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1.
Nat Commun ; 11(1): 3906, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764563

RESUMO

Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C-H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C-H activations beyond canonical oxygenase-dependent reactions.


Assuntos
Trifosfato de Adenosina/metabolismo , Betaproteobacteria/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Betaproteobacteria/genética , Carbono/química , Colestadienóis/química , Colestadienóis/metabolismo , Colesterol/química , Colesterol/metabolismo , Genes Bacterianos , Hidroliases/genética , Hidroliases/metabolismo , Hidroxilação , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Biológicos , Oxirredução , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Água/metabolismo
2.
Int J Nanomedicine ; 15: 5361-5376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801694

RESUMO

Background and Aim: Polymeric nanoparticles (NPs) have received much attention as promising carrier systems in lung cancer and brain metastases. Methods: Here, for the first time, we investigated the feasibility of using inhaled cholesterol-PEG co-modified poly (n-butyl) cyanoacrylate NPs (CLS-PEG NPs) of docetaxel (DTX) for sustained pulmonary drug delivery in cancer metastasis. Results: Spray-dried or freeze-dried NPs yielded sustained drug release in vitro. In vitro inhalation evaluation data indicated that the inhalation formulation had better inhalability. Compared with intravenous (IV) administration, pharmacokinetic data suggested that the inhalation formulation prolonged plasma concentration of DTX for greater than 24 h and is more quickly and completely absorbed into the rat lung after intratracheal (IT) administration. Furthermore, freeze-dried powders were found to increase the t1/2 and area under curve (AUC) by 2.3 and 6.5 fold compared to the free drug after IT administration, and spray-dried powders were found to increase the t1/2 and AUC by 3.4 and 8.8 fold, respectively. After pulmonary administration of the inhalation formulation, DTX appeared to prolong the pulmonary absorption time. In addition, the inhalation formulation was distributed to the brain in a sustained release manner. Conclusion: These experimental results demonstrated that freeze- and spray-dried powders have the potential for pulmonary sustained release, and they also have the potential to be used as a novel treatment for the delivery of drugs that pass through the air-blood barrier and enter the brain and are efficient carriers for the treatment of brain metastasis.


Assuntos
Antineoplásicos/administração & dosagem , Docetaxel/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/administração & dosagem , Células A549 , Administração por Inalação , Animais , Encéfalo/efeitos dos fármacos , Colesterol/química , Preparações de Ação Retardada , Docetaxel/farmacocinética , Portadores de Fármacos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Embucrilato/química , Feminino , Liofilização , Humanos , Pulmão/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/química , Tamanho da Partícula , Polímeros/química , Pós/química , Ratos Wistar , Distribuição Tecidual
3.
Food Chem ; 328: 127127, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32473492

RESUMO

Poor stability of fish hydrolyzed collagen (HC) hampers its applications, especially as food ingredients. The use of liposome as a vesicle can be a potential means to enhance bioactivities and stability of HC. HC from defatted Asian sea bass skin at different levels (0.25%-2%, w/v) were loaded into liposomes prepared from soy phosphatidylcholine (SPC) with various stabilizers (cholesterol (CHO) or glycerol (GLY)). The highest encapsulation efficiency (EE) was found in SPC-CHO-0.5%HC (P < 0.05) (85.42%), while liposome stabilized with GLY had the highest EE (74.54%) for SPC-GLY-0.25%HC (P < 0.05). After lyophilization, SPC-CHO-0.5%HC had higher EE than SPC-GLY-0.25%HC (P < 0.05). Increasing particle size and decreasing negative surface charge were found for both lyophilized samples. Lyophilized SPC-CHO-0.5%HC exhibited higher stability than lyophilized SPC-GLY-0.25%HC during storage at 25 °C for 28 days. Also, higher antioxidant activities in gastrointestinal track model system was found for SPC-CHO-0.5%HC. Thus, SPC-CHO liposome could be used as a promising carrier of HC.


