RESUMO
Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Biofilmes , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Tuberculose/patologia , Virulência , Fenômenos BiomecânicosRESUMO
The glycogenin knockout mouse is a model of Glycogen Storage Disease type XV. These animals show high perinatal mortality (90%) due to respiratory failure. The lungs of glycogenin-deficient embryos and P0 mice have a lower glycogen content than that of wild-type counterparts. Embryonic lungs were found to have decreased levels of mature surfactant proteins SP-B and SP-C, together with incomplete processing of precursors. Furthermore, non-surviving pups showed collapsed sacculi, which may be linked to a significantly reduced amount of surfactant proteins. A similar pattern was observed in glycogen synthase1-deficient mice, which are devoid of glycogen in the lungs and are also affected by high perinatal mortality due to atelectasis. These results indicate that glycogen availability is a key factor for the burst of surfactant production required to ensure correct lung expansion at the establishment of air breathing. Our findings confirm that glycogen deficiency in lungs can cause respiratory distress syndrome and suggest that mutations in glycogenin and glycogen synthase 1 genes may underlie cases of idiopathic neonatal death.
Assuntos
Glucosiltransferases/fisiologia , Glicogênio Sintase/fisiologia , Glicoproteínas/fisiologia , Surfactantes Pulmonares/metabolismo , Síndrome do Desconforto Respiratório/patologia , Animais , Animais Recém-Nascidos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/metabolismoRESUMO
By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.
Assuntos
Líquido Amniótico/química , Surfactantes Pulmonares/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Animais , Varredura Diferencial de Calorimetria , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lauratos/química , Lipídeos/química , Membranas , SuínosRESUMO
Obesity and type 2 diabetes are nutrition-related conditions associated with lung function impairment and pulmonary diseases; however, the underlying pathomechanisms are incompletely understood. Pulmonary surfactant is essential for lung function, and surfactant synthesis by AT2 (alveolar epithelial type 2) cells relies on nutrient uptake. We hypothesized that dietary amounts of carbohydrates or fat affect surfactant homeostasis and composition. Feeding mice a starch-rich diet (StD), sucrose-rich diet (SuD), or fat-rich diet (FaD) for 30 weeks resulted in hypercholesterolemia and hyperinsulinemia compared with a fiber-rich control diet. In SuD and FaD groups, lung mechanic measurements revealed viscoelastic changes during inspiration, indicating surfactant alterations, and interfacial adsorption of isolated surfactant at the air-liquid interface was decreased under FaD. The composition of characteristic phospholipid species was modified, including a shift from dipalmitoyl-phosphatidylcholine (PC16:0/16:0) to palmitoyl-palmitoleoyl-phosphatidylcholine (PC16:0/16:1) in response to carbohydrates and decreased myristic acid-containing phosphatidylcholine species (PC14:0/14:0; PC16:0/14:0) on excess fat intake, as well as higher palmitoyl-oleoyl-phosphatidylglycerol (PG16:0/18:1) and palmitoyl-linoleoyl-phosphatidylglycerol (PG16:0/18:2) fractions in StD, SuD, and FaD groups than in the control diet. Moreover, mRNA expression levels of surfactant synthesis-related proteins within AT2 cells were altered. Under the StD regimen, AT2 cells showed prominent lipid accumulations and smaller lamellar bodies. Thus, in an established mouse model, distinct diet-related surfactant alterations were subtle, yet detectable, and may become challenging under conditions of reduced respiratory capacity. Dietary fat was the only macronutrient significantly affecting surfactant function. This warrants future studies examining alimentary effects on lung surfactant, with special regard to pulmonary complications in obesity and type 2 diabetes.
Assuntos
Carboidratos da Dieta/efeitos adversos , Gorduras na Dieta/efeitos adversos , Surfactantes Pulmonares/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Fenômenos Biomecânicos , Forma Celular/efeitos dos fármacos , Glucose/metabolismo , Homeostase , Espaço Intracelular/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Pulmão/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Fosfolipídeos/sangueRESUMO
Lung surfactant (LS) is an outstanding example of how a highly regulated and dynamic membrane-based system has evolved to sustain a wealth of structural reorganizations in order to accomplish its biophysical function, as it coats and stabilizes the respiratory air-liquid interface in the mammalian lung. The present review dissects the complexity of the structure-function relationships in LS through an updated description of the lipid-protein interactions and the membrane structures that sustain its synthesis, secretion, interfacial performance and recycling. We also revise the current models and the biophysical techniques employed to study the membranous architecture of LS. It is important to consider that the structure and functional properties of LS are often studied in bulk or under static conditions, in spite that surfactant function is strongly connected with a highly dynamic behaviour, sustained by very polymorphic structures and lipid-lipid, lipid-protein and protein-protein interactions that reorganize in precise spatio-temporal coordinates. We have tried to underline the evidences available of the existence of such structural dynamism in LS. A last important aspect is that the synthesis and assembly of LS is a strongly regulated intracellular process to ensure the establishment of the proper interactions driving LS surface activity, while protecting the integrity of other cell membranes. The use of simplified lipid models or partial natural materials purified from animal tissues could be too simplistic to understand the true molecular mechanisms defining surfactant function in vivo. In this line, we will bring into the attention of the reader the methodological challenges and the questions still open to understand the structure-function relationships of LS at its full biological relevance.
Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , HumanosRESUMO
Pediatric (PARDS) and neonatal (NARDS) acute respiratory distress syndrome have different age-specific characteristics and definitions. Trials on surfactant for ARDS in children and neonates have been performed well before the PARDS and NARDS definitions and yielded conflicting results. This is mainly due to heterogeneity in study design reflecting historic lack of pathobiology knowledge. We reviewed the available clinical and preclinical data to create an expert consensus aiming to inform future research steps and advance the knowledge in this area. Eight trials investigated the use of surfactant for ARDS in children and ten in neonates, respectively. There were improvements in oxygenation (7/8 trials in children, 7/10 in neonates) and mortality (3/8 trials in children, 1/10 in neonates) improved. Trials were heterogeneous for patients' characteristics, surfactant type and administration strategy. Key pathobiological concepts were missed in study design. Consensus with strong agreement was reached on four statements: 1. There are sufficient preclinical and clinical data to support targeted research on surfactant therapies for PARDS and NARDS. Studies should be performed according to the currently available definitions and considering recent pathobiology knowledge. 2. PARDS and NARDS should be considered as syndromes and should be pre-clinically studied according to key characteristics, such as direct or indirect (primary or secondary) nature, clinical severity, infectious or non-infectious origin or patients' age. 3. Explanatory should be preferred over pragmatic design for future trials on PARDS and NARDS. 4. Different clinical outcomes need to be chosen for PARDS and NARDS, according to the trial phase and design, trigger type, severity class and/or surfactant treatment policy. We advocate for further well-designed preclinical and clinical studies to investigate the use of surfactant for PARDS and NARDS following these principles.
Assuntos
Prova Pericial , Pesquisa/tendências , Síndrome do Desconforto Respiratório/terapia , Tensoativos/uso terapêutico , Criança , Pré-Escolar , Previsões/métodos , Humanos , Lactente , Recém-Nascido , Pediatria/instrumentação , Pediatria/tendências , Síndrome do Desconforto Respiratório/mortalidade , Síndrome do Desconforto Respiratório/fisiopatologiaRESUMO
Surfactant protein (SP)-C deficiency is found in samples from patients with idiopathic pulmonary fibrosis, especially in familial forms of this disease. We hypothesized that SP-C may contribute to fibrotic remodeling in aging mice and alveolar lipid homeostasis. For this purpose, we analyzed lung function, alveolar dynamics, lung structure, collagen content, and expression of genes related to lipid and cholesterol metabolism of aging SP-C knockout mice. In addition, in vitro experiments with an alveolar macrophage cell line exposed to lipid vesicles with or without cholesterol and/or SP-C were performed. Alveolar dynamics showed progressive alveolar derecruitment with age and impaired oxygen saturation. Lung structure revealed that decreasing volume density of alveolar spaces was accompanied by increasing of the ductal counterparts. Simultaneously, septal wall thickness steadily increased, and fibrotic wounds appeared in lungs from the age of 50 weeks. This remarkable phenotype is unique to the 129Sv strain, which has an increased absorption of cholesterol, linking the accumulation of cholesterol and the absence of SP-C to a fibrotic remodeling process. The findings of this study suggest that overall loss of SP-C results in an age-dependent, complex, heterogeneous phenotype characterized by a combination of overdistended air spaces and fibrotic wounds that resembles combined emphysema and pulmonary fibrosis in patients with idiopathic pulmonary fibrosis. Addition of SP-C to cholesterol-laden lipid vesicles enhanced the expression of cholesterol metabolism and transport genes in an alveolar macrophage cell line, identifying a potential new lipid-protein axis involved in lung remodeling.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Colesterol/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Proteína C/metabolismo , Surfactantes Pulmonares/metabolismo , Idoso , Animais , Enfisema/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Pulmão/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Alvéolos Pulmonares/metabolismoRESUMO
