RESUMEN
The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.
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Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Membrana Externa Bacteriana/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Periplasma/metabolismo , Dominios Proteicos , Pliegue de ProteínaRESUMEN
Neurotransmitter analysis plays a pivotal role in diagnosing and managing neurodegenerative diseases, often characterized by disturbances in neurotransmitter systems. However, prevailing methods for quantifying neurotransmitters involve invasive procedures or require bulky imaging equipment, therefore restricting accessibility and posing potential risks to patients. The innovation of compact, in vivo instruments for neurotransmission analysis holds the potential to reshape disease management. This innovation can facilitate non-invasive and uninterrupted monitoring of neurotransmitter levels and their activity. Recent strides in microfabrication have led to the emergence of diminutive instruments that also find applicability in in vitro investigations. By harnessing the synergistic potential of microfluidics, micro-optics, and microelectronics, this nascent realm of research holds substantial promise. This review offers an overarching view of the current neurotransmitter sensing techniques, the advances towards in vitro microsensors tailored for monitoring neurotransmission, and the state-of-the-art fabrication techniques that can be used to fabricate those microsensors.
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Dispositivos Laboratorio en un Chip , Microfluídica , Humanos , Microfluídica/métodos , Microtecnología , Óptica y Fotónica , NeurotransmisoresRESUMEN
The eye is a complex sensory organ that enables visual perception of the world. The dysfunction of any of these tissues can impair vision. Conduction studies on laboratory animals are essential to ensure the safety of therapeutic products directly applied or injected into the eye to treat ocular diseases before eventually proceeding to clinical trials. Among these tissues, the cornea has unique homeostatic and regenerative mechanisms for maintaining transparency and refraction of external light, which are essential for vision. However, being the outermost tissue of the eye and directly exposed to the external environment, the cornea is particularly susceptible to injury and diseases. This review highlights the evidence for selecting appropriate animals to better understand and treat corneal diseases, which rank as the fifth leading cause of blindness worldwide. The development of reliable and human-relevant animal models is, therefore, a valuable research tool for understanding and translating fundamental mechanistic findings, as well as for assessing therapeutic potential in humans. First, this review emphasizes the unique characteristics of animal models used in ocular research. Subsequently, it discusses current animal models associated with human corneal pathologies, their utility in understanding ocular disease mechanisms, and their role as translational models for patients.
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Córnea , Enfermedades de la Córnea , Animales , Humanos , Córnea/patología , Enfermedades de la Córnea/tratamiento farmacológico , Modelos Animales , Ceguera , Susceptibilidad a EnfermedadesRESUMEN
Eye drops represent 90% of all currently used ophthalmic treatments. Only 0.02% of therapeutic molecules contained in eye drops reach the eye anterior chamber despite their high concentration. The tear film efficiently protects the cornea, reducing access to the target. Thereby, the increase in the drug bioavailability and efficiency must come from the mucoadhesion optimization of the drug delivery system. The gold nanoparticles, used as a drug delivery system in this study, already showcased ultrastable and mucoadhesive properties. The goal was to study the gold nanoparticles' ability to release two specific ophthalmic drugs, flurbiprofen and ketorolac. The parameters of interest were those involving the loading conditions, the gold nanoparticles properties, and the release experimental conditions. The drug release was measured using an in vitro model based on dialysis bags coupled with UV-visible spectroscopy. Gold nanoparticles showed an ability to release different molecules, whether hydrophobic or hydrophilic, in passive or active drug release environments. Based on these preliminary results, gold nanoparticles could represent a promising drug delivery system for ketorolac and flurbiprofen when topically applied through eye drops.
