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Lipid nanodiscs are popular mimetics of biological membranes for determining membrane protein structures. However, a recent study revealed that the choice of nanodisc scaffold directly influenced the structure of an ion channel. This finding prompts us to be cautious and calls for improved membrane mimetics for structure determination.
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Proteínas de Membrana , Nanoestruturas , Bicamadas Lipídicas/química , Lipídeos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nanoestruturas/química , Conformação ProteicaRESUMO
Biomolecular phase separation has emerged as an essential mechanism for cellular organization. How cells respond to environmental stimuli in a robust and sensitive manner to build functional condensates at the proper time and location is only starting to be understood. Recently, lipid membranes have been recognized as an important regulatory center for biomolecular condensation. However, how the interplay between the phase behaviors of cellular membranes and surface biopolymers may contribute to the regulation of surface condensation remains to be elucidated. Using simulations and a mean-field theoretical model, we show that two key factors are the membrane's tendency to phase-separate and the surface polymer's ability to reorganize local membrane composition. Surface condensate forms with high sensitivity and selectivity in response to features of biopolymer when positive co-operativity is established between coupled growth of the condensate and local lipid domains. This effect relating the degree of membrane-surface polymer co-operativity and condensate property regulation is shown to be robust by different ways of tuning the co-operativity, such as varying membrane protein obstacle concentration, lipid composition, and the affinity between lipid and polymer. The general physical principle emerged from the current analysis may have implications in other biological processes and beyond.
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Proteínas de Membrana , Polímeros , Membrana Celular , Membranas , LipídeosRESUMO
Cellular engulfment and uptake of macromolecular assemblies or nanoparticles via endocytosis can be associated to both healthy and disease-related biological processes as well as delivery of drug nanoparticles and potential nanotoxicity of pollutants. Depending on the physical and chemical properties of the system, the adsorbed particles may remain at the membrane surface, become wrapped by the membrane, or translocate across the membrane through an endocytosis-like process. In this paper, we address the question of how the wrapping of colloidal particles by lipid membranes can be controlled by the shape of the particles, the particle-membrane adhesion energy, the membrane phase behavior, and the membrane-bending rigidity. We use a model system composed of soft core-shell microgel particles with spherical and ellipsoidal shapes, together with phospholipid membranes with varying composition. Confocal microscopy data clearly demonstrate how tuning of these basic properties of particles and membranes can be used to direct wrapping and membrane deformation and the organization of the particles at the membrane. The deep-wrapped states are more favorable for ellipsoidal than for spherical microgel particles of similar volume. Theoretical calculations for fixed adhesion strength predict the opposite behavior-wrapping becomes more difficult with increasing aspect ratio. The comparison with the experiments implies that the microgel adhesion strength must increase with increasing particle stretching. Considering the versatility offered by microgels systems to be synthesized with different shapes, functionalizations, and mechanical properties, the present findings further inspire future studies involving nanoparticle-membrane interactions relevant for the design of novel biomaterials and therapeutic applications.
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Microgéis , Membrana Celular/química , Endocitose , Membranas , Lipídeos/químicaRESUMO
Liquid protein condensates play important roles in orchestrating subcellular organization and as biochemical reaction hubs. Recent studies have linked lipid membranes to proteins capable of forming liquid condensates, and shown that biophysical parameters, like protein enrichment and restricted diffusion at membranes, regulate condensate formation and size. However, the impact of membrane topography on liquid condensates remains poorly understood. Here, we devised a cell-free system to reconstitute liquid condensates on lipid membranes with microstructured topographies and demonstrated that lipid membrane topography is a significant biophysical regulator. Using membrane surfaces designed with microwells, we observed ordered condensate patterns. Furthermore, we demonstrate that membrane topographies influence the shape of liquid condensates. Finally, we show that capillary forces, mediated by membrane topographies, lead to the directed fusion of liquid condensates. Our results demonstrate that membrane topography is a potent biophysical regulator for the localization and shape of mesoscale liquid protein condensates.
