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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.

The disulfide bonds formed in the SAPA domain of a recombinant version of the NHsub2/sub-terminal propeptide (SP-BsubN/sub) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-BsubN/subwith GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cyssup32/sup-Cyssup49/supand Cyssup40/sup-Cyssup55/sup, and we propose a disulfide connectivity pattern of 1-3 and 2-4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z ∼ 2136 and ∼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptidessup24/supAWTTSSLACAQGPEsup37/supandsup45/supQALQCRsup50/suplinked by Cyssup32/sup-Cyssup49/supandsup38/supFWCQSLEsup44/supandsup51/supALGHCLQEsup58/suplinked by Cyssup40/sup-Cyssup55/suprespectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1-3/2-4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BsubN/subpropeptide, the MS analysis of both digests identified the bond Cyssup100/sup-Cyssup112/sup, numbered 7-8, which is coincident with the bond position in the kringle motif. SIGNIFICANCE: The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.

Lung surfactant (LS) stabilizes the respiratory surface by forming a film at the alveolar air-liquid interface that reduces surface tension and minimizes the work of breathing. Typically, this surface-active agent has been isolated from animal lungs both for research and biomedical applications. However, these materials are constituted by complex membranous architectures including surface-active and inactive lipid/protein assemblies. In this work, we describe the composition, structure and surface activity of discrete membranous entities that are part of a LS preparation isolated from bronchoalveolar lavages of porcine lungs. Seven different fractions could be resolved from whole surfactant subjected to sucrose density gradient centrifugation. Detailed compositional characterization revealed differences in protein and cholesterol content but no distinct saturated:unsaturated phosphatidylcholine ratios. Moreover, no significant differences were detected regarding apparent hydration at the headgroup region of membranes, as reported by the probe Laurdan, and lipid chain mobility analysed by electron spin resonance (ESR) in spite of the variety of membranous assemblies observed by transmission electron microscopy. In addition, six of the seven separated LS subfractions formed similar, essentially disordered-like, interfacial films and performed efficient surface activity, under physiologically relevant conditions. Altogether, our work show that a LS isolated from porcine lungs is comprised by a heterogenous population of membranous assemblies lacking freshly secreted unused LS complexes sustaining highly dehydrated and ordered membranous assemblies as previously reported. We propose that surfactant subfractions may illustrate intermediates in sequential structural steps within the structural transformations occurring along the respiratory compression-expansion cycles.

52 p.-9 fig.-3 tab., Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for the binding to the PHA granule. BioF has been exploited with 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 have analysed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain the affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on the PHA, stability on the surface of the PHA granule against pH, temperature and ionic strength, and their possible influence on the PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP), which retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing functionality and stability of the fusion proteins MinP-β-galactosidase and MinP-CueO from Escherichia coli., IMPORTANCE.Polyhydroxyalkanoates (PHAs) are biocompatible, non-toxic 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., This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme, grant agreement no. 633962 (P4SB), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R, BIO2016-79323-R, RTI2018-094564-B-I00) and the Community of Madrid (P2013/MIT2807, P2018/NMT4389). Francisco Blanco is a recipient of a predoctoral grant from the State 560 Program for the Promotion of Talent and its Employability in R&D&I (PRE-2018- 561 083859) from the Spanish Ministry of Science and Innovation.

This work is focused on the potential use of pulmonary surfactant to deliver full-length recombinant human surfactant protein SP-D (rhSP-D) using the respiratory air-liquid interface as a shuttle. Surfactant protein D (SP-D) is a collectin protein present in the pulmonary surfactant (PS) system, involved in innate immune defense and surfactant homeostasis. It has been recently suggested as a potential therapeutic to alleviate inflammatory responses and lung diseases in preterm infants suffering from respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD). However, none of the current clinical surfactants used for surfactant replacement therapy (SRT) to treat RDS contain SP-D. The interaction of SP-D with surfactant components, the potential of PS as a respiratory drug delivery system and the possibility to produce recombinant versions of human SP-D, brings the possibility of delivering clinical surfactants supplemented with SP-D. Here, we used an in vitro setup that somehow emulates the respiratory air-liquid interface to explore this novel approach. It consists in two different compartments connected with a hydrated paper bridge forming a continuous interface. We firstly analyzed the adsorption and spreading of rhSP-D alone from one compartment to another over the air-liquid interface, observing low interfacial activity. Then, we studied the interfacial spreading of the protein co-administered with PS, both at different time periods or as a mixed formulation, and which oligomeric forms of rhSP-D better traveled associated with PS. The results presented here demonstrated that PS may transport rhSP-D long distances over air-liquid interfaces, either as a mixed formulation or separately in a close window time, opening the doors to empower the current clinical surfactants and SRT.