No in vivo data are available regarding the effect of meconium on human surfactant in the early stages of severe meconium aspiration syndrome (MAS). In the present study, we sought to characterize the changes in surfactant composition, function, and structure during the early phase of meconium injury. We designed a translational prospective cohort study of nonbronchoscopic BAL of neonates with severe MAS (n = 14) or no lung disease (n = 18). Surfactant lipids were analyzed by liquid chromatography-high-resolution mass spectrometry. Secretory phospholipase A2 subtypes IB, V, and X and SP-A (surfactant protein A) were assayed by ELISA. SP-B and SP-C were analyzed by Western blotting under both nonreducing and reducing conditions. Surfactant function was assessed by adsorption test and captive bubble surfactometry, and lung aeration was evaluated by semiquantitative lung ultrasound. Surfactant nanostructure was studied using cryo-EM and atomic force microscopy. Several changes in phospholipid subclasses were detected during MAS. Lysophosphatidylcholine species released by phospholipase A2 hydrolysis were increased. SP-B and SP-C were significantly increased together with some shorter immature forms of SP-B. Surfactant function was impaired and correlated with poor lung aeration. Surfactant nanostructure was significantly damaged in terms of vesicle size, tridimensional complexity, and compactness. Various alterations of surfactant phospholipids and proteins were detected in the early phase of severe meconium aspiration and were due to hydrolysis and inflammation and a defensive response. This impairs both surfactant structure and function, finally resulting in reduced lung aeration. These findings support the development of new surfactant protection and antiinflammatory strategies for severe MAS.
Assuntos
Pulmão/efeitos dos fármacos , Síndrome de Aspiração de Mecônio/tratamento farmacológico , Surfactantes Pulmonares/farmacologia , Tensoativos/farmacologia , Anti-Inflamatórios/farmacologia , Humanos , Recém-Nascido , Pulmão/metabolismo , Síndrome de Aspiração de Mecônio/metabolismo , Síndrome de Aspiração de Mecônio/fisiopatologia , Fosfolipases A2/efeitos dos fármacos , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/metabolismoRESUMO
Morbidity and mortality rates in acute lung injury (ALI) increase with age. As alveolar epithelial type II cells (AE2) are crucial for lung function and repair, we hypothesized that aging promotes senescence in AE2 and contributes to the severity and impaired regeneration in ALI. ALI was induced with 2.5 µg lipopolysaccharide/g body weight in young (3 mo) and old (18 mo) mice that were euthanized 24 h, 72 h, and 10 days later. Lung function, pulmonary surfactant activity, stereology, cell senescence, and single-cell RNA sequencing analyses were performed to investigate AE2 function in aging and ALI. In old mice, surfactant activity was severely impaired. A 60% mortality rate and lung function decline were observed in old, but not in young, mice with ALI. AE2 of young mice adapted to injury by increasing intracellular surfactant volume and proliferation rate. In old mice, however, this adaptive response was compromised, and AE2 of old mice showed signs of cell senescence, increased inflammatory signaling, and impaired surfactant metabolism in ALI. These findings provide evidence that ALI promotes a limited proliferation rate, increased inflammatory response, and surfactant dysfunction in old, but not in young, mice, supporting an impaired regenerative capacity and reduced survival rate in ALI with advancing age.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Envelhecimento , Células Epiteliais Alveolares/metabolismo , Surfactantes Pulmonares/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismoRESUMO
Secreted phospholipase A2 hydrolyzes surfactant phospholipids and is crucial for the inflammatory cascade; preterm neonates are treated with exogenous surfactant, but the interaction between surfactant and phospholipase is unknown. We hypothesize that this interplay is complex and the enzyme plays a relevant role in neonates needing surfactant replacement. We aimed to: 1) identify phospholipases A2 isoforms expressed in preterm lung; 2) study the enzyme role on surfactant retreatment and function and the effect of exogenous surfactant on the enzyme system; and 3) verify whether phospholipase A2 is linked to respiratory outcomes. In bronchoalveolar lavages of preterm neonates, we measured enzyme activity (alone or with inhibitors), enzyme subtypes, surfactant protein-A, and inflammatory mediators. Surfactant function and phospholipid profile were also tested. Urea ratio was used to obtain epithelial lining fluid concentrations. Follow-up data were prospectively collected. Subtype-IIA is the main phospholipase isoform in preterm lung, although subtype-IB may be significantly expressed. Neonates needing surfactant retreatment have higher enzyme activity (P = 0.021) and inflammatory mediators (P always ≤ 0.001) and lower amounts of phospholipids (P always < 0.05). Enzyme activity was inversely correlated to surfactant adsorption (ρ = -0.6; P = 0.008; adjusted P = 0.009), total phospholipids (ρ = -0.475; P = 0.05), and phosphatidylcholine (ρ = -0.622; P = 0.017). Exogenous surfactant significantly reduced global phospholipase activity (P < 0.001) and subtype-IIA (P = 0.005) and increased dioleoylphosphatidylglycerol (P < 0.001) and surfactant adsorption (P < 0.001). Enzyme activity correlated with duration of ventilation (ρ = 0.679, P = 0.005; adjusted P = 0.04) and respiratory morbidity score at 12 mo postnatal age (τ-b = 0.349, P = 0.037; adjusted P = 0.043) but was not associated with mortality, bronchopulmonary dysplasia, or other long-term respiratory outcomes.