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Lipídeos , Membranas , Transporte Biológico , Biofísica , Sistema Livre de CélulasRESUMO
Coronavirus disease 2019 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2. The virus binds to angiotensinogen converting enzyme 2 (ACE2), which mediates viral entry into mammalian cells. COVID-19 is notably severe in the elderly and in those with underlying chronic conditions. The cause of selective severity is not well understood. Here we show cholesterol and the signaling lipid phosphatidyl-inositol 4,5 bisphosphate (PIP2) regulate viral infectivity through the localization of ACE2's into nanoscopic (<200 nm) lipid clusters. Uptake of cholesterol into cell membranes (a condition common to chronic disease) causes ACE2 to move from PIP2 lipids to endocytic ganglioside (GM1) lipids, where the virus is optimally located for viral entry. In mice, age and high-fat diet increase lung tissue cholesterol by up to 40%. And in smokers with chronic disease, cholesterol is elevated 2-fold, a magnitude of change that dramatically increases infectivity of virus in cell culture. We conclude increasing the ACE2 location near endocytic lipids increases viral infectivity and may help explain the selective severity of COVID-19 in aged and diseased populations.
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COVID-19 , Hipercolesterolemia , Animais , Camundongos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2 , Peptidil Dipeptidase A/metabolismo , Colesterol/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Mamíferos/metabolismoRESUMO
Chemokines are cytokines whose primary role is cellular activation and stimulation of leukocyte migration. They perform their various functions by interacting with G protein-coupled cell surface receptors (GPCRs) and are involved in the regulation of many biological processes such as apoptosis, proliferation, angiogenesis, hematopoiesis or organogenesis. They contribute to the maintenance of the homeostasis of lymphocytes and coordinate the function of the immune system. However, chemokines and their receptors are sometimes hijacked by some pathogens to infect the host organism. For a given chemokine receptor, there is a wide structural, organizational and conformational diversity. In this review, we describe the evidence for structural variety reported for the chemokine receptor CCR5, how this variability can be exploited by HIV-1 to infect its target cells and what therapeutic solutions are currently being developed to overcome this problem.
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HIV-1 , Apoptose , Membrana Celular , Movimento Celular , QuimiocinasRESUMO
Understanding interactions of inorganic nanoparticles with biomolecules is important in many biotechnology, nanomedicine, and toxicological research, however, the size of typical nanoparticles makes their direct modeling by atomistic simulations unfeasible. Here, we present a bottom-up coarse-graining approach for modeling titanium dioxide (TiO 2 ) nanomaterials in contact with phospholipids that uses the inverse Monte Carlo method to optimize the effective interactions from the structural data obtained in small-scale all-atom simulations of TiO 2 surfaces with lipids in aqueous solution. The resulting coarse-grained models are able to accurately reproduce the structural details of lipid adsorption on different titania surfaces without the use of an explicit solvent, enabling significant computational resource savings and favorable scaling. Our coarse-grained simulations show that small spherical TiO 2 nanoparticles ( r = 2 nm) can only be partially wrapped by a lipid bilayer with phosphoethanolamine headgroups, however, the lipid adsorption increases with the radius of the nanoparticle. The current approach can be used to study the effect of the size and shape of TiO 2 nanoparticles on their interactions with cell membrane lipids, which can be a determining factor in membrane wrapping as well as the recently discovered phenomenon of nanoquarantining, which involves the formation of layered nanomaterial-lipid structures.
