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Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores.
Juan Palacios-Ortega; Esperanza Rivera-De-Torre; Sara García-Linares; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte. Oligomerization of Sticholysins from Förster Resonance Energy Transfer. Biochemistry 2021, 60, 314 -323.
AMA StyleJuan Palacios-Ortega, Esperanza Rivera-De-Torre, Sara García-Linares, José G. Gavilanes, Álvaro Martínez-Del-Pozo, J. Peter Slotte. Oligomerization of Sticholysins from Förster Resonance Energy Transfer. Biochemistry. 2021; 60 (4):314-323.
Chicago/Turabian StyleJuan Palacios-Ortega; Esperanza Rivera-De-Torre; Sara García-Linares; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte. 2021. "Oligomerization of Sticholysins from Förster Resonance Energy Transfer." Biochemistry 60, no. 4: 314-323.
Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey–predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.
Esperanza Rivera-De-Torre; Juan Palacios-Ortega; J. Peter Slotte; José G. Gavilanes; Álvaro Martínez-Del-Pozo; Sara García-Linares. Functional and Structural Variation Among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus. International Journal of Molecular Sciences 2020, 21, 8915 .
AMA StyleEsperanza Rivera-De-Torre, Juan Palacios-Ortega, J. Peter Slotte, José G. Gavilanes, Álvaro Martínez-Del-Pozo, Sara García-Linares. Functional and Structural Variation Among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus. International Journal of Molecular Sciences. 2020; 21 (23):8915.
Chicago/Turabian StyleEsperanza Rivera-De-Torre; Juan Palacios-Ortega; J. Peter Slotte; José G. Gavilanes; Álvaro Martínez-Del-Pozo; Sara García-Linares. 2020. "Functional and Structural Variation Among Sticholysins, Pore-Forming Proteins from the Sea Anemone Stichodactyla helianthus." International Journal of Molecular Sciences 21, no. 23: 8915.
All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1–4, a phenomenon that occurs in hypoxia4–8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism. Na+ controls the function of the mitochondrial oxidative phosphorylation system and hypoxic redox signalling through an unexpected interaction with phospholipids.
Pablo Hernansanz; Carmen Choya-Foces; Susana Carregal-Romero; Elena Ramos; Tamara Oliva; Tamara Villa-Piña; Laura Moreno; Alicia Izquierdo-Álvarez; J. Daniel Cabrera-García; Ana Cortés; Ana Victoria Lechuga-Vieco; Pooja Jadiya; Elisa Navarro; Esther Parada; Alejandra Palomino-Antolín; Daniel Tello; Rebeca Acín-Pérez; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Ángel Cogolludo; Iván López-Montero; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. López; John W. Elrod; Jesús Ruíz-Cabello; Anna Bogdanova; José Antonio Enríquez; Antonio Martínez-Ruiz. Na+ controls hypoxic signalling by the mitochondrial respiratory chain. Nature 2020, 586, 287 -291.
AMA StylePablo Hernansanz, Carmen Choya-Foces, Susana Carregal-Romero, Elena Ramos, Tamara Oliva, Tamara Villa-Piña, Laura Moreno, Alicia Izquierdo-Álvarez, J. Daniel Cabrera-García, Ana Cortés, Ana Victoria Lechuga-Vieco, Pooja Jadiya, Elisa Navarro, Esther Parada, Alejandra Palomino-Antolín, Daniel Tello, Rebeca Acín-Pérez, Juan Carlos Rodríguez-Aguilera, Plácido Navas, Ángel Cogolludo, Iván López-Montero, Álvaro Martínez-Del-Pozo, Javier Egea, Manuela G. López, John W. Elrod, Jesús Ruíz-Cabello, Anna Bogdanova, José Antonio Enríquez, Antonio Martínez-Ruiz. Na+ controls hypoxic signalling by the mitochondrial respiratory chain. Nature. 2020; 586 (7828):287-291.
Chicago/Turabian StylePablo Hernansanz; Carmen Choya-Foces; Susana Carregal-Romero; Elena Ramos; Tamara Oliva; Tamara Villa-Piña; Laura Moreno; Alicia Izquierdo-Álvarez; J. Daniel Cabrera-García; Ana Cortés; Ana Victoria Lechuga-Vieco; Pooja Jadiya; Elisa Navarro; Esther Parada; Alejandra Palomino-Antolín; Daniel Tello; Rebeca Acín-Pérez; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Ángel Cogolludo; Iván López-Montero; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. López; John W. Elrod; Jesús Ruíz-Cabello; Anna Bogdanova; José Antonio Enríquez; Antonio Martínez-Ruiz. 2020. "Na+ controls hypoxic signalling by the mitochondrial respiratory chain." Nature 586, no. 7828: 287-291.
Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor.
Angélica Partida-Hanon; Moisés Maestro López; Stefania Vitale; David Turrà; Antonio Di Pietro; Álvaro Martínez-Del-Pozo; Marta Bruix. Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface. Frontiers in Microbiology 2020, 11, 1090 .
AMA StyleAngélica Partida-Hanon, Moisés Maestro López, Stefania Vitale, David Turrà, Antonio Di Pietro, Álvaro Martínez-Del-Pozo, Marta Bruix. Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface. Frontiers in Microbiology. 2020; 11 ():1090.
Chicago/Turabian StyleAngélica Partida-Hanon; Moisés Maestro López; Stefania Vitale; David Turrà; Antonio Di Pietro; Álvaro Martínez-Del-Pozo; Marta Bruix. 2020. "Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface." Frontiers in Microbiology 11, no. : 1090.
Actinoporins are a family of pore-forming toxins produced by sea anemones as part of their venomous cocktail. These proteins remain soluble and stably folded in aqueous solution, but when interacting with sphingomyelin-containing lipid membranes, they become integral oligomeric membrane structures that form a pore permeable to cations, which leads to cell death by osmotic shock. Actinoporins appear as multigenic families within the genome of sea anemones: several genes encoding very similar actinoporins are detected within the same species. The Caribbean Sea anemone Stichodactyla helianthus produces three actinoporins (sticholysins I, II and III; StnI, StnII and StnIII) that differ in their toxic potency. For example, StnII is about four-fold more effective than StnI against sheep erythrocytes in causing hemolysis, and both show synergy. However, StnIII, recently discovered in the S. helianthus transcriptome, has not been characterized so far. Here we describe StnIII's spectroscopic and functional properties and show its potential to interact with the other Stns. StnIII seems to maintain the well-preserved fold of all actinoporins, characterized by a high content of β-sheet, but it is significantly less thermostable. Its functional characterization shows that the critical concentration needed to form active pores is higher than for either StnI or StnII, suggesting differences in behavior when oligomerizing on membrane surfaces. Our results show that StnIII is an interesting and unexpected piece in the puzzle of how this Caribbean Sea anemone species modulates its venomous activity.
Esperanza Rivera-De-Torre; Juan Palacios-Ortega; Jessica E. Garb; J. Peter Slotte; José G. Gavilanes; Álvaro Martínez-Del-Pozo. Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus. Archives of Biochemistry and Biophysics 2020, 689, 108435 .
AMA StyleEsperanza Rivera-De-Torre, Juan Palacios-Ortega, Jessica E. Garb, J. Peter Slotte, José G. Gavilanes, Álvaro Martínez-Del-Pozo. Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus. Archives of Biochemistry and Biophysics. 2020; 689 ():108435.
Chicago/Turabian StyleEsperanza Rivera-De-Torre; Juan Palacios-Ortega; Jessica E. Garb; J. Peter Slotte; José G. Gavilanes; Álvaro Martínez-Del-Pozo. 2020. "Structural and functional characterization of sticholysin III: A newly discovered actinoporin within the venom of the sea anemone Stichodactyla helianthus." Archives of Biochemistry and Biophysics 689, no. : 108435.
Release of aqueous contents from model lipid vesicles has been a standard procedure to evaluate pore formation efficiency by actinoporins, such as sticholysin II (StnII), for the last few decades. However, regardless of the probe of choice, the results reported that StnII action was never able to empty the vesicles completely. This was hard to explain if StnII pores were to be stable and always leaky for the probes used. To address this question, we have used a variety of probes, including rhodamine 6G or Tb3+, to test the permeability of StnII's pores. Our results indicate that calcein was in fact too large to fit through StnII's pores, and that the standard method in the field is actually reporting StnII-induced transient permeation of the membrane rather than the passage of solutes through the stable assembled pores. In order to evaluate the permeability of these structures, we used a dithionite-based assay, which showed that the final pores were in fact open. Thus, our results indicate that the stable actinoporins' pores are open in spite of plateaued classic release curves. Besides the proper pore, the first stages of pore formation would inflict serious damage to living cells as well.
Juan Palacios-Ortega; Esperanza Rivera-De-Torre; José G. Gavilanes; J. Peter Slotte; Álvaro Martínez-Del-Pozo. Evaluation of different approaches used to study membrane permeabilization by actinoporins on model lipid vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes 2020, 1862, 183311 .