Assuntos
Antioxidantes/química , Colágeno/química , Lipossomos/química , Lipossomos/farmacologia , Animais , Antioxidantes/farmacologia , Bass , Colesterol/química , Excipientes/química , Armazenamento de Alimentos , Liofilização , Trato Gastrointestinal , Glicerol , Hidrólise , Lecitinas/química , Tamanho da Partícula , Fosfatidilcolinas/química , Pele/química , Temperatura
4.
Nature ; 581(7808): 339-343, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433613

RESUMO

Cholesterol is an essential component of mammalian cell membranes, constituting up to 50% of plasma membrane lipids. By contrast, it accounts for only 5% of lipids in the endoplasmic reticulum (ER)1. The ER enzyme sterol O-acyltransferase 1 (also named acyl-coenzyme A:cholesterol acyltransferase, ACAT1) transfers a long-chain fatty acid to cholesterol to form cholesteryl esters that coalesce into cytosolic lipid droplets. Under conditions of cholesterol overload, ACAT1 maintains the low cholesterol concentration of the ER and thereby has an essential role in cholesterol homeostasis2,3. ACAT1 has also been implicated in Alzheimer's disease4, atherosclerosis5 and cancers6. Here we report a cryo-electron microscopy structure of human ACAT1 in complex with nevanimibe7, an inhibitor that is in clinical trials for the treatment of congenital adrenal hyperplasia. The ACAT1 holoenzyme is a tetramer that consists of two homodimers. Each monomer contains nine transmembrane helices (TMs), six of which (TM4-TM9) form a cavity that accommodates nevanimibe and an endogenous acyl-coenzyme A. This cavity also contains a histidine that has previously been identified as essential for catalytic activity8. Our structural data and biochemical analyses provide a physical model to explain the process of cholesterol esterification, as well as details of the interaction between nevanimibe and ACAT1, which may help to accelerate the development of ACAT1 inhibitors to treat related diseases.


Assuntos
Microscopia Crioeletrônica , Esterol O-Aciltransferase/química , Esterol O-Aciltransferase/ultraestrutura , Ureia/análogos & derivados , Colesterol/química , Colesterol/metabolismo , Histidina/química , Histidina/metabolismo , Holoenzimas/química , Holoenzimas/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Multimerização Proteica , Eletricidade Estática , Ureia/química
5.
J Food Sci ; 85(6): 1668-1674, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32458493

RESUMO

We used ultrasound-microwave comodification and enzyme modification (cellulase and hemicellulase) methods to extract soluble dietary fibers (SDFs) from black soybean hulls. Moreover, the structure, physical, and chemical properties, as well as the cholesterol-binding capacity of SDFs before and after modification were analyzed. The average molecular weight of SDFs extracted from raw black soybean hulls was 2.815 × 105  Da. By comparison, the average molecular weight of SDFs from ultrasound-microwave comodified hulls and enzyme-modified hulls decreased by 33.21% and 45.29%, respectively. The water-holding capacity (WHC), water-swelling capacity (WSC), and oil-holding capacity (OHC) of the extracted SDFs modified by the ultrasound-microwave method were 3.79 g/g, 1.39 mL/g, and 1.14 g/g, respectively, a 9.54%, 23.01%, and 17.53% increase from the values of raw SDF. The WHC, WSC, and OHC of SDFs modified via the enzyme method were 3.59 g/g, 1.25 mL/g, and 1.03 g/g, respectively, with a 3.76%, 10.62%, and 6.19% increase when compared to raw SDFs. The cholesterol-binding capacity of SDFs modified via the ultrasound-microwave and enzyme methods was 13.82 and 12.34 mg/g, respectively, with an increase of 47.98% and 32.20% when compared to raw SDFs. The changes in structure and physical and chemical properties were shown to be closely related to the significantly improved cholesterol-binding capacity of the SDFs from modified black soybean hulls. This provides a theoretical basis for subsequent research and development of black soybean hulls products. PRACTICAL APPLICATION: At present, the black soybean hull, a byproduct of general grains, is usually abandoned, but black soybean hull is rich in dietary fiber. Enzymatic modification and ultrasound-microwave comodification were used to treat black soybean hull to prepare small molecular weight, highly active soluble dietary fiber. This research is of great significance to the deep processing of black soybean hull and improvement of the economic benefits of black soybean byproducts.


Assuntos
Colesterol/química , Fibras na Dieta/análise , Soja/química , Biocatálise , Celulase/química , Colesterol/metabolismo , Cor , Peso Molecular , Ligação Proteica
6.
Gen Physiol Biophys ; 39(2): 107-122, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32329439