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OBJECTIVES: Potassium channels in the endoplasmic reticulum (ER) are crucial for maintaining calcium balance during calcium fluxes. Disruption in ER calcium balance leads to ER stress, implicated in diseases like diabetes and Alzheimer's disease (AD). However, limited data exists on ER potassium channels in excitable tissues such as the brain. To fill this gap, we aimed to evaluate potassium currents in rat brain rough endoplasmic reticulum (RER). METHODS: Rats were euthanized under deep anesthesia and their brains were immediately removed. The brains were then homogenized in ice-cold sucrose buffer, followed by the extraction of RER microsomes through a series of centrifugation processes. Purity of sample was evaluated using western blotting technique. Single channel recordings were done in voltage steps from +50 to -60 mV following incorporation of rat brain RER vesicles into planar bilayers. RESULTS: We observed a voltage-dependent potassium channel with an approximate conductance of 188 pS. Channel open probability was low at negative voltages, increasing at positive voltages. The channel was blocked by Charybdotoxin but not by Iberiotoxin. Additionally, TRAM-34, a specific KCa3.1 channel blocker, suppressed channel current amplitude and open probability. Western blot analysis revealed specific bands for anti-KCa3.1 antibody, approximately 50 kDa in brain homogenate and RER fraction. CONCLUSION: Our study provides strong evidence for the presence of an KCa3.1 channel on the RER membrane in rat brain, exhibiting distinct electro-pharmacological profile compared to plasma membrane and other organelles.
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Encéfalo , Animais , Ratos , Encéfalo/metabolismo , Masculino , Ratos Wistar , Canais de Potássio Cálcio-Ativados/metabolismo , Retículo Endoplasmático/metabolismoRESUMO
Acute rhabdomyolysis (RM) constitutes a life-threatening emergency resulting from the (acute) breakdown of skeletal myofibers, characterized by a plasma creatine kinase (CK) level exceeding 1000 IU/L in response to a precipitating factor. Genetic predisposition, particularly inherited metabolic diseases, often underlie RM, contributing to recurrent episodes. Both sporadic and congenital forms of RM share common triggers. Considering the skeletal muscle's urgent need to rapidly adjust to environmental cues, sustaining sufficient energy levels and functional autophagy and mitophagy processes are vital for its preservation and response to stressors. Crucially, the composition of membrane lipids, along with lipid and calcium transport, and the availability of adenosine triphosphate (ATP), influence membrane biophysical properties, membrane curvature in skeletal muscle, calcium channel signaling regulation, and determine the characteristics of autophagic organelles. Consequently, a genetic defect involving ATP depletion, aberrant calcium release, abnormal lipid metabolism and/or lipid or calcium transport, and/or impaired anterograde trafficking may disrupt autophagy resulting in RM. The complex composition of lipid membranes also alters Toll-like receptor signaling and viral replication. In response, infections, recognized triggers of RM, stimulate increased levels of inflammatory cytokines, affecting skeletal muscle integrity, energy metabolism, and cellular trafficking, while elevated temperatures can reduce the activity of thermolabile enzymes. Overall, several mechanisms can account for RMs and may be associated in the same disease-causing RM.
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Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. The toxicity of amyloid to neuronal cell surfaces arises from interactions between small intermediate aggregates, namely amyloid oligomers, and the cell membrane. The nature of these interactions changes with age and disease progression. In our previous work, we demonstrated that both membrane composition and nanoscale structure play crucial roles in amyloid toxicity, and that membrane models mimicking healthy neuron were less affected by amyloid than model membranes mimicking AD neuronal membranes. This understanding introduces the possibility of modifying membrane properties with membrane-active molecules, such as melatonin, to protect them from amyloid-induced damage. In this study, we employed atomic force microscopy and localized surface plasmon resonance to investigate the protective effects of melatonin. We utilized synthetic lipid membranes that mimic the neuronal cellular membrane at various stages of AD and explored their interactions with amyloid-ß(1-42) in the presence of melatonin. Our findings reveal that the early diseased membrane model is particularly vulnerable to amyloid binding and subsequent damage. However, melatonin exerts its most potent protective effect on this early-stage membrane. These results suggest that melatonin could act at the membrane level to alleviate amyloid toxicity, offering the most protection during the initial stages of AD.