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Flurbiprofeno , Nanopartículas del Metal , Nanopartículas , Oro , Liberación de Fármacos , Ketorolaco , Diálisis Renal , Sistemas de Liberación de Medicamentos , Córnea , Nanopartículas/química , Antiinflamatorios , Soluciones OftálmicasRESUMEN
Neurotransmitter sensing in the brain is crucial for the understanding of neuro-degenerative diseases. Most modern methods for the purpose rely on bulky instruments or are disruptive to the neurotransmitter medium. In this work, we describe and evaluate the design of a novel, compact and non-invasive instrument for neurotransmitter detection based on the colorimetric sensing method. The instrument includes a grism-based spectrometer that measures the wavelength shift of gold nanoparticles that are functionalized with aptamers to act as neurotransmitter-specific markers. It also includes microfluidic and electronic subsystems for sample preparation and control, and processing of the obtained signal. The instrument is tested with gold nanoparticles and its performance is compared to that of a commercial instrument, showing that the designed prototype matches the commercial instrument in performance while being much smaller, and it can surpass it with further improvements. This article is part of the theme issue 'Advanced neurotechnologies: translating innovation for health and well-being'.
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Oro , Nanopartículas del Metal , Colorimetría/métodosRESUMEN
Preparation of drug delivery systems and nanomedicines necessitates the use of biocompatible excipients that are readily eliminated from the body. The systematic preclinical development of novel materials requires tools to evaluate their pharmacokinetics, biodistribution and excretion. Herein, we propose a technique called Size Exclusion of Radioactive Polymers (SERP) to trail the disposition of a radiolabeled polymer and its nanoparticles using chromatography in the presence of complex biological media such as blood, urine and feces. Trimethyl chitosan (TMC) is a polysaccharide of natural origin showing promise for controlled and targeted drug delivery applications. SERP was used to monitor degradation of radiolabeled TMC and its nanoparticles in vitro in the presence of strong acid, enzymes released by macrophages, as well as in vivo after administration to rats. Excretion of the radiolabeled TMC nanoparticles in urine and feces was monitored for 14 days after dosing to healthy rats, confirming that the polymer could be readily eliminated from the body. This work demonstrates the ability of SERP to understand the biological journey of biomaterials in vivo. Paving the way to understand the fate of polymers and nanoparticles in complex environments, the technique might facilitate the development of safer and better tolerated nanomedicines.
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Quitosano , Nanopartículas , Animales , Quitosano/química , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Polímeros , Ratas , Distribución TisularRESUMEN
Cervical cancer is the fourth most common malignancy among women. Compared to other types of cancer, therapeutic agents can be administrated locally at the mucosal vaginal membrane. Thermosensitive gels have been developed over the years for contraception or for the treatment of bacterial, fungal, and sexually transmitted infections. These formulations often carry therapeutic nanoparticles and are now being considered in the arsenal of tools for oncology. They can also be three-dimensionally (3D) printed for a better geometrical adjustment to the anatomy of the patient, thus enhancing the local delivery treatment. In this study, a localized delivery system composed of a Pluronic F127-alginate hydrogel with efficient nanoparticle (NP) release properties was prepared for intravaginal application procedures. The kinetics of hydrogel degradation and its NP releasing properties were demonstrated with ultrasmall gold nanoparticles (â¼80% of encapsulated AuNPs released in 48 h). The mucoadhesive properties of the hydrogel formulation were assayed by the periodic acid/Schiff reagent staining, which revealed that 19% of mucins were adsorbed on the gel's surface. The hydrogel formulation was tested for cytocompatibility in three cell lines (HeLa, CRL 2616, and BT-474; no sign of cytotoxicity revealed). The release of AuNPs from the hydrogel and their accumulation in vaginal membranes were quantitatively measured in vitro/ex vivo with positron emission tomography, a highly sensitive modality allowing real-time imaging of nanoparticle diffusion (lag time to start of permeation of 3.3 h, 47% of AuNPs accumulated in the mucosa after 42 h). Finally, the potential of the AuNP-containing Pluronic F127-alginate hydrogel for 3D printing was demonstrated, and the geometrical precision of the 3D printed systems was measured by magnetic resonance imaging (<0.5 mm precision; deviation from the design values <2.5%). In summary, this study demonstrates the potential of Pluronic F127-alginate formulations for the topical administration of NP-releasing gels applied to vaginal wall therapy. This technology could open new possibilities for photothermal and radiosensitizing oncology applications.