AMA StyleJuan Palacios-Ortega, Esperanza Rivera-De-Torre, José G. Gavilanes, J. Peter Slotte, Álvaro Martínez-Del-Pozo. Evaluation of different approaches used to study membrane permeabilization by actinoporins on model lipid vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2020; 1862 (9):183311.
Chicago/Turabian StyleJuan Palacios-Ortega; Esperanza Rivera-De-Torre; José G. Gavilanes; J. Peter Slotte; Álvaro Martínez-Del-Pozo. 2020. "Evaluation of different approaches used to study membrane permeabilization by actinoporins on model lipid vesicles." Biochimica et Biophysica Acta (BBA) - Biomembranes 1862, no. 9: 183311.
Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.
Rodrigo Lázaro Gorines; J. Ruiz-De-La-Herrán; Rocío Navarro; Laura Sanz; Luis Alvarez-Vallina; A. Martínez-Del-Pozo; J. G. Gavilanes; J. Lacadena. A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts. Scientific Reports 2019, 9, 1 -13.
AMA StyleRodrigo Lázaro Gorines, J. Ruiz-De-La-Herrán, Rocío Navarro, Laura Sanz, Luis Alvarez-Vallina, A. Martínez-Del-Pozo, J. G. Gavilanes, J. Lacadena. A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts. Scientific Reports. 2019; 9 (1):1-13.
Chicago/Turabian StyleRodrigo Lázaro Gorines; J. Ruiz-De-La-Herrán; Rocío Navarro; Laura Sanz; Luis Alvarez-Vallina; A. Martínez-Del-Pozo; J. G. Gavilanes; J. Lacadena. 2019. "A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts." Scientific Reports 9, no. 1: 1-13.
Animal venoms are complex mixtures of highly specialized toxic molecules. Cnidarians and arachnids produce pore-forming proteins (PFPs) directed against the plasma membrane of their target cells. Among PFPs from cnidarians, actinoporins stand out for their small size and molecular simplicity. While native actinoporins require only sphingomyelin for membrane binding, engineered chimeras containing a recognition antibody-derived domain fused to an actinoporin isoform can nonetheless serve as highly specific immunotoxins. Examples of such constructs targeted against malignant cells have been already reported. However, PFPs from arachnid venoms are less well-studied from a structural and functional point of view. Spiders from the Latrodectus genus are professional insect hunters that, as part of their toxic arsenal, produce large PFPs known as latrotoxins. Interestingly, some latrotoxins have been identified as potent and highly-specific insecticides. Given the proteinaceous nature of these toxins, their promising future use as efficient bioinsecticides is discussed throughout this Perspective. Protein engineering and large-scale recombinant production are critical steps for the use of these PFPs as tools to control agriculturally important insect pests. In summary, both families of PFPs, from Cnidaria and Arachnida, appear to be molecules with promising biotechnological applications.
Esperanza Rivera-De-Torre; Juan Palacios-Ortega; José Gavilanes; Álvaro Martínez-Del-Pozo; Sara García-Linares. Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools. Toxins 2019, 11, 370 .
AMA StyleEsperanza Rivera-De-Torre, Juan Palacios-Ortega, José Gavilanes, Álvaro Martínez-Del-Pozo, Sara García-Linares. Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools. Toxins. 2019; 11 (6):370.
Chicago/Turabian StyleEsperanza Rivera-De-Torre; Juan Palacios-Ortega; José Gavilanes; Álvaro Martínez-Del-Pozo; Sara García-Linares. 2019. "Pore-Forming Proteins from Cnidarians and Arachnids as Potential Biotechnological Tools." Toxins 11, no. 6: 370.
Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp) mutants to study StnII/bilayer interactions. First, we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done because both Chol and OCer have a 1-hydroxyl, which helps to orient the molecule in the bilayer (although OCer has additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization; OCer failed to activate it. To further examine possible Chol/StnII interactions, we measured Förster resonance energy transfer between Trp in StnII and cholestatrienol, a fluorescent analog of Chol. We could show higher Förster resonance energy transfer efficiency between cholestatrienol and Trps in position 100 and 114 of StnII when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII.
Juan Palacios-Ortega; Sara García-Linares; Esperanza Rivera-De-Torre; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte; Esperanza Rivera-De-Torre. Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction. Biophysical Journal 2019, 116, 2253 -2265.