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Peptídeos beta-Amiloides , Melatonina , Microscopia de Força Atômica , Ressonância de Plasmônio de Superfície , Melatonina/farmacologia , Melatonina/química , Microscopia de Força Atômica/métodos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Bicamadas Lipídicas/química , Doença de Alzheimer/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/químicaRESUMO
The unique properties of water are critical for life. Water molecules have been reported to hydrate cations and anions asymmetrically in bulk water, being a key element in the balance of biochemical interactions. We show here that this behavior extends to charged lipid nanoscale interfaces. Charge hydration asymmetry was investigated by using nonlinear light scattering methods on lipid nanodroplets and liposomes. Nanodroplets covered with negatively charged lipids induce strong water ordering, while droplets covered with positively charged lipids induce negligible water ordering. Surprisingly, this charge-induced hydration asymmetry is reversed around liposomes. This opposite behavior in charge hydration asymmetry is caused by a delicate balance of electrostatic and hydrogen-bonding interactions. These findings highlight the importance of not only the charge state but also the specific distribution of neutral and charged lipids in cellular membranes.
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Gotículas Lipídicas , Lipossomos , Lipossomos/química , Membrana Celular , Lipídeos , Água/químicaRESUMO
Numerous studies highlight the therapeutic potential of G protein-coupled receptor (GPCR) heterodimers, emphasizing their significance in various pathological contexts. Despite extensive basic research and promising outcomes in animal models, the translation of GPCR heterodimer-targeting drugs into clinical use remains limited. The complexities of in vivo conditions, particularly within thecomplex central nervous system, pose challenges in fully replicating physiological environments, hindering clinical success. This review discusses examples of the most studied heterodimers, their involvement in nervous system pathology, and the available data on their potential ligands. In addition, this review highlights the intricate interplay between lipids and GPCRs as a potential key factor in understanding the complexity of cell signaling. The multifaceted role of lipids in modulating the dynamics of GPCR dimerization is explored, shedding light on the elaborate molecular mechanisms governing these interactions.
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Receptores Acoplados a Proteínas G , Transdução de Sinais , Animais , Dimerização , Receptores Acoplados a Proteínas G/metabolismo , Membrana Celular/metabolismo , LipídeosRESUMO
High concentrations of acrolein (2-propenal) are found in polluted air and cigarette smoke, and may also be generated endogenously. Acrolein is also associated with the induction and progression of many diseases. The high reactivity of acrolein towards the thiol and amino groups of amino acids may cause damage to cell proteins. Acrolein may be responsible for the induction of oxidative stress in cells. We hypothesized that acrolein may contribute to the protein damage in erythrocytes, leading to the disruption of the structure of cell membranes. The lipid membrane fluidity, membrane cytoskeleton, and osmotic fragility were measured for erythrocytes incubated with acrolein for 24 h. The levels of thiol, amino, and carbonyl groups were determined in cell membrane and cytosol proteins. The level of non-enzymatic antioxidant potential (NEAC) and TBARS was also measured. The obtained research results showed that the exposure of erythrocytes to acrolein causes changes in the cell membrane and cytosol proteins. Acrolein stiffens the cell membrane of erythrocytes and increases their osmotic sensitivity. Moreover, it has been shown that erythrocytes treated with acrolein significantly reduce the non-enzymatic antioxidant potential of the cytosol compared to the control.
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Acroleína , Citosol , Membrana Eritrocítica , Eritrócitos , Acroleína/farmacologia , Acroleína/toxicidade , Acroleína/metabolismo , Citosol/metabolismo , Citosol/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Humanos , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Proteínas de Membrana/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Fluidez de Membrana/efeitos dos fármacos , Fragilidade Osmótica/efeitos dos fármacosRESUMO
We report electrochemical impedance spectroscopy measurements to characterize the membrane-disruptive properties of medium-chain fatty acid and monoglyceride mitigants interacting with tethered bilayer lipid membrane (tBLM) platforms composed of E. coli bacterial lipid extracts. The tested mitigants included capric acid (CA) and monocaprin (MC) with 10-carbon long hydrocarbon chains, and lauric acid (LA) and glycerol monolaurate (GML) with 12-carbon long hydrocarbon chains. All four mitigants disrupted E. coli tBLM platforms above their respective critical micelle concentration (CMC) values; however, there were marked differences in the extent of membrane disruption. In general, CA and MC caused larger changes in ionic permeability and structural damage, whereas the membrane-disruptive effects of LA and GML were appreciably smaller. Importantly, the distinct magnitudes of permeability changes agreed well with the known antibacterial activity levels of the different mitigants against E. coli, whereby CA and MC are inhibitory and LA and GML are non-inhibitory. Mechanistic insights obtained from the EIS data help to rationalize why CA and MC are more effective than LA and GML at disrupting E. coli membranes, and these measurement capabilities support the potential of utilizing bacterial lipid-derived tethered lipid bilayers for predictive assessment of antibacterial drug candidates and mitigants.