Therapeutic hypothermia (TH) enhances pulmonary surfactant performance in vivo by molecular mechanisms still unknown. Here, the interfacial structure and the composition of lung surfactant films have been analysed in vitro under TH as well as the molecular basis of its improved performance both under physiological and inhibitory conditions. The biophysical activity of a purified porcine surfactant was tested under slow and breathing-like dynamics by constrained drop surfactometry (CDS) and in the captive bubble surfactometer (CBS) at both 33 and 37 °C. Additionally, the temperature-dependent surfactant activity was also analysed upon inhibition by plasma and subsequent restoration by further surfactant supplementation. Interfacial performance was correlated with lateral structure and lipid composition of films made of native surfactant. Lipid/protein mixtures designed as models to mimic different surfactant contexts were also studied. The capability of surfactant to drastically reduce surface tension was enhanced at 33 °C. Larger DPPC-enriched domains and lower percentages of less active lipids were detected in surfactant films exposed to TH-like conditions. Surfactant resistance to plasma inhibition was boosted and restoration therapies were more effective at 33 °C. This may explain the improved respiratory outcomes observed in cooled patients with acute respiratory distress syndrome and opens new opportunities in the treatment of acute lung injury.

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.

Surfactant protein C (SP-C) is a protein present in the pulmonary surfactant system that is involved in the biophysical properties of this lipoprotein complex, but it also has a role in lung defense and homeostasis. In this article, we propose that the link between both functions could rely on the ability of SP-C to induce fragmentation of phospholipid membranes and generate small vesicles that serve as support to present different ligands to cells in the lungs. Our results using bimolecular fluorescence complementation and tunable resistive pulse sensing setups suggest that SP-C oligomerization could be the triggering event that causes membrane budding and nanovesiculation. As shown by fluorescence microscopy and flow cytometry, these vesicles are differentially assimilated by alveolar macrophages and alveolar type II cells, indicating distinct roles of these alveoli-resident cells in the processing of the SP-C- induced vesicles and their cargo. These results depict a more accurate picture of the mechanisms of this protein, which could be relevant for the comprehension of pulmonary pathologies and the development of new therapeutic approaches.

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.

Surfactant protein B (SP-B) is essential in transferring surface-active phospholipids from membrane-based surfactant complexes into the alveolar air-liquid interface. This allows maintaining the mechanical stability of the surfactant film under high pressure at the end of expiration; therefore, SP-B is crucial in lung function. Despite its necessity, the structure and the mechanism of lipid transfer by SP-B have remained poorly characterized. Earlier, we proposed higher-order oligomerization of SP-B into ring-like supramolecular assemblies. In the present work, we used coarse-grained molecular dynamics simulations to elucidate how the ring-like oligomeric structure of SP-B determines its membrane binding and lipid transfer. In particular, we explored how SP-B interacts with specific surfactant lipids, and how consequently SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. Based on these studies, there are specific lipid-protein interactions leading to perturbation and reorganization of pulmonary surfactant layers. Especially, we found compelling evidence that anionic phospholipids and cholesterol are needed or even crucial in the membrane binding and lipid transfer function of SP-B. Also, on the basis of the simulations, larger oligomers of SP-B catalyze lipid transfer between adjacent surfactant layers. Better understanding of the molecular mechanism of SP-B will help in the design of therapeutic SP-B-based preparations and novel treatments for fatal respiratory complications, such as the acute respiratory distress syndrome. (C) 2020 The Author(s). Published by Elsevier Ltd.

