Bárbara Olmeda

Structure–function correlations of pulmonary surfactant protein SP-B and the saposin-like family of proteins

Pulmonary surfactant is a lipid-protein complex secreted by the respiratory epithelium of mammalian lungs, which plays an essential role in stabilising the alveolar surface and so reducing the work of breathing. The surfactant protein SP-B is part of this complex, and is strictly required for the assembly of pulmonary surfactant and its extracellular development to form stable surface-active films at the air–liquid alveolar interface, making the lack of SP-B incompatible with life. In spite of its physiological importance, a model for the structure and the mechanism of action of SP-B is still needed. The sequence of SP-B is homologous to that of the saposin-like family of proteins, which are membrane-interacting polypeptides with apparently diverging activities, from the co-lipase action of saposins to facilitate the degradation of sphingolipids in the lysosomes to the cytolytic actions of some antibiotic proteins, such as NK-lysin and granulysin or the amoebapore of Entamoeba histolytica. Numerous studies on the interactions of these proteins with membranes have still not explained how a similar sequence and a potentially related fold can sustain such apparently different activities. In the present review, we have summarised the most relevant features of the structure, lipid-protein and protein–protein interactions of SP-B and the saposin-like family of proteins, as a basis to propose an integrated model and a common mechanistic framework of the apparent functional versatility of the saposin fold.

Pulmonary surfactant layers accelerate O2 diffusion through the air-water interface

During respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid-protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air-liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface.

Lamellar Bodies Form Solid Three-dimensional Films at the Respiratory Air-Liquid Interface

Pulmonary surfactant is essential for lung function. It is assembled, stored and secreted as particulate entities (lamellar body-like particles; LBPs). LBPs disintegrate when they contact an air-liquid interface, leading to an instantaneous spreading of material and a decline in surface tension. Here, we demonstrate that the film formed by the adsorbed material spontaneously segregate into distinct ordered and disordered lipid phase regions under unprecedented near-physiological conditions and, unlike natural surfactant purified from bronchoalveolar lavages, dynamically reorganized into highly viscous multilayer domains with complex three-dimensional topographies. Multilayer domains, in coexistence with liquid phases, showed a progressive stiffening and finally solidification, probably driven by a self-driven disassembly of LBPs from a sub-surface compartment. We conclude that surface film formation from LBPs is a highly dynamic and complex process, leading to a more elaborated scenario than that observed and predicted by models using reconstituted, lavaged, or fractionated preparations.

A model for the structure and mechanism of action of pulmonary surfactant protein B

Surfactant protein B (SP‐B), from the saposin‐like family of proteins, is essential to facilitate the formation and proper performance of surface active films at the air‐liquid interface of mammalian lungs, and lack of or deficiency in this protein is associated with lethal respiratory failure. Despite its importance, neither a structural model nor a molecular mechanism of SP‐B is available. The purpose of the present work was to purify and characterize native SP‐B supramolecular assemblies to provide a model supporting structure‐function features described for SP‐B. Purification of porcine SP‐B using detergent‐solubilized surfactant reveals the presence of 10 nm ring‐shaped particles. These rings, observed by atomic force and electron microscopy, would be assembled by oligomerization of SP‐B as a multimer of dimers forming a hydrophobically coated ring at the surface of phospholipid membranes or monolayers. Docking of rings from neighboring membranes would lead to formation of SP‐B‐based hydrophobic tubes, competent to facilitate the rapid flow of surface active lipids both between membranes and between surfactant membranes and the interface. A similar sequential assembly of dimers, supradimeric oligomers and phospholipid‐loaded tubes could explain the activity of other saposins with colipase, cytolysin, or antibiotic activities, offering a common framework to understand the range of functions carried out by saposins.—Olmeda, B., García‐Álvarez, B., Gómez, M. J., Martínez‐Calle, M., Cruz, A., Pérez‐Gil, J. Amodel for the structure and mechanism of action of pulmonary surfactant protein B. FASEB J. 29, 4236‐4247 (2015). www.fasebj.org

Pulmonary surfactant metabolism in the alveolar airspace: Biogenesis, extracellular conversions, recycling ☆

Pulmonary surfactant is a lipid-protein complex that lines and stabilizes the respiratory interface in the alveoli, allowing for gas exchange during the breathing cycle. At the same time, surfactant constitutes the first line of lung defense against pathogens. This review presents an updated view on the processes involved in biogenesis and intracellular processing of newly synthesized and recycled surfactant components, as well as on the extracellular surfactant transformations before and after the formation of the surface active film at the air-water interface. Special attention is paid to the crucial regulation of surfactant homeostasis, because its disruption is associated with several lung pathologies.

A small key unlocks a heavy door: the essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

Effect of hypoxia on lung gene expression and proteomic profile: Insights into the pulmonary surfactant response

UNLABELLED Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72h) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins. BIOLOGICAL SIGNIFICANCE This work reveals that hypoxia induces extensive changes in the proteomic profile of lung bronchoalveolar lavage, including the presence of proteins related with inflammation both in lung tissue and lavage, and a significant increase in the synthesis and secretion by the lung tissue of different forms of hemoglobin. The level of specific pulmonary surfactant-associated proteins is not substantially altered due to hypoxia, but hypoxia-adapted surfactant exhibits an enhanced ability to form surface-active films at the air-liquid interface. The increased amount of β-globin integrated into the operative surfactant complexes obtained from hypoxic rats is a relevant feature that points to the existence of adaptive responses coupling surfactant function and oxygen availability.

Surfactant protein B (SP-B) enhances the cellular siRNA delivery of proteolipid coated nanogels for inhalation therapy

Despite the many advantages of small interfering RNA (siRNA) inhalation therapy and a growing prevalence of respiratory pathologies, its clinical translation is severely hampered by inefficient intracellular delivery. To this end, we previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). Interestingly, the PS shell was shown to markedly improve particle stability as well as intracellular siRNA delivery in vitro and in vivo. The major aim of this work was to identify the key molecular components of PS responsible for the enhanced siRNA delivery and evaluate how the complexity of the PS coat could be reduced. We identified surfactant protein B (SP-B) as a potent siRNA delivery enhancer when reconstituted in proteolipid coated hydrogel nanocomposites. Improved cytosolic siRNA delivery was achieved by inserting SP-B into a simplified phospholipid mixture prior to nanogel coating. This effect was observed both in vitro (lung epithelial cell line) and in vivo (murine acute lung injury model), albeit that distinct phospholipids were required to achieve these results. Importantly, the developed nanocomposites have a low in vivo toxicity and are efficiently taken up by resident alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Our results demonstrate the potential of the endogenous protein SP-B as an intracellular siRNA delivery enhancer, paving the way for future design of nanoformulations for siRNA inhalation therapy. STATEMENT OF SIGNIFICANCE Despite the therapeutic potential of small interfering RNA (siRNA) and a growing prevalence of lung diseases for which innovative therapies are needed, a safe and effective siRNA inhalation therapy remains non-existing due to a lack of suitable formulations. We identified surfactant protein B (SP-B) as a potent enhancer of siRNA delivery by proteolipid coated nanogel formulations in vitro in a lung epithelial cell line. The developed nanocomposites have a low in vivo toxicity and show a high uptake by alveolar macrophages, a main target cell type for treatment of inflammatory pulmonary pathologies. Importantly, in vivo SP-B is also critical for the developed formulation to obtain a significant silencing of TNFα in a murine LPS-induced acute lung injury model.