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Ácidos Graxos , Monoglicerídeos , Monoglicerídeos/farmacologia , Ácidos Graxos/farmacologia , Escherichia coli , Bicamadas Lipídicas , Antibacterianos/farmacologia , Bactérias , Lisados Bacterianos , Carbono , HidrocarbonetosRESUMO
The catalytic properties of cytochrome c (Cc) have captured great interest in respect to mitochondrial physiology and apoptosis, and hold potential for novel enzymatic bioremediation systems. Nevertheless, its contribution to the metabolism of environmental toxicants remains unstudied. Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with impactful diseases, and animal models have unveiled concerning signs of PAHs' toxicity to mitochondria. In this work, a series of eight PAHs with ionization potentials between 7.2 and 8.1 eV were used to challenge the catalytic ability of Cc and to evaluate the effect of vesicles containing cardiolipin mimicking mitochondrial membranes activating the peroxidase activity of Cc. With moderate levels of H2O2 and at pH 7.0, Cc catalyzed the oxidation of toxic PAHs, such as benzo[a]pyrene, anthracene, and benzo[a]anthracene, and the cardiolipin-containing membranes clearly increased the PAH conversions. Our results also demonstrate for the first time that Cc and Cc-cardiolipin complexes efficiently transformed the PAH metabolites 2-hydroxynaphthalene and 1-hydroxypyrene. In comparison to horseradish peroxidase, Cc was shown to reach more potent oxidizing states and react with PAHs with ionization potentials up to 7.70 eV, including pyrene and acenaphthene. Spectral assays indicated that anthracene binds to Cc, and docking simulations proposed possible binding sites positioning anthracene for oxidation. The results give support to the participation of Cc in the metabolism of PAHs, especially in mitochondria, and encourage further investigation of the molecular interaction between PAHs and Cc.
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Hidrocarbonetos Policíclicos Aromáticos , Animais , Humanos , Hidrocarbonetos Policíclicos Aromáticos/química , Citocromos c , Cardiolipinas , Peróxido de Hidrogênio , AntracenosRESUMO
Advances in structural biology have bestowed insights into the pleiotropic effects of neurokinin 1 receptors (NK1R) in diverse patho-physiological processes, thereby highlighting the potential therapeutic value of antagonists directed against NK1R. Herein, we investigate the mode of antagonist recognition to discern the obscure atomic facets germane for the function and molecular determinants of NK1R. To commence discernment of potent antagonists and the conformational changes in NK1R, induced upon antagonist binding, state-of-the-art classical all-atoms molecular dynamics (MD) simulations in lipid mimetic bilayers have been utilized. MD simulations of structural ensembles reveals the involvement of TM5 and TM6 in tight anchoring of antagonists through a network of interhelical hydrogen-bonds, while, the extracellular loop 2 (ECL2) governs the overall size and nature of the pocket, thereby modulating NK1R. Consistent comparison between experiments and MD simulation results discerns the predominant role of TM3, TM4, and TM6 in lipid-NK1R interaction. Correlation between hydrophobic index and helicity of TM domains elucidates their importance in maintaining the structural stability in addition to regulating NK1R antagonism. Taken together, we anticipate that our computational study marks a comprehensive structural basis of NK1R antagonism in lipid bilayers, which may facilitate designing of new therapeutics against associated diseases targeting human neurokinin receptors.