Pulmonary surfactant is a complex of lipids and proteins assembled and secreted by the alveolar epithelium into the thin layer of fluid coating the respiratory surface of lungs. There, surfactant forms interfacial films at the air-water interface, reducing dramatically surface tension and thus stabilizing the air-exposed interface to prevent alveolar collapse along respiratory mechanics. The absence or deficiency of surfactant produces severe lung pathologies. This review describes some of the most important surfactant-related pathologies, which are a cause of high morbidity and mortality in neonates and adults. The review also updates current therapeutic approaches pursuing restoration of surfactant operative films in diseased lungs, mainly through supplementation with exogenous clinical surfactant preparations. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

Pulmonary surfactant is a lipid-protein complex which coats lung alveoli. It displays the essential function of reducing surface tension at the air-liquid interface, avoiding alveolar collapse during expiration. The optimized biophysical properties of surfactant rely on its defined composition, constituted mainly by phospholipids and tiny amounts of lipid-associated specific proteins. Due to the highly hydrophobic nature of surfactant, organic solvents have been traditionally employed to obtain and characterize surfactant lipids and proteins, very likely leading to disruption of native interactions among its components. In the present work we have addressed the search of native protein complexes in pulmonary surfactant, which could have an essential role in the optimal function of the system. By solubilizing native lipid-protein membranes of surfactant with non-denaturing detergents, and with the use of a two-dimensional electrophoresis strategy, we have been able to detect the presence of supramolecular complexes composed of surfactant proteins SP-A, SP-B and SP-C. Furthermore, by co-immunoprecipitation assays, we have confirmed for the first time the existence of a direct interaction between SP-A and SP-B, an important feature which could explain the known functional cooperation of both proteins in several aspects of surfactant biology. Significance This paper deepens for the first time in the existence of complex interaction networks of surfactant proteins in native surfactant membranes. By the use of non-denaturing detergents, two-dimensional electrophoresis and immunoprecipitation, we have been able to make progress in the elucidation of native protein complexes in this essential system, that had been previously hindered by the classical purification protocols employing organic solvents. In this work, we have described the presence of interactions between SP-B and SP-A, two important proteins whose functional cooperation has been broadly reported in the literature. Pioneer determination of such native complexes could have potential implications for understanding the wide variety of roles of pulmonary surfactant system.

Pulmonary surfactant (PS) is a lipid-protein complex essential to stabilise the delicate structure of mammalian alveoli along with successive compression-expansion respiratory cycles. To do so, surfactant reduces dramatically surface tension at the air-liquid interface, an activity that depends critically on a proper lipid composition and the presence of some specific surfactant proteins. Lack or dysfunction of this system is associated with severe respiratory pathologies, which are in some cases treated by supplementation with exogenous surfactant materials. The biophysical function and performance of PS, in health and disease, are directly influenced by its composition, structure and mechanical properties. This review summarises the main biophysics concepts behind the mechanisms that define surfactant function in a healthy lung and in pathological situations. It also revises some of the most useful biophysical techniques that provide information about surfactant-related processes. Finally, translational biophysics will be invoked to illustrate how biophysical studies may contribute to understand the role of surfactant in health and disease and to design better surfactant-based therapeutic approaches.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

With the intention to move away from animal testing for the toxicological evaluation of chemicals comes the need to develop new approach methodologies which are mechanism-anchored and target relevant key events leading to an adverse outcome. To date, no validated alternative methods are available for studying the acute inhalation toxicity potential of airborne chemicals but the constrained drop surfactometer measuring the surface tension of a drop of lung surfactant presents as a promising candidate. Indeed, the correlation of the increase in minimum surface tension of lung surfactant in vitro with changes in the breathing patterns of mice after inhalation of test compounds has been shown in multiple studies. However, the causal factors leading to lung surfactant inactivation remain speculative. This paper combines molecular and biophysical methods (constrained drop and captive bubble surfactometers, Langmuir-Blodgett balance, epifluorescence microscopy, cryogenic transmission electron microscopy, and differential scanning calorimetry) applied to purified porcine lung surfactant and dipalmitoylphosphatidylcholine interfacial films to gain insights into the disruption of lung surfactant function by three chemicals known to show acute inhalation toxicity (trimethoxyoctylsilane, methyl 3-oxo-2-pentylcyclopentaneacetate, and diisopentyl ether). The results of this study suggest that the test chemicals intercalate between the phospholipids at the air-liquid interface, reduce the stability of the films, and decrease the cohesivity of interface-associated multilayered structures thereby perturbing the lung surfactant surface activity. These findings contribute to a better understanding of chemically-induced lung surfactant function disruption.