RESUMO

Solubilisation of model membranes of dioleoylphosphatidylcholine (DOPC) and DOPCcholesterol (CHOL) induced by surfactant N,N-dimethyl-1-dodecanamine-N-oxide (DDAO) was studied. At the maintained pH ~ 7.5, the DDAO molecules are in their neutral state with respect to the pK ~ 5. Pore formation in lipid bilayer was studied by fluorescence probe leakage method. The changes in the size of lipid aggregates upon increasing DDAO concentration were followed turbidimetrically. Effective ratio Re at different steps of the solubilisation process was determined. The molar partition coefficient of DDAO in case of the DOPC membrane is Kp = 2262 ± 379, for DOPC-CHOL membrane Kp = 2092 ± 594. Within the experimental error, the partition coefficient, as well as effective ratios Re, are not considerably influenced when one third of DOPC molecules is substituted with CHOL (DOPC:CHOL = 2:1). Constituents of buffer (50 mmol/dm3 PBS, 150 mmol/dm3 NaCl) caused aggregation of DOPC and DOPC-CHOL unilamellar liposomes at zero and low DDAO concentration, as was shown by SANS, turbidimetry and DIC microscopy. After solubilisation of bilayer structures by surfactant, mixed DOPC-DDAO and DOPC-CHOL-DDAO micelles with the shape of cylinders with elliptical cross section were detected.


Assuntos
Colesterol/química , Dimetilaminas/química , Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Tensoativos/química , Lipossomos , Micelas
7.
Gen Physiol Biophys ; 39(2): 135-144, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32329441

RESUMO

The structure and dynamics of membranes depend on many external and internal factors that in turn determine their biological functions. One of the widely accepted and studied characteristics of biomembranes is their fluidity. We research a simple system with variable fluidity tweakable via its composition. The addition of cholesterol is employed to increase the order of lipid chains, thus decreasing the membrane fluidity, while melatonin is shown to elevate the chain disorder, thus also the membrane fluidity. We utilize the densitometric measurements to show a shift of studied systems closer or further from the gel-to-fluid phase transition. The structural changes represented by changes to membrane thickness are evaluated from small angle neutron scattering. Finally, we look at the ability of the two additives to control the interactions between membrane and amyloid-beta peptides. Our results suggest that fluidizing effect of melatonin can promote an insertion of peptide within the membrane interior. Intriguingly, the latter structure relates possibly to an Alzheimer's disease preventing mechanism postulated in the case of melatonin.


Assuntos
Colesterol/química , Melatonina/química , Fluidez de Membrana , Peptídeos beta-Amiloides/química , Bicamadas Lipídicas/química , Conformação Molecular , Fosfatidilcolinas/química
8.
Nat Microbiol ; 5(7): 929-942, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32284563

RESUMO

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.


Assuntos
Bactérias/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Imunidade Inata/efeitos dos fármacos , Oxisteróis/farmacologia , Infecções Bacterianas/tratamento farmacológico , Colesterol/química , Citocinas/metabolismo , Células Epiteliais/microbiologia , Humanos , Interferons/metabolismo , Listeria/efeitos dos fármacos , Listeria/imunologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Oxisteróis/química , Oxisteróis/metabolismo , Shigella/efeitos dos fármacos , Shigella/imunologia , Esterol O-Aciltransferase/metabolismo , Relação Estrutura-Atividade
9.
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
10.
Toxicol Appl Pharmacol ; 393: 114941, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126212

RESUMO

Incidence of nonalcoholic fatty liver disease is increasing worldwide. Activation of the NLRP3 inflammasome is central to the development of diet-induced nonalcoholic steatohepatitis (NASH). We investigated whether benzyl isothiocyanate (BITC) ameliorates diet-induced NASH and the mechanisms involved. C57BL/6 J mice fed a high-fat diet containing cholesterol and cholic acid (HFCCD) and Kupffer cells stimulated with LPS and cholesterol crystals (CC) were studied. LPS/CC increased the expression of the active form of caspase 1 (p20) and the secretion of IL-1ß by Kupffer cells, and these changes were reversed by MCC950, an NLRP3 inflammasome inhibitor. LPS/CC-induced NLRP3 inflammasome activation and IL-1ß production were dose-dependently attenuated by BITC. BITC decreased cathepsin B release from lysosomes and binding to NLRP3 induced by LPS/CC. Compared with a normal diet, the HFCCD increased serum levels of ALT, AST, total cholesterol, and IL-1ß and hepatic contents of triglycerides and total cholesterol. BITC administration (0.1% in diet) reversed the increase in AST and hepatic triglycerides in the HFCCD group. Moreover, BITC suppressed lipid accumulation, macrophage infiltration, fibrosis, crown-like structure formation, and p20 caspase 1 and p17 IL-1ß expression in liver in the HFCCD group. These results suggest that BITC ameliorates HFCCD-induced steatohepatitis by inhibiting the activation of NLRP3 inflammasome in Kupffer cells and may protect against diet-induced NASH.