AMA StyleJuan Palacios-Ortega, Sara García-Linares, Esperanza Rivera-De-Torre, José G. Gavilanes, Álvaro Martínez-Del-Pozo, J. Peter Slotte, Esperanza Rivera-De-Torre. Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction. Biophysical Journal. 2019; 116 (12):2253-2265.
Chicago/Turabian StyleJuan Palacios-Ortega; Sara García-Linares; Esperanza Rivera-De-Torre; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte; Esperanza Rivera-De-Torre. 2019. "Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction." Biophysical Journal 116, no. 12: 2253-2265.
Filamentous fungi are an invaluable source for biocontrol strategies and for production and development of different antifungal polypeptides. Within this context, cysteine-rich antifungal AFP-like peptides stand out among many different antimicrobial compounds given their production easiness, stability, versatility, and efficacy. AFP from Aspergillus giganteus represents the hallmark of this still increasing family of antifungal polypeptides. Close in silico analyses of the Fusarium graminearum genome revealed the presence of an AFP-like peptide, here designated as FgAFP. This new peptide was cloned, produced in the yeast Pichia pastoris, and characterized. The results obtained showed its strong and specific antifungal activity against several well-recognized maize pathogens, but inefficacy against F. oxysporum, which has not been described as a natural biological competitor of other fungal pathogens assayed. All results together suggest that this small peptide is an important factor for the fungal interplays involved in maize infection and reveals unforeseen potential biotechnological applications for FgAFP in maize production and storage.
Belén Patiño; Covadonga Vázquez; James M. Manning; María Isabel G. Roncero; Dolores Córdoba-Cañero; Antonio Di Pietro; Álvaro Martínez-Del-Pozo. Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens. International Journal of Food Microbiology 2018, 283, 45 -51.
AMA StyleBelén Patiño, Covadonga Vázquez, James M. Manning, María Isabel G. Roncero, Dolores Córdoba-Cañero, Antonio Di Pietro, Álvaro Martínez-Del-Pozo. Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens. International Journal of Food Microbiology. 2018; 283 ():45-51.
Chicago/Turabian StyleBelén Patiño; Covadonga Vázquez; James M. Manning; María Isabel G. Roncero; Dolores Córdoba-Cañero; Antonio Di Pietro; Álvaro Martínez-Del-Pozo. 2018. "Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens." International Journal of Food Microbiology 283, no. : 45-51.
All metazoans depend on O2 delivery and consumption by the mitochondrial oxidative phosphorylation (OXPHOS) system to produce energy. A decrease in O2 availability (hypoxia) leads to profound metabolic rewiring. In addition, OXPHOS uses O2 to produce reactive oxygen species (ROS) that can drive cell adaptations through redox signalling, but also trigger cell damage1–4, and both phenomena occur in hypoxia4–8. However, the precise mechanism by which acute hypoxia triggers mitochondrial ROS production is still unknown. Ca2+ is one of the best known examples of an ion acting as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential and collaborating in ion transport10. Here we show that Na+ acts as a second messenger regulating OXPHOS function and ROS production by modulating fluidity of the inner mitochondrial membrane (IMM). We found that a conformational shift in mitochondrial complex I during acute hypoxia11 drives the acidification of the matrix and solubilization of calcium phosphate precipitates. The concomitant increase in matrix free-Ca2+ activates the mitochondrial Na+/Ca2+ exchanger (NCLX), which imports Na+ into the matrix. Na+ interacts with phospholipids reducing IMM fluidity and mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III, generating a redox signal. Inhibition of mitochondrial Na+ import through NCLX is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ import into the mitochondrial matrix controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences in cellular metabolism.
Pablo Hernansanz-Agustín; Carmen Choya-Foces; Susana Carregal-Romero; Elena Ramos; Tamara Oliva; Tamara Villa-Piña; Laura Moreno; Alicia Izquierdo-Álvarez; J. Daniel Cabrera-García; Ana Cortés; Ana Victoria Lechuga-Vieco; Pooja Jadiya; Elisa Navarro; Esther Parada; Alejandra Palomino-Antolín; Daniel Tello; Rebeca Acín-Pérez; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Ángel Cogolludo; Iván López-Montero; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. López; John W. Elrod; Jesús Ruiz-Cabello; Anna Bogdanova; José Antonio Enríquez; Antonio Martínez-Ruiz; Pooya Jadiya; Izaskun Buendia. Mitochondrial Na+ controls oxidative phosphorylation and hypoxic redox signalling. 2018, 385690 .