Pulmonary surfactant is a lipoprotein complex that reduces surface tension to prevent alveolar collapse and contributes to the protection of the respiratory surface from the entry of pathogens. Surfactant protein A (SP-A) is a hydrophilic glycoprotein of the collectin family, and its main function is related to host defense. However, previous studies have shown that SP-A also aids in the formation and biophysical properties of pulmonary surfactant films at the air-water interface. Humans, unlike rodents, have two genes, SFTPA1 and SFTPA2. The encoded proteins, SP-A1 and SP-A2, differ quantitatively or qualitatively in function. It has been shown that both gene products are necessary for tubular myelin formation, an extracellular structural form of lung surfactant. The goal of this study was to investigate potential differences in the biophysical properties of surfactants containing human SP-A1, SP-A2, or both. For this purpose, we have studied for the first time, to our knowledge, the biophysical properties of pulmonary surfactant from individual humanized transgenic mice expressing human SP-A1, SP-A2, or both SP-A1 and SP-A2, in the captive bubble surfactometer. We observed that pulmonary surfactant containing SP-A1 reaches lower surface tension after postexpansion interfacial adsorption than surfactants containing no SP-A or only SP-A2. Under interfacial compression-expansion cycling conditions, surfactant films containing SP-A1 also performed better, particularly with respect to the reorganization of the films that takes place during compression. On the other hand, addition of recombinant SP-A1 to a surfactant preparation reconstituted from the hydrophobic fraction of a porcine surfactant made it more resistant to inhibition by serum than the addition of equivalent amounts of SP-A2. We conclude that the presence of SP-A1 allows pulmonary surfactant to adopt a particularly favorable structure with optimal biophysical properties.

Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages. SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency. In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein., This work has been supported by grants from the Spanish Ministry of Science and Innovation (RTI2018-094564-B-I00 to J.P.-G) and the Regional Government of Madrid (P2018/NMT­4389 to J.P.-G.) F.M.-H. Acknowledges support from the Spanish Ministry of Science Innovation and Universities (project BFU2017-83794-P, (AEI/FEDER, UE)) and from the European Research Council (ERC) under the European Union Horizon 2020 research and innovation (grant agreement No 681299). R.A. acknowledges an FPU PhD fellowship from the Spanish Ministry of Education.

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways. This work was supported by SAF2014-53209-R and SAF2017-86483-R grants from the Ministerio de Economía y Competitividadto R.B. and M.V., and to M.V., respectively, RTI2018-094564-B-I00 from the Spanish Ministry of Science and Universities andP2018/NMT4389 from the Regional Government of Madrid to J.P-G. and A.C., and RIRAAF Network RD12/0013/0015 grant and ARADyAL Network RD16/0006 grant from the Instituto de Salud Carlos III (ISCIII) co-founded by Fondo Europeo de Desarrollo Regional -FEDER-for the Thematic Networks and Co-operative Research Centres.A.J. and C.M. acknowledge PI-01119-2016 from the Consejería de Salud (Junta de Andalucía) and the Alergosur Foundation. JC.L-R., P.SS-A. and C.G-M. are recipients of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. We also gratefully acknowledge the financial support from theAES-ISCIII for the PI17CIII/00045 grant to R.B. We also thank the excellent technical support of Sara Abián. Sí