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Nanopartículas del Metal , Neoplasias del Cuello Uterino , Alginatos , Femenino , Oro , Humanos , Hidrogeles , Masculino , Nanopartículas del Metal/uso terapéutico , Poloxámero , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
Protein S100A10 participates in different cellular mechanisms and has different functions, especially at the membrane. Among those, it forms a ternary complex with annexin A2 and the C-terminal of AHNAK and then joins the dysferlin membrane repair complex. Together, they act as a platform enabling membrane repair. Both AHNAK and annexin A2 have been shown to have membrane binding properties. However, the membrane binding abilities of S100A10 are not clear. In this paper, we aimed to study the membrane binding of S100A10 in order to better understand its role in the cell membrane repair process. S100A10 was overexpressed by E. coli and purified by affinity chromatography. Using a Langmuir monolayer as a model membrane, the binding parameters and ellipsometric angles of the purified S100A10 were measured using surface tensiometry and ellipsometry, respectively. Phosphorus-31 solid-state nuclear magnetic resonance spectroscopy was also used to study the interaction of S100A10 with lipid bilayers. In the presence of a lipid monolayer, S100A10 preferentially interacts with unsaturated phospholipids. In addition, its behavior in the presence of a bilayer model suggests that S100A10 interacts more with the negatively charged polar head groups than the zwitterionic ones. This work offers new insights on the binding of S100A10 to different phospholipids and advances our understanding of the parameters influencing its membrane behavior.
RESUMEN
Polyethylene glycol (PEG) is considered the gold standard to prepare long circulating nanoparticles. The hydrophilic layer that sterically protects PEGylated nanomedicines also impedes their separation from biological media. In this study, we describe an immunoprecipitation method using AntiPEG antibodies cross-linked to magnetic beads to extract three types of radiolabeled PEGylated systems: polymeric nanoparticles, liposomes, and therapeutic proteins. The potential of the method is emphasized by isolating these systems after in vivo administration and ex vivo incubation in human biological fluids. Immunoprecipitation also allows a unique perspective on the size distribution of nanoparticles in the bloodstream after intravenous and intraperitoneal administrations. Further, we highlight the potential of the approach to inform on nanomaterial-associated drug in plasma as well as help characterize the protein corona. Altogether, we believe this method answers an unmet need in nanomedicine research and will contribute a fresh perspective on the interactions of nanomedicines with biological systems.
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Nanopartículas , Corona de Proteínas , Humanos , Inmunoprecipitación , Nanomedicina , PolietilenglicolesRESUMEN
Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.
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Córnea/citología , Lesiones de la Cornea/terapia , Traumatismos Ocupacionales/terapia , Ingeniería de Tejidos/métodos , Trasplante de Córnea , Humanos , Modelos Biológicos , Andamios del TejidoRESUMEN
The dysferlin membrane repair complex contains a small complex, S100A10-annexin A2, which initiates membrane repair by recruiting the protein AHNAK to the membrane, where it interacts via binding sites in the C-terminal region. However, no molecular data are available for the membrane binding of the various proteins involved in this complex. Therefore, the present study investigated the membrane binding of AHNAK to elucidate its role in the cell membrane repair process. A chemically synthesized peptide (pAHNAK), comprising the 20 amino acids in the C-terminal domain of AHNAK, was applied to Langmuir monolayer models, and the binding parameters and insertion angles were measured with surface tensiometry and ellipsometry. The interaction of pAHNAK with lipid bilayers was studied using 31P solid-state nuclear magnetic resonance. pAHNAK preferentially and strongly interacted with phospholipids that comprised negatively charged polar head groups with unsaturated lipids. This finding provides a better understanding of AHNAK membrane behavior and the parameters that influence its function in membrane repair.
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Membrana Dobles de Lípidos/química , Proteínas de la Membrana/química , Proteínas de Neoplasias/química , Fosfolípidos/química , Humanos , Unión ProteicaRESUMEN
As a member of the S100 protein family, S100A10 has already been purified. However, its purity, or even yield, have often not been reported in the literature. To facilitate future biophysical experiments with S100A10, we aimed to obtain it at a purity of at least 95% in a reasonably large amount. Here, we report optimized conditions for the transformation, overexpression and purification of the protein. We obtained a purity of 97% and performed stability studies by circular dichroism. Our data confirmed that the S100A10 obtained is suitable for experiments to be performed at room temperature up to several days.
