Person: Cruz Rodríguez, Antonio
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First Name
Antonio
Last Name
Cruz Rodríguez
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
22 results
Search Results
Now showing 1 - 10 of 22
Item In Vitro functional and structural characterization of a synthetic clinical pulmonary surfactant with enhanced resistance to inhibition(Scientific Reports, 2020) Echaide Torreguitar, Mercedes; Autilio, Chiara; López-Rodríguez, Elena; Cruz Rodríguez, Antonio; Pérez-Gil, JesúsCHF5633 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 purifed 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 fuorescence 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 flms formed in a Langmuir-Blodgett balance coupled with epifuorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than flms 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 diferential 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-defned composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung infammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.Item Functional organization of the HIV lipid envelope(Scientific Reports, 2016) Huarte, Nerea; Carravilla, Pablo; Cruz Rodríguez, Antonio; Lorizate, Maier; Nieto-Garay, Jon A.; Kräusslich, Hans-Georg; Pérez-Gil, Jesús; Requejo Isidro, José; Nieva, José L.The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.Item Effects of HIV-1 gp41-Derived Virucidal Peptides on Virus-like Lipid Membranes(Biophysical Journal, 2017) Carravilla, Pablo; Cruz Rodríguez, Antonio; Martín-Ugarte, Itziar; Oar-Arteta, Itziar R.; Torralba, Johanna; Apellaniz, Beatriz; Pérez-Gil, Jesús; Requejo Isidro, José; Huarte, Nerea; Nieva, José L.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.Item A model for the structure and mechanism of action of pulmonary surfactant protein B(The FASEB Journal, 2015) Olmeda Lozano, Bárbara; García Álvarez, María Begoña; Gómez, Manuel J.; Martínez Calle, Marta; Cruz Rodríguez, Antonio; Pérez Gil, JesúsSurfactant 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 structuralmodel nor amolecular 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 detergentsolubilized surfactant reveals the presence of 10 nm ringshaped 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., Perez-Gil, J. A model for the structure and mechanism of action of pulmonary surfactant protein B. FASEB J. 29, 4236–4247 (2015). www.fasebj.orgItem Pulmonary surfactant and drug delivery: Vehiculization of a tryptophan-tagged antimicrobial peptide over the air-liquid interfacial highway(European Journal of Pharmaceutics and Biopharmaceutics, 2022) García-Mouton, Cristina; Parra Ortiz, Elisa; Malmsten, Martin; Cruz Rodríguez, Antonio; Pérez Gil, JesúsThis work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically endtagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air–liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.Item Pulmonary surfactant and nanocarriers: toxicity versus combined nanomedical applications(Biochimica et Biophysica Acta - Biomembranes, 2017) Hidalgo Román, Alberto; Cruz Rodríguez, Antonio; Pérez-Gil, JesúsPulmonary 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.Item Surface activity as a crucial factor of the biological actions of Ole e1, the main aeroallergen of olive tree (Olea europaea) pollen(Langmuir, 2016) López-Rodríguez, Juan C.; Barderas Manchado, Rodrigo; Echaide Torreguitar, Mercedes; Pérez-Gil, Jesús; Villalba, Mayte; Batanero Cremades, Eva; Cruz Rodríguez, AntonioAeroallergens are airborne substancesmainly proteinscapable of triggering Th2-immune responses in respiratory allergies. They enter into the body through the upper airways, reaching the mucosa afterward. Mucosae lining at the luminal side consists of an epithelial barrier completely covered by mucus and pulmonary surfactant. Both pulmonary surfactant and plasma membrane of the epithelial cells represent two physiological phospholipid-based barriers where allergens first impact before triggering their biological effects. The interaction of allergens with lipids at relevant physiological surfaces could promote structural changes on the molecule, resulting on a potential modification of its allergenic properties. In this work, we have first described the surface and phospholipid interaction capabilities of the clinically relevant aeroallergen Ole e 1, the main allergen of olive tree pollen. By using epifluorescence microscopy of Langmuir transferred films, we observed that lipid-packed ordered domains may function as a preferential location for allergen to accumulate at the air−liquid interface, an effect that is abolished in the presence of cholestenone. The possible implications of phospholipid-interfacial effects in the modification of allergen structural and functional properties will be discussedItem Molecular and biophysical mechanisms behind the enhancement of lung surfactant function during controlled therapeutic hypothermia(Scientific Reports, 2020) Autilio, Chiara; Echaide Torreguitar, Mercedes; Cruz Rodríguez, Antonio; Hidalgo, A.; Da Silva, E.; De Luca, Daniele; Sørli, Jorid B.; Pérez-Gil, JesúsTherapeutic hypothermia (TH) enhances pulmonary surfactant performance in vivo by molecular mechanisms still unknown. Here, the interfacial structure and the composition of lung surfactant flms 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 purifed 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 temperaturedependent 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 flms made of native surfactant. Lipid/protein mixtures designed as models to mimic diferent 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 flms exposed to TH-like conditions. Surfactant resistance to plasma inhibition was boosted and restoration therapies were more efective 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.Item Efficient interfacially driven vehiculization of corticosteroids by pulmonary surfactant(Langmuir, 2017) Hidalgo, Alberto; Salomone, Fabrizio; Fresno, Nieves; Orellana Moraleda, Guillermo; Cruz Rodríguez, Antonio; Pérez-Gil, JesúsPulmonary surfactant is a crucial system to stabilize the respiratory air-liquid interface. Furthermore, pulmonary surfactant has been proposed as an effective method for targeting drugs to the lungs. However, few studies have examined in detail the mechanisms of incorporation of drugs into surfactant, the impact of the presence of drugs on pulmonary surfactant performance at the interface under physiologically meaningful conditions, or the ability of pulmonary surfactant to use the air-liquid interface to vehiculise drugs to long distances. This study focuses on the ability of pulmonary surfactant to interfacially vehiculize corticosteroids such as beclomethasone dipropionate (BDP) or Budesonide (BUD) as model drugs. The main objectives have been to (a) characterize the incorporation of corticosteroids into natural and synthetic surfactants, (b) evaluate whether the presence of corticosteroids affects surfactant functionality, and (c) determine whether surfactant preparations enable the efficient spreading and distribution of BDP and BUD along the air-liquid interface. We have compared the performance of a purified surfactant from porcine lungs and two clinical surfactants: Poractant alfa, a natural surfactant of animal origin extensively used to treat premature babies, and CHF5633, a new synthetic surfactant preparation currently under clinical trials. Both, natural and clinical surfactants spontaneously incorporated corticosteroids up to at least 10% by mass with respect to phospholipid content. The presence of the drugs did not interfere with their ability to efficiently adsorb into air-liquid interfaces and form surface active films able to reach and sustain very low surface tensions (<2 mN/m) under compression-expansion cycling mimicking breathing dynamics. Furthermore, the combination of clinical surfactant with corticosteroids efficiently promoted the active diffusion of the drug to long distances along the air-liquid interface. This effect could not be mimicked by vehiculisation of corticosteroids in liposomes or in micellar emulsions similar to the formulations currently in use to deliver anti-inflammatory corticosteroids through inhalation.Item Structural hallmarks of lung surfactant: Lipid-protein interactions, membrane structure and future challenges(Archives of Biochemistry and Biophysics, 2021) Castillo Sánchez, José Carlos; Cruz Rodríguez, Antonio; Pérez Gil, JesúsLung 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.
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