Pulmonary surfactant protein D (SP-D) is a glycoprotein from the collectin family that takes part in the lung surfactant system. It exhibits host defense and immune regulatory functions within the lung. It is known that SP-D monomer forms trimers, which associate into higher order oligomers. However, the pathway and the interactions involved in the assembly of SP-D oligomers are not clearly understood. In the current study, a recombinant form of human SP-D (rhSP-D) has been studied by Atomic Force Microscopy (AFM) and electrophoresis, with the goal of analyzing, qualitative and quantitatively, the conformational diversity and the determinants defining the oligomerization pathway of the protein. The studied rhSP-D is a mixture of trimers, hexamers, dodecamers and higher order oligomeric species, with dodecamers accounting for more than 50% of the protein by mass. Similar structures were also found in hSP-D obtained from proteinosis patients. The proportion of dodecamer is increased under acidic conditions, accompanied by a conformational change into more compact configurations. Dodecamers were found to be necessarily formed by two hexamers joined through non-covalent ionic and hydrophobic interactions between the N-terminal domains and the nearby area of SP-D collagen stems., MINECO. Spanish Ministry of Economy (BIO2015-67930-R). Regional Government of Madrid (S2013/MlT-2807). Spanish Ministry of Education. European Research Council (ERC). European Union’s Horizon2020 Research and Innovation Program (grant agreement No 681299).

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.-Martinez-Calle, M., Olmeda, B., Dietl, P., Frick, M., Perez-Gil, J. Pulmonary surfactant protein SP-B promotes exocytosis of lamellar bodies in alveolar type II cells.

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.

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to development of mammalian lungs, and great efforts have been done to understand its structure–function relationships. Previous evidence has outlined the importance of SP-C palmitoylation to sustain the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification aids SP-C to stabilize the interfacial surfactant film along the compression–expansion breathing cycles, is still unrevealed. In this work we have compared the structure, orientation and lipid–protein interactions of a native palmitoylated SP-C with those of a non-palmitoylated recombinant SP-C (rSP-C) form in air-exposed multilayer membrane environments, by means of ATR-FTIR spectroscopy. Palmitoylation does not affect the secondary structure of the protein, which exhibits a full α-helical conformation in partly dehydrated phospholipid multilayer films. However, differences between the Amide I band of the IR spectrum of palmitoylated and non-palmitoylated proteins suggest subtle differences affecting the environment of their helical component. These differences are accompanied by differential effects on the IR bands from phospholipid phosphates, indicating that palmitoylation modulates lipid–protein interactions at the headgroup region of phospholipid layers. On the other hand, the relative dichroic absorption of polarized IR has allowed calculating that the palmitoylated protein adopts a more tilted transmembrane orientation than the non-palmitoylated SP-C, likely contributing to more compact, dehydrated and possibly stable multilayer lipid–protein films. As a whole, the behavior of multilayer films containing palmitoylated SP-C may reflect favorable structural properties for surfactant reservoirs at the air–liquid respiratory interface.

Pulmonary surfactant is a membrane-based lipid-protein system essential for the process of breathing, which coats and stabilizes the whole respiratory surface and possesses exceptional biophysical properties. It constitutes the first barrier against the entry of pathogens and harmful particles in the alveolar region, extended through the lungs, but on the other hand, it can offer novel possibilities as a shuttle for the delivery of drugs and nanocarriers. The advances in nanotechnology are opening the doors to new diagnostic and therapeutic avenues, which are not accessible by means of the current approaches. In this context, the pulmonary route is called to become a powerful way of entry for innovative treatments based on nanotechnology. In this review, the anatomy of the respiratory system and its properties for drug entry are first revisited, as well as some current strategies that use the respiratory route for both local and peripheral action. Then, a brief overview is presented on what pulmonary surfactant is, how it works and why it could be used as a drug delivery vehicle. Finally, the review is closed with a description of the development of nanocarriers in the lung context and their interaction with endogenous and clinical pulmonary surfactants. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escriba.