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Biotecnología/métodos , Proteínas S100/aislamiento & purificación , Dicroismo Circular , Estabilidad Proteica , TemperaturaRESUMEN
Nanotechnologies are increasingly being developed for medical purposes. However, these nanomaterials require ultrastability for better control of their pharmacokinetics. The present study describes three types of ultrastable gold nanoparticles stabilized by thiolated polyethylene glycol groups remaining intact when subjected to some of the harshest conditions described thus far in the literature, such as autoclave sterilization, heat and freeze-drying cycles, salts exposure, and ultracentrifugation. Their stability is characterized by transmission electron microscopy, UV-visible spectroscopy, and dynamic light scattering. For comparison purposes, two conventional nanoparticle types were used to assess their colloidal stability under all conditions. The ability of ultrastable gold nanoparticles to encapsulate bimatoprost, a drug for glaucoma treatment, is demonstrated. MTS assays on human corneal epithelial cells is assessed without changing cell viability. The impact of ultrastable gold nanoparticles on wound healing dynamics is assessed on tissue engineered corneas. These results highlight the potential of ultrastable gold nanoparticles as a drug delivery system in ocular therapy.
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Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Oro , Nanopartículas del Metal , Línea Celular , Supervivencia Celular , Fenómenos Químicos , Técnicas de Química Sintética , Portadores de Fármacos/química , Oro/química , Humanos , Ligandos , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Análisis Espectral , Cicatrización de HeridasRESUMEN
Many research projects are underway to improve the diagnosis and therapy in ophthalmology. Indeed, visual acuity deficits affect 285 million people worldwide and different strategies are being developed to strengthen patient care. One of these strategies is the use of gold nanoparticles (GNP) for their multiple properties and their ability to be used as both diagnosis and therapy tools. This review exhaustively details research developing GNPs for use in ophthalmology. The toxicity of GNPs and their distribution in the eye are described through in vitro and in vivo studies. All publications addressing the pharmacokinetics of GNPs administered in the eye are extensively reviewed. In addition, their use as biosensors or for imaging with optical coherence tomography is illustrated. The future of GNPs for ophthalmic therapy is also discussed. GNPs can be used to deliver genes or drugs through different administration routes. Their antiangiogenic and anti-inflammatory properties are of great interest for different ocular pathologies. Finally, GNPs can be used to improve stereotactic radiosurgery, brachytherapy, and photothermal therapy because of their many properties.
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Oro/química , Nanopartículas del Metal/química , Oftalmología , Animales , Sistemas de Liberación de Medicamentos , Ojo/efectos de los fármacos , Oro/toxicidad , Humanos , Nanopartículas del Metal/toxicidad , Nanopartículas del Metal/ultraestructuraRESUMEN
Neurocalcin delta (NCALD) is a member of the neuronal calcium sensors protein family. In the retina, NCALD is expressed by ganglion and amacrine cells. NCALD is composed of 4 EF-hand motifs but only 3 of them may bind calcium. The binding of calcium induces a conformational change of the protein which leads to the extrusion of its N-terminal myristoyl group as well as some hydrophilic residues. This mechanism, named calcium-myristoyl switch, is presumably involved in its membrane binding. The parameters responsible for the interaction of NCALD with membranes are only partially known. The purpose of this study was thus to gather more information on the membrane binding behavior of NCALD using lipid monolayers, including the influence of the lipid composition, the calcium and the myristoyl group. NCALD was injected underneath different lipid monolayers and this model membrane allowed the determination of the binding parameters as maximum insertion pressure (MIP) and synergy. The values of MIP are larger when monolayers were composed of a saturated phospholipid with phosphoethanolamine polar head. This trend is confirmed by polarization modulation infrared reflection absorption spectroscopy measurements. Moreover, the observations by fluorescence microscopy show that NCALD preferentially interacts with phospholipids which are in the liquid-condensed physical state, as found in membrane microdomains. This observation could explain the changes of NCALD expression level in the brains of patients suffering from Alzheimer's disease because of the alteration of lipid composition in microdomains structures.