Assuntos
Recém-Nascido Prematuro/fisiologia , Fosfolipases A2 Secretórias/metabolismo , Surfactantes Pulmonares/metabolismo , Respiração , Líquido da Lavagem Broncoalveolar , Células Epiteliais/metabolismo , Feminino , Humanos , Recém-Nascido , Masculino , Fosfolipases A2 Secretórias/antagonistas & inibidores , FosfolipídeosRESUMO
Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.
Assuntos
Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Líquido Amniótico/metabolismo , Asparagina/metabolismo , Ligação Competitiva , Cromatografia de Afinidade , Feminino , Glicosilação , Humanos , Polissacarídeos/metabolismo , Gravidez , Ligação Proteica , Multimerização Proteica , Proteinose Alveolar Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP-ß-galactosidase and MinP-CueO from Escherichia coliIMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas putida/metabolismo , Biotecnologia , Enzimas Imobilizadas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/metabolismoRESUMO
Pulmonary surfactant is a lipid/protein complex synthesized by the alveolar epithelium and secreted into the airspaces, where it coats and protects the large respiratory air-liquid interface. Surfactant, assembled as a complex network of membranous structures, integrates elements in charge of reducing surface tension to a minimum along the breathing cycle, thus maintaining a large surface open to gas exchange and also protecting the lung and the body from the entrance of a myriad of potentially pathogenic entities. Different molecules in the surfactant establish a multivalent crosstalk with the epithelium, the immune system and the lung microbiota, constituting a crucial platform to sustain homeostasis, under health and disease. This review summarizes some of the most important molecules and interactions within lung surfactant and how multiple lipid-protein and protein-protein interactions contribute to the proper maintenance of an operative respiratory surface.
Assuntos
Células Epiteliais Alveolares/metabolismo , Homeostase , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Células Epiteliais Alveolares/microbiologia , Células Epiteliais Alveolares/fisiologia , Animais , Humanos , Metabolismo dos LipídeosRESUMO
TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.
Assuntos
Cromatografia em Camada Fina , Eletroforese em Gel de Poliacrilamida , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Lipídeos de Membrana/isolamento & purificação , Proteínas de Membrana/isolamento & purificaçãoRESUMO
Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.
Assuntos
Bicamadas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Mapas de Interação de Proteínas , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Animais , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos , Multimerização Proteica , Proteína B Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/química , SuínosRESUMO
The release of pulmonary surfactant by alveolar type II (ATII) cells is essential for lowering surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to alveolar collapse. Hydrophobic surfactant protein (SP)-B ensures the proper packing of newly synthesized surfactant particles, promotes the formation of the surface active film at the alveolar air-liquid interface and maintains its proper structure along the respiratory dynamics. We report that membrane-associated SP-B efficiently induces secretion of pulmonary surfactant by ATII cells, at the same level as potent secretagogues such as ATP. The presence in the extracellular medium of lipid-protein complexes containing SP-B activates the P2Y2 purinergic signaling pathway that ultimately triggers exocytosis of lamellar bodies by ATII cells. Our data suggest that SP-B prompts Ca2+-dependent surfactant secretion via ATP release from ATII cells. This result implies that SP-B is not only an essential component for the biophysical function of surfactant but is also a central element in the alveolar homeostasis by eliciting autocrine and paracrine cell stimulation.-Martínez-Calle, M., Olmeda, B., Dietl, P., Frick, M., Pérez-Gil, J. Pulmonary surfactant protein SP-B promotes exocytosis of lamellar bodies in alveolar type II cells.