Membrane fusion induced by the envelope glycoprotein enables the intracellular replication of HIV-1; hence, this process constitutes a major target for antiretroviral compounds. It has been proposed that peptides having propensity to interact with membrane interfaces might exert broad antiviral activity against enveloped viruses. To test this hypothesis, in this contribution we have analyzed the antiviral effects of peptides derived from the membrane-proximal external region and the transmembrane domain of the envelope glycoprotein subunit gp41, which display different degrees of interfacial hydrophobicity. Our data support the virucidal activity of a region that combines hydrophobic-at-interface membrane-proximal external region aromatics with hydrophobic residues of the transmembrane domain, and contains the absolutely conserved 679LWYIK/R683 sequence, proposed to embody a "cholesterol recognition/interaction amino acid consensus" motif. We further sought to correlate the antiviral activity of these peptides and their effects on membranes that mimic lipid composition and biophysical properties of the viral envelope. The data revealed that peptides endowed with virucidal activity were membrane active and induced permeabilization and fusion of virus-like lipid vesicles. In addition, they modulated lipid packing and miscibility of laterally segregated liquid domains, two properties that depend on the high cholesterol content of the viral membrane. Thus, the overall experimental evidence is consistent with a pattern of HIV inhibition that involves direct alteration of the physical chemistry of the virus membrane. Furthermore, the sequence-dependent effects observed might guide the development of new virucidal peptides.

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.

Pulmonary surfactant (PS) is a surface active complex of lipids and proteins that prevents the alveolar structures from collapsing and reduces the work of breathing by lowering the surface tension at the alveolar air–liquid interface (ALI). Surfactant is synthesized by the alveolar type II (AT II) cells, and it is stored in specialized organelles, the lamellar bodies (LBs), as tightly packed lipid bilayers. Upon secretion into the alveolar lining fluid, a large fraction of these particles retain most of their packed lamellar structure, giving rise to the term lamellar body like-particles (LBPs). Due to their stability in aqueous media, freshly secreted LBPs can be harvested from AT II cell preparations. However, when LBPs get in contact with an ALI, they quickly and spontaneously adsorb into a highly organized surface film. In the present study we investigated the adsorptive capacity of LBPs at an ALI under relevant physiological parameters that characterize the alveolar environment in homeostatic or in pathological conditions. Adsorption of LBPs at an ALI is highly sensitive to pH, temperature and albumin concentration and to a relatively lesser extent to changes in osmolarity or Ca2 + concentrations in the physiological range. Furthermore, proteolysis of LBPs significantly decreases their adsorptive capacity confirming the important role of surfactant proteins in the formation of surface active films.

The outstanding properties of gold nanoparticles (NPs) make them very attractive for biomedical applications. In particular, the inhalation route has gained considerable interest as an innovative strategy for diagnosis and treatment of pulmonary diseases. It is, therefore, important to scrutinise the potentially deleterious or side effects of NPs on lung epithelium. The present study investigates, for the first time, the impact of polyethylene glycol (PEG)-coated NPs on freshly purified primary cultures of rat alveolar type II (ATII) cells. These cells play a central role in the respiratory function of the lungs. They are responsible for synthesizing and secreting pulmonary surfactant (PS), which is required to stabilise the respiratory surface during breathing dynamics. Cytotoxicity and cellular uptake of NPs was evaluated by analysing morphology, viability and exocytotic activity of ATII cells (PS secretion). The impact of ATII cells' exposure to NPs was studied in a wide range of gold concentration with particles sizes of 15 and 100 nm. The results show that PEG-coated NPs are very modestly internalised by ATII cells and it neither leads to detectable morphological changes nor to decreased cell viability nor to alterations in basic functional parameters such as PS secretion, even on exposure to high gold concentration (~0.2 mM) during relatively long periods of time (24-48 h).