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Antagonistas dos Receptores de Neurocinina-1 , Receptores da Neurocinina-1 , Humanos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores da Neurocinina-1/metabolismo , Simulação de Dinâmica Molecular , LipídeosRESUMO
Membrane fusion is essential for the basal functionality of eukaryotic cells. In physiological conditions, fusion events are regulated by a wide range of specialized proteins, operating with finely tuned local lipid composition and ionic environment. Fusogenic proteins, assisted by membrane cholesterol and calcium ions, provide the mechanical energy necessary to achieve vesicle fusion in neuromediator release. Similar cooperative effects must be explored when considering synthetic approaches for controlled membrane fusion. We show that liposomes decorated with amphiphilic Au nanoparticles (AuLips) can act as minimal tunable fusion machinery. AuLips fusion is triggered by divalent ions, while the number of fusion events dramatically changes with, and can be finely tuned by, the liposome cholesterol content. We combine quartz-crystal-microbalance with dissipation monitoring (QCM-D), fluorescence assays, and small-angle X-ray scattering (SAXS) with molecular dynamics (MD) at coarse-grained (CG) resolution, revealing new mechanistic details on the fusogenic activity of amphiphilic Au nanoparticles (AuNPs) and demonstrating the ability of these synthetic nanomaterials to induce fusion regardless of the divalent ion used (Ca2+ or Mg2+ ). The results provide a novel contribution to developing new artificial fusogenic agents for next-generation biomedical applications that require tight control of the rate of fusion events (e.g., targeted drug delivery).
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Lipossomos , Nanopartículas Metálicas , Ouro , Espalhamento a Baixo Ângulo , Difração de Raios X , Proteínas , Colesterol , ÍonsRESUMO
Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.
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COVID-19 , Nanopartículas , Humanos , Bicamadas Lipídicas , Dióxido de Silício , Tamanho da PartículaRESUMO
Trodusquemine is an aminosterol with a variety of biological and pharmacological functions, such as acting as an antimicrobial, stimulating body weight loss and interfering with the toxicity of proteins involved in the development of Alzheimer's and Parkinson's diseases. The mechanisms of interaction of aminosterols with cells are, however, still largely uncharacterized. Here, by using fluorescently labeled trodusquemine (TRO-A594 and TRO-ATTO565), we show that trodusquemine binds initially to the plasma membrane of living cells, that the binding affinity is dependent on cholesterol, and that trodusquemine is then internalized and mainly targeted to lysosomes after internalization. We also found that TRO-A594 is able to strongly and selectively bind to myelinated fibers in fixed mouse brain slices, and that it is a marker compatible with tissue clearing and light-sheet fluorescence microscopy or expansion microscopy. In conclusion, this work contributes to further characterize the biology of aminosterols and provides a new tool for nerve labeling suitable for the most advanced microscopy techniques.
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Colestanos , Animais , Camundongos , Colestanos/farmacologia , Espermina/farmacologia , Microscopia de Fluorescência/métodos , ColesterolRESUMO
In this study, we investigated the effects of drugs on membrane function in which lipid peroxidation was inhibited by the antioxidant Trolox (TRO) in liposomes containing egg yolk lecithin. Local anesthetics (LAs), such as lidocaine (LID) and dibucaine (DIB), were used as model drugs. The effect of LAs on the inhibitory activity of TRO was evaluated by calculating the pI50 from the inhibition constant K calculated by curve fitting. pI50TRO indicates the strength of TRO membrane protective function. pI50LA indicates the strength of LA activity. LAs inhibited lipid peroxidation in a dose-dependent manner and decreased pI50TRO. The effect of DIB on pI50TRO was 1.9 times more than that of LID. This result indicated that LA may improve the fluidity of the membrane, which may facilitate the migration of TRO from the membrane to the liquid phase. As a result, TRO is less likely to suppress lipid peroxidation within the lipid membrane, possibly resulting in a decrease in pI50TRO. The effect of TRO on pI50LA was found to be similar in both, indicating that it did not depend on the type of the model drug. These results suggest that our developed procedure successfully quantified the effects of LAs on lipid membrane functions. We were able to obtain the characteristics of model drugs independent of TRO by simultaneously measuring and analyzing the lipid peroxidation inhibitory activities of TRO and model drugs in liposomes.