AMA StylePablo Hernansanz-Agustín, Carmen Choya-Foces, Susana Carregal-Romero, Elena Ramos, Tamara Oliva, Tamara Villa-Piña, Laura Moreno, Alicia Izquierdo-Álvarez, J. Daniel Cabrera-García, Ana Cortés, Ana Victoria Lechuga-Vieco, Pooja Jadiya, Elisa Navarro, Esther Parada, Alejandra Palomino-Antolín, Daniel Tello, Rebeca Acín-Pérez, Juan Carlos Rodríguez-Aguilera, Plácido Navas, Ángel Cogolludo, Iván López-Montero, Álvaro Martínez-Del-Pozo, Javier Egea, Manuela G. López, John W. Elrod, Jesús Ruiz-Cabello, Anna Bogdanova, José Antonio Enríquez, Antonio Martínez-Ruiz, Pooya Jadiya, Izaskun Buendia. Mitochondrial Na+ controls oxidative phosphorylation and hypoxic redox signalling. . 2018; ():385690.
Chicago/Turabian StylePablo Hernansanz-Agustín; Carmen Choya-Foces; Susana Carregal-Romero; Elena Ramos; Tamara Oliva; Tamara Villa-Piña; Laura Moreno; Alicia Izquierdo-Álvarez; J. Daniel Cabrera-García; Ana Cortés; Ana Victoria Lechuga-Vieco; Pooja Jadiya; Elisa Navarro; Esther Parada; Alejandra Palomino-Antolín; Daniel Tello; Rebeca Acín-Pérez; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Ángel Cogolludo; Iván López-Montero; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. López; John W. Elrod; Jesús Ruiz-Cabello; Anna Bogdanova; José Antonio Enríquez; Antonio Martínez-Ruiz; Pooya Jadiya; Izaskun Buendia. 2018. "Mitochondrial Na+ controls oxidative phosphorylation and hypoxic redox signalling." , no. : 385690.
Transcriptomic profiling of venom producing tissues from different animals is an effective approach for discovering new toxins useful in biotechnological and pharmaceutical applications, as well in evolutionary comparative studies of venomous animals. Stichodactyla helianthus is a Caribbean sea anemone which produces actinoporins as part of its toxic venom. This family of pore forming toxins is multigenic and at least two different isoforms, encoded by separate genes, are produced by S. helianthus. These isoforms, sticholysins I and II, share 93% amino acid identity but differ in their pore forming activity and act synergistically. This observation suggests that other actinoporin isoforms, if present in the venomous mixture, could offer an advantageous strategy to modulate whole venom activity. Using high-throughput sequencing we generated a de novo transcriptome of S. helianthus and determined the relative expression of assembled transcripts using RNA-Seq to better characterize components of this species' venom, focusing on actinoporin diversity. Applying this approach, we have discovered at least one new actinoporin variant from S. helianthus in addition to several other putative venom components.
Esperanza Rivera-De-Torre; Álvaro Martínez-Del-Pozo; Jessica E. Garb. Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform. Toxicon 2018, 150, 105 -114.
AMA StyleEsperanza Rivera-De-Torre, Álvaro Martínez-Del-Pozo, Jessica E. Garb. Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform. Toxicon. 2018; 150 ():105-114.
Chicago/Turabian StyleEsperanza Rivera-De-Torre; Álvaro Martínez-Del-Pozo; Jessica E. Garb. 2018. "Stichodactyla helianthus' de novo transcriptome assembly: Discovery of a new actinoporin isoform." Toxicon 150, no. : 105-114.
Pablo Hernansanz-Agustín; Elena Ramos; Tamara Villa-Piña; Elisa Navarro; Esther Parada; Laura Moreno; Alicia Izquierdo-Álvarez; Tamara Oliva; J. Daniel Cabrera-García; Ana Cortés; Daniel Tello; Rebeca Acín-Pérez; Izaskun Buendía; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Angel Cogolludo; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. Lopez; Anna Bogdanova; José Antonio Enriquez; Antonio Martínez-Ruiz. Mitochondrial Na + import controls oxidative phosphorylation and hypoxic redox signaling. Free Radical Biology and Medicine 2018, 120, S29 .