Pulmonary surfactant is a complex mixture of lipids and specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C, in charge of stabilizing the respiratory surface of mammalian lungs. The combined action of both proteins is responsible for the proper structure and dynamics of membrane arrays in the pulmonary surfactant network that covers the respiratory surface. In this study, we explore the possibility that proteins SP-B and SP-C induce the permeabilization of phospholipid membranes via pore formation. To this end, electrophysiological measurements have been carried out in planar lipid membranes prepared with different lipid/protein mixtures. Our main result is that channel-like structures are detected in the presence of SP-B, SP-C, or the native mixture of both proteins. Current traces show a high variety of conductance states (from pS to nS) that are dependent both on the lipid composition and the applied potential. We also show that the type of host lipid crucially determines the ionic selectivity of the observed pores: the anionic selectivity observed in zwitterionic membranes is inverted to cationic selectivity in the presence of negatively charged lipids. All those results suggest that SP-B and SP-C proteins promote the formation of proteolipid channels in which lipid molecules are functionally involved. We propose that proteolipidic membrane-permeabilizing structures may have an important role to tune ionic and lipidic flows through the pulmonary surfactant membrane network at the alveolar surfaces.

Surfacen® is a clinical surfactant preparation of porcine origin, partly depleted of cholesterol, which is widely used in Cuba to treat pre-term babies at risk or already suffering neonatal respiratory distress. In the present study we have characterized the interfacial behavior of Surfacen in several in vitro functional models, including spreading and compression–expansion cycling isotherms in surface balances and in a captive bubble surfactometer, in comparison with the functional properties of whole native surfactant purified from porcine lungs and its reconstituted organic extract, the material from which Surfacen is derived. Surfacen exhibited similar properties to native porcine surfactant or its organic extract to efficiently form stable surface active films at the air–liquid interface, able to consistently reach surface tensions below 5mN/m upon repetitive compression–expansion cycling. Surfacen films, however, showed a substantially larger and stable compression-driven segregation of condensed lipid phases than exhibited by films formed by native surfactant or its organic extract. In spite of structural differences observed at microscopic level, Surfacen membranes showed a similar thermotropic behavior to membranes from native surfactant or its organic extract, characterized by calorimetry or fluorescence spectroscopy of samples doped with the Laurdan probe. On the other hand, analysis by atomic force microscopy of films formed by Surfacen or by the organic extract of native porcine surfactant revealed a similar network of interconnected condensed nanostructures, suggesting that the organization of the films at the submicroscopic level is the essential feature to support the proper stability and mechanical properties permitting the interfacial surfactant films to facilitate the work of breathing.

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant's interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.

Contradictory results have been reported with respect to the depth of penetration and the orientation of pulmonary surfactant protein SP-B in phospholipid membranes and its relative selectivity to interact with anionic over zwitterionic phospholipid species. In the present study we have re-evaluated lipid–protein interactions of SP-B by analysing Forster resonance energy transfer (FRET) efficiencies, obtained from time-resolved measurements, from the single tryptophan in SP-B to different fluorescently labelled phospholipids in matrix bilayers made of either pure phosphatidylcholine (POPC) or the full lipid extract obtained from purified surfactant. In the background of POPC membranes SP-B exhibits a certain level of selectivity for anionic fluorescent phospholipids over the corresponding zwitterionic analogues, but apparently no preference for phosphatidylglycerol over other anionic species such as phosphatidylserine. No selectivity was detected in membranes made of full surfactant lipids, indicating that specific lipid–protein binding sites could already be occupied by endogenous anionic phospholipids. Furthermore, we have analysed the fit of two different models of how SP-B could be orientated with respect to phospholipid membrane surfaces to the FRET data. The FRET results are consistent with topology models in which the protein has a superficial orientation, with no regions of exclusion by the protein to the access of phospholipids, both in POPC membranes and in membranes made of the whole surfactant lipid fraction. This discards a deep penetration of the protein into the core of bilayers and suggests that most hydrophobic segments of SP-B could participate in protein–protein instead of lipid–protein interactions.

