-bound cell wall precursors as specific targets explains the specificity of daptomycin for bacterial cells. Our work reconciles apparently inconsistent previous results, and supports a concise model for the mode of action of daptomycin.


Assuntos
Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Daptomicina/farmacologia , Lipídeos de Membrana/metabolismo , Vias Biossintéticas/efeitos dos fármacos , Parede Celular/metabolismo , Humanos , Membranas Artificiais , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Fosfatidilgliceróis/metabolismo , Fosfatos de Poli-Isoprenil/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
19.
PLoS Biol ; 18(3): e3000618, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32182233

RESUMO

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.


Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/farmacologia , Membrana Celular/metabolismo , Animais , Sítios de Ligação , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Células Cultivadas , Cristalografia por Raios X , Feminino , Gangliosídeos/metabolismo , Lipídeos de Membrana/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paralisia/induzido quimicamente , Engenharia de Proteínas , Ratos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Sinaptotagminas/metabolismo , Triptofano/química , Triptofano/metabolismo
20.
Sci Adv ; 6(8): eaay5736, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128410

RESUMO

Association of peripheral proteins with lipid bilayers regulates membrane signaling and dynamics. Pleckstrin homology (PH) domains bind to phosphatidylinositol phosphate (PIP) molecules in membranes. The effects of local PIP enrichment on the interaction of PH domains with membranes is unclear. Molecular dynamics simulations allow estimation of the binding energy of GRP1 PH domain to PIP3-containing membranes. The free energy of interaction of the PH domain with more than two PIP3 molecules is comparable to experimental values, suggesting that PH domain binding involves local clustering of PIP molecules within membranes. We describe a mechanism of PH binding proceeding via an encounter state to two bound states which differ in the orientation of the protein relative to the membrane, these orientations depending on the local PIP concentration. These results suggest that nanoscale clustering of PIP molecules can control the strength and orientation of PH domain interaction in a concentration-dependent manner.


Assuntos
Sítios de Ligação , Membrana Celular/química , Lipídeos/química , Fosfatidilinositóis/química , Domínios de Homologia à Plecstrina , Algoritmos , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Teóricos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo
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