Assuntos
Colesterol na Dieta/efeitos adversos , Colesterol/química , Ácido Cólico/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Inflamassomos/efeitos dos fármacos , Isotiocianatos/uso terapêutico , Macrófagos do Fígado/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Colesterol/sangue , Relação Dose-Resposta a Droga , Interleucina-1beta/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Testes de Função Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/etiologia , Triglicerídeos/metabolismo
11.
Biochim Biophys Acta Biomembr ; 1862(7): 183254, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32135144

RESUMO

BACKGROUND: Oxidation of small dense low-density lipoprotein (sdLDL) and membranes is causally related to atherosclerosis. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA, 20:5, ω-3) significantly reduced oxidized LDL in patients with hypertriglyceridemia by unknown mechanisms. We compared EPA effects to related FAs of varying chain length and unsaturation on oxidation of sdLDL and model membranes, and on cholesterol crystal domains. We compared EPA to the FAs: stearic (SA, 18:0), oleic (OA, 18:1, ω-9), linoleic (LA, 18:2, ω-6), alpha-linolenic (ALA, 18:3, ω-3), eicosanoic (EA, 20:0), eicosatrienoic (ETE, 20:3, ω-3), arachidonic (AA, 20:4, ω-6), docosapentaenoic (DPA, 22:5, ω-3), and docosahexaenoic (DHA, 22:6, ω-3). METHODS: Human sdLDL or model membranes of cholesterol and 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine [18:2(cis)PC or DLPC] were preincubated with FAs followed by copper-induced oxidation. Malondialdehyde (MDA) or lipid hydroperoxides (LOOH) levels measured oxidation; small-angle X-ray diffraction assessed cholesterol domain formation. RESULTS: After 40 min, EPA reduced MDA levels 70% compared to vehicle (p < 0.001). Lesser inhibition was observed with DHA, DPA, ETE, and ALA (33%, 34%, 32%, and 16%, respectively; all p < 0.001 versus vehicle). Similar relative FA effects were observed in model membranes where EPA more substantially inhibited cholesterol crystal domain formation. CONCLUSION: We observed relationships between hydrocarbon length and unsaturation with antioxidant activity and membrane cholesterol domain formation. EPA had the most favorable molecular structure, likely contributing to membrane stability, improved lipoprotein clearance, and reduced inflammation. GENERAL SIGNIFICANCE: Insight is provided into FA hydrocarbon length and unsaturation relationships with antioxidant activity in lipoproteins and membranes, and cholesterol crystal domains formation.


Assuntos
Colesterol/química , Ácido Eicosapentaenoico/química , Ácidos Graxos Ômega-3/química , Ácidos Graxos/química , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/metabolismo , Humanos , Lipoproteínas LDL/química , Oxirredução , Triglicerídeos/química
12.
Biochim Biophys Acta Biomembr ; 1862(7): 183253, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142820

RESUMO

Membrane lipids are inherently highly dynamic molecules. Currently, it is difficult to probe the structures of individual lipids experimentally at the timescales corresponding to atomic motions, and consequently molecular dynamics simulations are used widely. In our previous work, we have introduced the principal component analysis (PCA) as a convenient framework for comprehensive quantitative description of lipid motions. Here, we present a newly developed open source script, PCAlipids, which automates the analysis and allows us to refine the approach and test its limitations. We use PCAlipids to determine the influence of temperature, cholesterol and curvature on individual lipids, and show that the most prominent lipid tail scissoring motion is strongly affected by these factors and allows tracking of phase transition. Addition of cholesterol affects the conformations and selectively changes the dynamics of lipid molecules, impacting the large-amplitude motions. Introduction of curvature biases the conformational ensembles towards more extended structures. We hope that the developed approach will be useful for understanding the molecular basis of different processes occurring in lipid membrane systems and will stimulate development of complementary experimental techniques probing the conformations of individual lipid molecules.