The breathing function is sustained by Lung Surfactant (LS), a macromolecular complex lining the alveolar air-liquid interface. In the context of the physiology of the LS system, the absence or dysfunction of surfactant protein SP-C is associated with several lung diseases. With the main goal of developing new therapeutic tools for the treatment of patients with respiratory dysfunction, we have evaluated different methodologies from the field of nanotechnology using scaffolding molecules that break membranes in nanoscale particles incorporating membrane proteins in their natural environment. We have explored the scaffolding reversible encapsulation of lipid bilayers incorporating either palmitoylated or a non-palmitoylated recombinant version of SP-C for the study of its structure-function relationships. In this work, SP-C has been solubilized using nanoparticles made with different lipid composition (POPC, POPC/POPG, POPC/POPG/DPPC or the Native Surfactant Lipid Fraction), assessing them as a potential native-like environment in order to preserve the tridimensional structure and functional properties of the protein. The nanoparticles behave themselves as a highly adsorptive material in the air-liquid interface, where they inhibited pulmonary surfactant adsorption. When SP-C is incorporated into the nanoparticles the interfacial adsorption capability of the complex increased slightly depending on the palmitoylation state of the protein. We have also analyzed the interaction of the complexes with surfactant monolayers and bilayers. Fluorescently labeled nanocomplexes seem to associate to both lipid structures with no apparent lipid transfer. These nanostructures could then constitute an efficient vehicle to direct molecules such as proteins or drugs to the respiratory air-liquid interface.

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air–liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37 °C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25 °C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37 °C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37 °C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37 °C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.

Mechanisms for meconium-induced inactivation of pulmonary surfactant as part of the meconium aspiration syndrome in newborn infants, to our knowledge, are not clearly understood. Here we have studied the biophysical mechanisms of how meconium affects surface activity of pulmonary surfactant and whether the membrane-perturbing effects of meconium can be mimicked by exposure of surfactant to a mixture of bile acids and cholesterol. Surface activity of pulmonary surfactant complexes purified from animal lungs was analyzed in the absence and in the presence of meconium in standard surface balances and in a captive bubble surfactometer. We have also evaluated accumulation of surfactant at the air-liquid interface by what we believe to be a novel microtiter plate fluorescent assay, and the effect of meconium components on surfactant membrane fluidity using Laurdan fluorescence thermotropic profiles and differential scanning calorimetry thermograms. Rapid interfacial adsorption, low surface tension upon film compression, efficient film replenishment upon expansion, and thermotropic properties of surfactant complexes are all adversely affected by meconium, and, in a similar manner, they are affected by cholesterol/taurocholate mixtures but not by taurocholate alone. We conclude that inhibition of surfactant by meconium can be mimicked by a bile salt-promoted incorporation of excess cholesterol into surfactant complexes. These results highlight the potential pathogenic role of cholesterol-mobilizing agents as a crucial factor resulting in cholesterol induced alterations of structure and dynamics of surfactant membranes and films.

Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.

Background Whole-body hypothermia (WBH) is used to improve neurological outcomes in perinatal asphyxia. Recent studies suggested a beneficial effect of hypothermia for some types of acute respiratory failure. However, no data are available about the biophysical function of human surfactant during WBH. We investigated whether WBH improves surfactant biophysical properties in asphyxiated neonates with or without meconium aspiration syndrome (MAS). Methods Non-bronchoscopic bronchoalveolar lavage (BAL) has been collected from 10 asphyxiated neonates (2 with MAS, 8 with no lung disease (NLD)) at different time-points (pre-WBH, 24h, 48h, 72h of WBH and post-WBH). Surfactant was extracted and tested by captive bubble surfactometry (CBS) in triplicate, at 37°C and 33.5°C, through initial adsorption and dynamic compression-expansion cycling. Phosphatidylcholine and cholesterol were assayed using enzymatic methods. Clinical data were recorded in real-time. Results Minimum surface tension under dynamic testing was significantly improved as assessed at 33.5°C compared with its behavior at 37°C in NLD neonates: the difference was evident after at least 72h of WBH and remained significant at 6h after rewarming (72h: p = 0.009; rewarming: p = 0.040). Similar results were obtained in MAS patients whose surfactant activity improved already at 48h of hypothermia. Total cholesterol showed a trend to increase at the first 24-48h of hypothermia in NLD patients. Conversely, hypothermia seemed to reduce the excess of exogenous cholesterol in MAS surfactant. Conclusions Surfactant biophysical properties may improve after 48-72h of WBH in asphyxiated neonates and the improvement is maintained shortly after rewarming. Due to study limitations, further studies are warranted to better clarify the effects of hypothermia on surfactant activity.