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Membranas Artificiales , Neurocalcina/química , Sitios de Unión , Calcio/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Fluorescente , Ácido Mirístico/química , Neurocalcina/genética , Neurocalcina/aislamiento & purificación , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Spectrometers are widely used in molecular detection. However most of them are bulky, power consuming, and quite expensive. This work presents the prototype of a compact visible spectrometer alternative that is battery-operated, and designed for autonomous operation and quick spectrum detection. It targets spherical gold nanoparticles spectroscopy, but other applications are possible thanks to a high-precision mechanism to move the sensor, which allows the spectrometer to cover a broad range of wavelengths in the visible spectrum.
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Nanopartículas del Metal , Refractometría , Color , Oro , LuzRESUMEN
A microfluidic-based spectrophotometer for neurotransmitters sensing is presented in this paper. In addition, a neurotransmitter photo-fingerprint is analyzed to evaluate the feasibility of selective neurotransmitter detection using optical techniques. The aim of this work is to detect major neurotransmitters (NTs) using a compact, portable and cost effective optical system for selective and real time NT concentration monitoring. Micro-spectroscopic detection of NTs is challenging because most of them are transparent to visible light. Nevertheless, they interfere with the absorption spectrum of gold nanoparticles (Au-NP), which exhibit maximum absorbance in the range of 520 nm. We observed an Au-NP maximum absorbance shift of up to 4nm in presence of NTs. Based on this shift, it is possible to detect NTs using visible ligh by using vertical-cavity surface-emitting laser (VCSEL) as light sources and an integrated system-on-chip (SoC) spectrophotometer.
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Oro , Nanopartículas del Metal , Color , Microfluídica , NeurotransmisoresRESUMEN
A large number of drugs are administered on different mucosal surfaces. However, due to the poor mucoadhesion of the current formulations, their bioavailability is often very low. The development of efficient mucoadhesive drug delivery systems is thus crucial for improving the performance of these drugs. The mucoadhesive properties of gold nanoparticles were investigated. First, two types of gold nanoparticles were synthesized: AuNP1 and AuNP2. AuNP1 only contain internal thiol groups on their metallic core, and AuNP2 contain both internal and peripheral thiol groups. Different protocols based on an adapted quantitative colorimetric method, UV-visible and fluorescence spectroscopies were then developed to gather information on their mucoadhesive properties. Moreover, a global correction factor for the inner filter effect in spectrofluorimetry was proposed, and the data obtained were compared to those commonly used in the literature. Mucins deeply interact with AuNP1, perturbing their core, whereas they remain at the periphery of AuNP2. The quantitative method suggests that a larger number of mucins interact with AuNP2. The establishment of this protocol could be applied to assess the mucoadhesive properties of other stable molecules. This mucoadhesive property of gold nanoparticles could be combined with their drug delivery ability in order to improve the medication administered on mucosa.
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Portadores de Fármacos/química , Oro/química , Nanopartículas del Metal/química , Membrana Mucosa/química , Adhesividad , Calibración , Ligandos , Tamaño de la PartículaRESUMEN
Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.
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Leishmania donovani/metabolismo , Microcuerpos/metabolismo , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/química , Fosfatidilgliceroles/química , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Sitios de Unión , Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Fraccionamiento Celular , Colesterol/química , Colesterol/metabolismo , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Leishmania donovani/genética , Fluidez de la Membrana , Microcuerpos/química , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/genética , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceroles/metabolismo , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
This review presents data on the influence of various experimental parameters on the binding of proteins onto Langmuir lipid monolayers. The users of the Langmuir methodology are often unaware of the importance of choosing appropriate experimental conditions to validate the data acquired with this method. The protein Retinitis pigmentosa 2 (RP2) has been used throughout this review to illustrate the influence of these experimental parameters on the data gathered with Langmuir monolayers. The methods detailed in this review include the determination of protein binding parameters from the measurement of adsorption isotherms, infrared spectra of the protein in solution and in monolayers, ellipsometric isotherms and fluorescence micrographs.