AMA StylePablo Hernansanz-Agustín, Elena Ramos, Tamara Villa-Piña, Elisa Navarro, Esther Parada, Laura Moreno, Alicia Izquierdo-Álvarez, Tamara Oliva, J. Daniel Cabrera-García, Ana Cortés, Daniel Tello, Rebeca Acín-Pérez, Izaskun Buendía, Juan Carlos Rodríguez-Aguilera, Plácido Navas, Angel Cogolludo, Álvaro Martínez-Del-Pozo, Javier Egea, Manuela G. Lopez, Anna Bogdanova, José Antonio Enriquez, Antonio Martínez-Ruiz. Mitochondrial Na + import controls oxidative phosphorylation and hypoxic redox signaling. Free Radical Biology and Medicine. 2018; 120 ():S29.
Chicago/Turabian StylePablo Hernansanz-Agustín; Elena Ramos; Tamara Villa-Piña; Elisa Navarro; Esther Parada; Laura Moreno; Alicia Izquierdo-Álvarez; Tamara Oliva; J. Daniel Cabrera-García; Ana Cortés; Daniel Tello; Rebeca Acín-Pérez; Izaskun Buendía; Juan Carlos Rodríguez-Aguilera; Plácido Navas; Angel Cogolludo; Álvaro Martínez-Del-Pozo; Javier Egea; Manuela G. Lopez; Anna Bogdanova; José Antonio Enriquez; Antonio Martínez-Ruiz. 2018. "Mitochondrial Na + import controls oxidative phosphorylation and hypoxic redox signaling." Free Radical Biology and Medicine 120, no. : S29.
Fungal ribotoxins constitute a family of extracellular ribonucleases with exquisite specificity against rRNA (ribonucleic acid). They induce apoptotic death of cells after inhibiting protein translation. Ribosomes become functionally incompetent because ribotoxins cleave one single phosphodiester bond, located at a unique and universally conserved loop, needed for elongation factors function. As secreted proteins, ribotoxins need to cross the membrane of their target cells in order to exert their catalytic activity, and they do it without receptor mediation. Using lipid model systems, it has been shown that they are able to enter cells with membranes enriched in acidic phospholipids. Both membrane-interacting and ribosomal-recognition activities are characterised by distinct structural features. Even though the natural function of ribotoxins is not known yet, their production by entomopathogenic fungi has suggested their insecticidal role. After decades of detailed study, the biotechnological potential of ribotoxins in pest control and as antitumour agents is becoming evident.
Lucía García-Ortega; Juan Palacios-Ortega; Alvaro Martinez-Del-Pozo. Fungal Ribotoxins. eLS 2018, 1 -9.
AMA StyleLucía García-Ortega, Juan Palacios-Ortega, Alvaro Martinez-Del-Pozo. Fungal Ribotoxins. eLS. 2018; ():1-9.
Chicago/Turabian StyleLucía García-Ortega; Juan Palacios-Ortega; Alvaro Martinez-Del-Pozo. 2018. "Fungal Ribotoxins." eLS , no. : 1-9.
Sticholysins I and II (StnI and StnII), α-pore forming toxins from the sea anemone Stichodactyla helianthus, are water-soluble toxic proteins which upon interaction with lipid membranes of specific composition bind to the bilayer, extend and insert their N-terminal α-helix, and become oligomeric integral membrane structures. The result is a pore that leads to cell death by osmotic shock. StnI and StnII show 93% of sequence identity, but also different membrane pore-forming activities. The hydrophobicity profile along the first 18 residues revealed differences which were canceled by substituting StnI amino acids 2 and 9. Accordingly, the StnID9A mutant, and the corresponding StnIE2AD9A variant, showed enhanced hemolytic activity. They also revealed a key role for an exposed salt bridge between Asp9 and Lys68. This interaction is not possible in StnII but appears conserved in the other two well-characterized actinoporins, equinatoxin II and fragaceatoxin C. The StnII mutant A8D showed that this single replacement was enough to transform StnII into a version with impaired pore-forming activity. Overall, the results show the key importance of this salt bridge linking the N-terminal stretch to the β-sandwich core. A conclusion of general application for the understanding of salt bridges role in protein design, folding and stability.
Esperanza Rivera-De-Torre; Juan Palacios-Ortega; Sara García-Linares; José G. Gavilanes; Álvaro Martínez-Del-Pozo. One single salt bridge explains the different cytolytic activities shown by actinoporins sticholysin I and II from the venom of Stichodactyla helianthus. Archives of Biochemistry and Biophysics 2017, 636, 79 -89.
AMA StyleEsperanza Rivera-De-Torre, Juan Palacios-Ortega, Sara García-Linares, José G. Gavilanes, Álvaro Martínez-Del-Pozo. One single salt bridge explains the different cytolytic activities shown by actinoporins sticholysin I and II from the venom of Stichodactyla helianthus. Archives of Biochemistry and Biophysics. 2017; 636 ():79-89.