Assuntos
Colesterol/química , Lipídeos de Membrana/química , Conformação Molecular , Software , Biologia Computacional/métodos , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Movimento (Física) , Análise de Componente Principal , Temperatura
13.
Cell ; 180(4): 655-665.e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32004463

RESUMO

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Receptor CB1 de Canabinoide/química , Receptor CB2 de Canabinoide/química , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Animais , Células CHO , Agonistas de Receptores de Canabinoides/química , Canabinoides/química , Canabinoides/farmacologia , Linhagem Celular Tumoral , Colesterol/química , Colesterol/farmacologia , Cricetinae , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Células Sf9 , Spodoptera
14.
Phys Rev Lett ; 124(3): 038001, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-32031854

RESUMO

Cholesterol is a crucial component of mammalian cell membranes that takes part in many vital processes. It is generally accepted that cholesterol stabilizes the membrane and induces transitions into ordered states. In contrast to expectations, we demonstrate that cholesterol can destabilize the membrane by creating a nanodomain around a perpendicularly embedded ultrashort carbon nanotube (CNT), and we show that cholesterol triggers the translocation of an ultrashort CNT through the cell membrane. Using atomistic simulations, we report the existence of a nanoscale domain around an ultrashort carbon nanotube within a crossover distance of 0.9 nm from the surface of the nanotube, where the properties of the bilayer are different from the bulk: the domain is characterized by increased fluctuations, increased thickness, and increased order of the lipids with respect to the bulk. Cholesterol decreases the thickness and order of lipids and increases the fluctuations with respect to a pure lipid bilayer. Experimentally, we confirm that cholesterol nanodomains provoke spontaneous translocation of nanotubes through a lipid bilayer even for low membrane tensions. A specially designed microfluidic device allows us to trace the kinetic pathway of the translocation process and establish the threshold cholesterol concentration of 20% for translocation. The reported nanoscale cholesterol-induced membrane restructuring near the ultrashort CNT in lipid membranes enables precise control and specific targeting of a membrane using cholesterol. As an example, it may allow for specific targeting between cholesterol-rich mammalian cells and cholesterol-poor bacterial cells.


Assuntos
Membrana Celular/química , Colesterol/química , Lipídeos de Membrana/química , Modelos Químicos , Nanotubos de Carbono/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Biológicos
15.
Proc Natl Acad Sci U S A ; 117(9): 4749-4757, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071249

RESUMO

Biological membranes exhibit a great deal of compositional and phase heterogeneity due to hundreds of chemically distinct components. As a result, phase separation processes in cell membranes are extremely difficult to study, especially at the molecular level. It is currently believed that the lateral membrane heterogeneity and the formation of domains, or rafts, are driven by lipid-lipid and lipid-protein interactions. Nevertheless, the underlying mechanisms regulating membrane heterogeneity remain poorly understood. In the present work, we combine inelastic X-ray scattering with molecular dynamics simulations to provide direct evidence for the existence of strongly coupled transient lipid pairs. These lipid pairs manifest themselves experimentally through optical vibrational (a.k.a. phononic) modes observed in binary (1,2-dipalmitoyl-sn-glycero-3-phosphocholine [DPPC]-cholesterol) and ternary (DPPC-1,2-dioleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine [DOPC/POPC]-cholesterol) systems. The existence of a phononic gap in these vibrational modes is a direct result of the finite size of patches formed by these lipid pairs. The observation of lipid pairs provides a spatial (subnanometer) and temporal (subnanosecond) window into the lipid-lipid interactions in complex mixtures of saturated/unsaturated lipids and cholesterol. Our findings represent a step toward understanding the lateral organization and dynamics of membrane domains using a well-validated probe with a high spatial and temporal resolution.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Membrana Celular/metabolismo , Fenômenos Químicos , Colesterol/química , Fônons
16.
Nat Commun ; 11(1): 983, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080183

RESUMO

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.


Assuntos
Colesterol/análogos & derivados , Lipídeos/química , Nanopartículas/química , RNA Mensageiro/administração & dosagem , Animais , Transporte Biológico Ativo , Linhagem Celular , Colesterol/química , Microscopia Crioeletrônica , Endossomos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Nanopartículas/ultraestrutura , Células RAW 264.7 , RNA Mensageiro/genética , Sitosteroides/química , Transfecção , Difração de Raios X
17.
Chem Commun (Camb) ; 56(21): 3147-3150, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32057047

RESUMO

Amyloid fibrils, implicated in health and diseases, commonly exhibit a periodic twist trait relevant to the structures and dynamics of the fibrils. However, the origins and modulations of fibril twist in complex in vivo environments are not yet fully understood. Here we highlight an important factor that causes twist variations in amyloid fibril structures-the presence of surrounding surfaces. Using cholesterol-containing lipid bilayers with varying cholesterol contents, we have demonstrated via atomic force microscopy that amyloid-ß peptide fibrils initiated on membranes increase their average pitch size of twisting periodicity as the cholesterol content increases. These surface-induced twist variations arise from the enhanced hydrophobic interactions between the fibril and the surface distorting the torsional elastic energy of the fibril twisting as supported by a theory of an elastic model. These findings not only provide an important insight into fibril polymorphism phenomena resulting from the surface effects but also suggest a novel solution to modulate filament twisting on the nanoscale for biomaterials applications involving nanoscale features.