Assuntos
Exocitose/fisiologia , Pulmão/metabolismo , Pulmão/fisiologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/fisiologia , Proteína B Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/fisiologia , Animais , Cálcio/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/fisiologia , SuínosRESUMO
Phasins, the major proteins coating polyhydroxyalkanoate (PHA) granules, have been proposed as suitable biosurfactants for multiple applications because of their amphiphilic nature. In this work, we analyzed the interfacial activity of the amphiphilic α-helical phasin PhaF from Pseudomonas putida KT2440 at different hydrophobic-hydrophilic interfacial environments. The binding of PhaF to surfaces containing PHA or phospholipids, postulated as structural components of PHA granules, was confirmed in vitro using supported lipid bilayers and confocal microscopy, with polyhydroxyoctanoate- co-hexanoate P(HO- co-HHx) and Escherichia coli lipid extract as model systems. The surfactant-like capabilities of PhaF were determined by measuring changes in surface pressure in Langmuir devices. PhaF spontaneously adsorbed at the air-water interface, reducing the surface tension from 72 mN/m (water surface tension at 25 °C) to 50 mN/m. The differences in the adsorption of the protein in the presence of different phospholipid films showed a marked preference for phosphatidylglycerol species, such as 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoglycerol. The PHA-binding domain of PhaF (BioF) conserved a similar surface activity to PhaF, suggesting that it is responsible for the surfactant properties of the whole protein. These new findings not only increase our knowledge about the role of phasins in the PHA machinery but also open new outlooks for the application of these proteins as biosurfactants.
Assuntos
Proteínas de Bactérias/química , Pseudomonas putida/química , Tensoativos/química , Adsorção , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Domínios Proteicos , Tensoativos/isolamento & purificação , Lipossomas Unilamelares/químicaRESUMO
Secreted pulmonary surfactant phosphatidylcholine (PC) has a complex intra-alveolar metabolism that involves uptake and recycling by alveolar type II epithelial cells, catabolism by alveolar macrophages, and loss up the bronchial tree. We compared the in vivo metabolism of animal-derived poractant alfa (Curosurf) and a synthetic surfactant (CHF5633) in adult male C57BL/6 mice. The mice were dosed intranasally with either surfactant (80 mg/kg body weight) containing universally 13C-labeled dipalmitoyl PC (DPPC) as a tracer. The loss of [U13C]DPPC from bronchoalveolar lavage and lung parenchyma, together with the incorporation of 13C-hydrolysis fragments into new PC molecular species, was monitored by electrospray ionization tandem mass spectrometry. The catabolism of CHF5633 was considerably delayed compared with poractant alfa, the hydrolysis products of which were cleared more rapidly. There was no selective resynthesis of DPPC and, strikingly, acyl remodeling resulted in preferential synthesis of polyunsaturated PC species. In conclusion, both surfactants were metabolized by similar pathways, but the slower catabolism of CHF5633 resulted in longer residence time in the airways and enhanced recycling of its hydrolysis products into new PC species.
Assuntos
Produtos Biológicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , Proteína C Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Produtos Biológicos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/farmacologia , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/farmacologia , Fosfolipídeos/farmacologia , Proteína B Associada a Surfactante Pulmonar/farmacologia , Proteína C Associada a Surfactante Pulmonar/farmacologia , Surfactantes Pulmonares/farmacologiaRESUMO
Lung surfactant (LS) is an essential system supporting the respiratory function. Cholesterol can be deleterious for LS function, a condition that is reversed by the presence of the lipopeptide SP-C. In this work, the structure of LS-mimicking membranes has been analyzed under the combined effect of SP-C and cholesterol by deuterium NMR and phosphorus NMR and by electron spin resonance. Our results show that SP-C induces phase segregation at 37°C, resulting in an ordered phase with spectral features resembling an interdigitated state enriched in dipalmitoylphosphatidylcholine, a liquid-crystalline bilayer phase, and an extremely mobile phase consistent with small vesicles or micelles. In the presence of cholesterol, POPC and POPG motion seem to be more hindered by SP-C than dipalmitoylphosphatidylcholine. The use of deuterated cholesterol did not show signs of specific interactions that could be attributed to SP-C or to the other hydrophobic surfactant protein SP-B. Palmitoylation of SP-C had an indirect effect on the extent of protein-lipid perturbations by stabilizing SP-C structure, and seemed to be important to maximize differences among the lipids participating in each phase. These results shed some light on how SP-C-induced lipid perturbations can alter membrane structure to sustain LS functionality at the air-liquid interface.