Chicago/Turabian StyleEsperanza Rivera-De-Torre; Juan Palacios-Ortega; Sara García-Linares; José G. Gavilanes; Álvaro Martínez-Del-Pozo. 2017. "One single salt bridge explains the different cytolytic activities shown by actinoporins sticholysin I and II from the venom of Stichodactyla helianthus." Archives of Biochemistry and Biophysics 636, no. : 79-89.
In this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using di-monounsaturated phosphatidylcholine (PC) analogs (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUV) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn-LUV interaction, we observed that the bilayer affinity constant (Ka) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins' N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC.
Juan Palacios-Ortega; Sara García-Linares; Esperanza Rivera-De-Torre; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte. Differential Effect of Bilayer Thickness on Sticholysin Activity. Langmuir 2017, 33, 11018 -11027.
AMA StyleJuan Palacios-Ortega, Sara García-Linares, Esperanza Rivera-De-Torre, José G. Gavilanes, Álvaro Martínez-Del-Pozo, J. Peter Slotte. Differential Effect of Bilayer Thickness on Sticholysin Activity. Langmuir. 2017; 33 (41):11018-11027.
Chicago/Turabian StyleJuan Palacios-Ortega; Sara García-Linares; Esperanza Rivera-De-Torre; José G. Gavilanes; Álvaro Martínez-Del-Pozo; J. Peter Slotte. 2017. "Differential Effect of Bilayer Thickness on Sticholysin Activity." Langmuir 33, no. 41: 11018-11027.
Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: Hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins
Moisés Maestro-López; Miriam Olombrada; Lucia Garcia-Ortega; Daniel Serrano-González; Javier Lacadena; Mercedes Oñaderra; José G. Gavilanes; Álvaro Martínez-Del-Pozo. Minimized natural versions of fungal ribotoxins show improved active site plasticity. Archives of Biochemistry and Biophysics 2017, 619, 45 -53.
AMA StyleMoisés Maestro-López, Miriam Olombrada, Lucia Garcia-Ortega, Daniel Serrano-González, Javier Lacadena, Mercedes Oñaderra, José G. Gavilanes, Álvaro Martínez-Del-Pozo. Minimized natural versions of fungal ribotoxins show improved active site plasticity. Archives of Biochemistry and Biophysics. 2017; 619 ():45-53.
Chicago/Turabian StyleMoisés Maestro-López; Miriam Olombrada; Lucia Garcia-Ortega; Daniel Serrano-González; Javier Lacadena; Mercedes Oñaderra; José G. Gavilanes; Álvaro Martínez-Del-Pozo. 2017. "Minimized natural versions of fungal ribotoxins show improved active site plasticity." Archives of Biochemistry and Biophysics 619, no. : 45-53.
During sexual development ascomycete fungi produce two types of peptide pheromones termed a and α. The α pheromone from the budding yeast Saccharomyces cerevisiae, a 13-residue peptide that elicits cell cycle arrest and chemotropic growth, has served as paradigm for the interaction of small peptides with their cognate G protein-coupled receptors. However, no structural information is currently available for α pheromones from filamentous ascomycetes, which are significantly shorter and share almost no sequence similarity with the S. cerevisiae homolog. High resolution structure of synthetic α-pheromone from the plant pathogenic ascomycete Fusarium oxysporum revealed the presence of a central β-turn resembling that of its yeast counterpart. Disruption of the-fold by d-alanine substitution of the conserved central Gly6-Gln7 residues or by random sequence scrambling demonstrated a crucial role for this structural determinant in chemoattractant activity. Unexpectedly, the growth inhibitory effect of F. oxysporum α-pheromone was independent of the cognate G protein-coupled receptors Ste2 and of the central β-turn but instead required two conserved Trp1-Cys2 residues at the N terminus. These results indicate that, despite their reduced size, fungal α-pheromones contain discrete functional regions with a defined secondary structure that regulate diverse biological processes such as polarity reorientation and cell division.
Stefania Vitale; Angelica Partida-Hanon; Soraya Serrano; Alvaro Martinez-Del-Pozo; Antonio Di Pietro; David Turrà; Marta Bruix. Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum. Journal of Biological Chemistry 2017, 292, 3591 -3602.
AMA StyleStefania Vitale, Angelica Partida-Hanon, Soraya Serrano, Alvaro Martinez-Del-Pozo, Antonio Di Pietro, David Turrà, Marta Bruix. Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum. Journal of Biological Chemistry. 2017; 292 (9):3591-3602.