Assuntos
Amiloide/química , Colesterol/química , Bicamadas Lipídicas/química , Microscopia de Força Atômica , Tamanho da Partícula , Conformação Proteica , Propriedades de Superfície
18.
Parasite ; 27: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31934848

RESUMO

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MßCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Exoesqueleto/química , Membrana Celular/química , Colesterol/química , Resistência a Medicamentos , Haemonchus/química , Haemonchus/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Membrana Celular/efeitos dos fármacos , Fluidez de Membrana , Xenobióticos/farmacologia
19.
Nanotechnology ; 31(19): 195101, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31958777

RESUMO

Natural products have been successfully used to treat various ailments since ancient times and currently several anticancer agents based on natural products are used as the main therapy to treat cancer patients, or as a complimentary treatment to chemotherapy or radiation. Balanocarpol, which is a promising natural product that has been isolated from Hopea dryobalanoides, has been studied as a potential anticancer agent but its application is limited due to its high toxicity, low water solubility, and poor bioavailability. Therefore, the aim of this study is to improve the characteristics of balanocarpol and increase its anticancer activity through its encapsulation in a bilayer structure of a lipid-based nanoparticle drug delivery system where the application of nanotechnology can help improve the limitations of balanocarpol. The compound was first extracted and isolated from H. dryobalanoides. Niosome nanoparticles composed of Span 80 (SP80) and cholesterol were formulated through an innovative microfluidic mixing method for the encapsulation and delivery of balanocarpol. The prepared particles were spherical, small, and uniform with an average particles size and polydispersity index ∼175 nm and 0.088, respectively. The encapsulation of balanocarpol into the SP80 niosomes resulted in an encapsulation efficiency of ∼40%. The niosomes formulation loaded with balanocarpol showed a superior anticancer effect over the free compound when tested in vitro on human ovarian carcinoma (A2780) and human breast carcinoma (ZR-75-1). This is the first study to report the use of SP80 niosomes for the successful encapsulation and delivery of balanocarpol into cancer cells.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Dipterocarpaceae/química , Neoplasias Ovarianas/tratamento farmacológico , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Cápsulas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/química , Feminino , Hexoses/química , Humanos , Lipossomos , Extratos Vegetais/química , Polifenóis/química
20.
Biochim Biophys Acta Biomembr ; 1862(5): 183196, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958437

RESUMO

Lipid bilayers form the basis of cell membranes and the phase behaviour of the membrane has been linked to proper cell function. Model membranes composed of relatively simple mixtures of phospholipids and cholesterol can already exhibit complex phase behaviour. Specifically, liquid ordered-liquid disordered fluid phase coexistence occurs in mixtures which contain one saturated long chain phospholipid and one unsaturated long chain phospholipid and cholesterol. This fluid-fluid two phase region persists over a broad range of temperatures and sample compositions and can be observed experimentally in various sample preparations including multilamellar dispersions, bicelles, and multi-lamellae stacked on glass slides. In order to explore the practicality of using oriented samples with different concentrations of the peptide, we investigated the effect of the addition of a synthetic 22 residue amphiphilic peptide on the orientability and phase behaviour of the lipid mixtures, as well as the orientation and dynamics of the peptide itself via 2H NMR. Increasing the peptide concentration promoted the formation of the liquid ordered phase, suggesting a preferential interaction of the peptide with the thicker ordered phase. However, higher peptide content (> 4 mol%) had a significant negative effect on the alignment of bicelles with their bilayer normal perpendicular to the external magnetic field. In the stacked bilayer samples, 6 mol% peptide eliminated the two phase coexistence region altogether and a single liquid ordered phase was observed from 285 to 311 K. Even so, 2H spectra of the peptide itself did not reveal any preference for the peptide to partition into either the liquid disordered or liquid ordered phase and we found two populations of the peptide, one which undergoes rapid axial reorientation about the bilayer normal and a second (powder component) which does not.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Membrana Celular/metabolismo , Colesterol/metabolismo , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Membranas/metabolismo , Orientação Espacial , Peptídeos/química , Fosfatidilcolinas/química , Fosfolipídeos/metabolismo , Tensoativos/química , Temperatura
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