Chicago/Turabian StyleStefania Vitale; Angelica Partida-Hanon; Soraya Serrano; Alvaro Martinez-Del-Pozo; Antonio Di Pietro; David Turrà; Marta Bruix. 2017. "Structure-Activity Relationship of α Mating Pheromone from the Fungal Pathogen Fusarium oxysporum." Journal of Biological Chemistry 292, no. 9: 3591-3602.
Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.
Miriam Olombrada; Rodrigo Lázaro Gorines; Juan C. López-Rodríguez; Álvaro Martínez-Del-Pozo; Mercedes Oñaderra; Moisés Maestro-López; Javier Lacadena; José G. Gavilanes; Lucía García-Ortega. Fungal Ribotoxins: A Review of Potential Biotechnological Applications. Toxins 2017, 9, 71 .
AMA StyleMiriam Olombrada, Rodrigo Lázaro Gorines, Juan C. López-Rodríguez, Álvaro Martínez-Del-Pozo, Mercedes Oñaderra, Moisés Maestro-López, Javier Lacadena, José G. Gavilanes, Lucía García-Ortega. Fungal Ribotoxins: A Review of Potential Biotechnological Applications. Toxins. 2017; 9 (2):71.
Chicago/Turabian StyleMiriam Olombrada; Rodrigo Lázaro Gorines; Juan C. López-Rodríguez; Álvaro Martínez-Del-Pozo; Mercedes Oñaderra; Moisés Maestro-López; Javier Lacadena; José G. Gavilanes; Lucía García-Ortega. 2017. "Fungal Ribotoxins: A Review of Potential Biotechnological Applications." Toxins 9, no. 2: 71.
Sea anemone actinoporins constitute a protein family of multigene pore-forming toxins (PFT). Equinatoxin II (EqtII), fragaceatoxin C (FraC), and sticholysins I and II (StnI and StnII, respectively), produced by three different sea anemone species, are the only actinoporins whose molecular structures have been studied in depth. These four proteins show high sequence identities and practically coincident three-dimensional structures. However, their pore-forming activity can be quite different depending on the model lipid system employed, a feature that has not been systematically studied before. Therefore, the aim of this work was to evaluate and compare the influence of several distinct membrane conditions on their particular pore-forming behavior. Using a complex model membrane system, such as sheep erythrocytes, StnII showed hemolytic activity much higher than those of the other three actinoporins studied. In lipid model systems, pore-forming ability when assayed against 4:1 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin (SM) vesicles, with the membrane binding being the rate-limiting step, decreased in the following order: StnI > StnII > EqtII > FraC. When using 1:1:1 DOPC/SM/cholesterol LUVs, the presence of Chol not only enhanced membrane binding affinities by ∼2 orders of magnitude but also revealed how StnII was much faster than the other three actinoporins in producing calcein release. This ability agrees with the proposal that explains this behavior in terms of their high sequence variability along their first 30 N-terminal residues. The influence of interfacial hydrogen bonding in SM- or dihydro-SM-containing bilayers was also shown to be a generalized feature of the four actinoporins studied. It is finally hypothesized that this observed variable ability could be explained as a consequence of their distinct specificities and/or membrane binding affinities. Eventually, this behavior can be modulated by the nature of their natural target membranes or the interaction with not yet characterized isotoxin forms from the same sea anemone species.
Sara García-Linares; Esperanza Rivera-De-Torre; Koldo Morante; Kouhei Tsumoto; Jose M. M. Caaveiro; José G. Gavilanes; J. Peter Slotte; Álvaro Martínez-Del-Pozo. Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins. Biochemistry 2016, 55, 6630 -6641.
AMA StyleSara García-Linares, Esperanza Rivera-De-Torre, Koldo Morante, Kouhei Tsumoto, Jose M. M. Caaveiro, José G. Gavilanes, J. Peter Slotte, Álvaro Martínez-Del-Pozo. Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins. Biochemistry. 2016; 55 (48):6630-6641.
Chicago/Turabian StyleSara García-Linares; Esperanza Rivera-De-Torre; Koldo Morante; Kouhei Tsumoto; Jose M. M. Caaveiro; José G. Gavilanes; J. Peter Slotte; Álvaro Martínez-Del-Pozo. 2016. "Differential Effect of Membrane Composition on the Pore-Forming Ability of Four Different Sea Anemone Actinoporins." Biochemistry 55, no. 48: 6630-6641.