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Ítem Acceso Abierto Two phosphoenolpyruvate carboxykinases with differing biochemical properties in Chlamydomonas reinhardtii(Wiley, 2023-02) Torresi, Florencia; Rodríguez, Fernanda; Gómez Casati, Diego F.; Martín, MarianaPhosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible reac-tion of decarboxylation and phosphorylation of oxaloacetate (OAA) to gener-ate phosphoenolpyruvate (PEP) and CO2 playing mainly a gluconeogenic rolein green algae. We found two PEPCK isoforms in Chlamydomonas rein-hardtii and we cloned, purified and characterised both enzymes. Chlre-PEPCK1 is more active as decarboxylase than ChlrePEPCK2.ChlrePEPCK1 is hexameric and its activity is affected by citrate, phenylala-nine and malate, while ChlrePEPCK2 is monomeric and it is regulated bycitrate, phenylalanine and glutamine. We postulate that the two PEPCK iso-forms found originate from alternative splicing of the gene or regulated prote-olysis of the enzyme. The presence of these two isoforms would be part of amechanism to finely regulate the biological activity of PEPCKs.Ítem Acceso Abierto Activation of spleen tyrosine kinase (Syk) at fertilization in Rhinella arenarum eggs(University of the Basque Country Press (UBC Press), 2015-07-02) Mouguelar, Valeria Soraya; Coux, GabrielaRecently, we have provided evidence for the involvement of a cytosolic tyrosine–phosphorylatable 70 kDa oocyte protein in Rhinella arenarum (Anura: Bufonidae) fertilization. The aim of the present work was to characterize its phosphorylation, determine the identity of this protein and establish its biological role during the fertilization process. Tyrosine phosphorylation of the 70 kDa protein was not observed in eggs activated with the calcium ionophore A23187. Pretreatment of oocytes with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation of the 70 kDa protein. In order to identify this protein, we examined the presence in amphibian oocytes of non-receptor 70 kDa tyrosine kinase members of the Syk/Zap70 and Tec families by RT-PCR using degenerate primers. We found that R. arenarum oocytes contain the transcripts coding for Syk and Tec kinases. Western blot analysis confirmed the presence of Syk protein in unfertilized oocytes and eggs. Studies using phospho-Syk specific antibodies showed that fertilization rapidly (less than 10 minutes) induces phosphorylation on Syk tyrosine residues (352 and 525/526) that are necessary for the activation of the enzyme. Finally, specific inhibition of Syk with the R406 compound provoked a diminished fertilization score, thereby confirming a functional role of the Syk protein during R. arenarum fertilization. To our knowledge this is the first time that Syk is described as a player in the signaling cascade activated after fertilization.Ítem Acceso Abierto Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity(Wiley, 2014-06-02) Debernardi, Juan Manuel; Mecchia, Martín Alejandro; Vercruyssen, Liesbeth; Smaczniak, Cezary; Kaufmann, Kerstin; Inze, Dirk; Rodríguez, Ramiro Esteban; Palatnik, Javier F.; Wang, Jia-Wei: provide the vectors containing the AS1 and ANT promoter; Weigel, Detlef: provide the vectors containing the AS1 and ANT promoterThe growth-regulating factors (GRFs) are plant-specific transcription factors. They form complexes with GRF-interacting factors (GIFs), a small family of transcriptional co-activators. In Arabidopsis thaliana, seven out of the nine GRFs are controlled by microRNA miR396. Analysis of Arabidopsis plants carrying a GRF3 allele insensitive to miR396 revealed a strong boost in the number of cells in leaves, which was further enhanced synergistically by an additional increase of GIF1 levels. Genetic experiments revealed that GRF3 can still increase cell number in gif1 mutants, albeit to a much lesser extent. Genome-wide transcript profiling indicated that the simultaneous increase of GRF3 and GIF1 levels causes additional effects in gene expression compared to either of the transgenes alone. We observed that GIF1 interacts in vivo with GRF3, as well as with chromatin-remodeling complexes, providing a mechanistic explanation for the synergistic activities of a GRF3–GIF1 complex. Interestingly, we found that, in addition to the leaf size, the GRF system also affects the organ longevity. Genetic and molecular analysis revealed that the functions of GRFs in leaf growth and senescence can be uncoupled, demonstrating that the miR396-GRF-GIF network impinges on different stages of leaf development. Our results integrate the post-transcriptional control of the GRF transcription factors with the progression of leaf development.Ítem Acceso Abierto On the offensive: the role of outer membrane vesicles in the successful dissemination of New Delhi Metallo-β-lactamase (NDM-1)(American Society for Microbiology, 2021-09-28) Martínez, Melina María Belén; Bonomo, Robert A.; Vila, Alejandro J.; Maffía, Paulo César; González, Lisandro Javier; https://orcid.org/0000-0002-4007-8721; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0001-7423-2646; https://orcid.org/0000-0002-0575-1810The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE: Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.Ítem Acceso Abierto TcHRG plays a central role in orchestrating heme uptake in Trypanosoma cruzi epimastigotes(Wiley, 2023-04-05) Tevere, Evelyn; Di Capua, Cecilia Beatriz; Chasen, Nathan Michael; Etheridge, Ronald Drew; Cricco, Julia Alejandra; https://orcid.org/0000-0001-9215-5015; https://orcid.org/0000-0002-8982-3489; https://orcid.org/0000-0002-9144-287X; https://orcid.org/0000-0002-8993-3880Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating TcHRG expression when a free heme source is added to axenic culture. Herein, we explore the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. It was found that the parasite’s endogenous TcHRG (protein and mRNA) responds similarly to bound (hemoglobin) and free (hemin) heme. Additionally, the overexpression of TcHRG leads to an increase in intracellular heme content. The localization of TcHRG is also not affected in parasites supplemented with hemoglobin as the sole heme source. Endocytic null epimastigotes do not show a significant difference in growth profile, intracellular heme content and TcHRG protein accumulation compared to WT when feeding with hemoglobin or hemin as a source of heme. These results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum, T. cruzi epimastigotes controls heme homeostasis by modulating TcHRG expression independently of the source of available heme.Ítem Acceso Abierto Structural requirements for the antifungal activities of natural drimane sesquiterpenes and analogues, supported by conformational and electronic studies(MDPI, 2013-02-05) Derita, Marcos Gabriel; Montenegro, Iván; Garibotto, Francisco; Enriz, Ricardo Daniel; Cuellar Fritis, Mauricio; Zacchino, Susana; https://orcid.org/0000-0002-7112-5500Seventeen drimanes including polygodial (1), isopolygodial (2), drimenol (3) and confertifolin (4) obtained from natural sources and the semi-synthetic derivatives 5–17 obtained from 1–3, were evaluated in vitro for antifungal properties against a unique panel of fungi with standardized procedures by using two end-points, MIC100 and MIC50. A SAR analysis of the whole series, supported by conformational and electronic studies, allowed us to show that the Δ7,8 -double bond would be one of the key structural features related to the antifungal activity. The MEPs obtained for active compounds exhibit a clear negative minimum value (deep red zone) in the vicinity of the Δ7,8 -double bond, which is not present in the inactive ones. Apart of this negative zone, a positive region (deep blue) appears in 1, which is not observed either in its epimer 2 nor in the rest of the active compounds. The LogP of active compounds varies between 2.33 and 3.84, but differences in MICs are not correlated with concomitant variations in LogP values.Ítem Acceso Abierto Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies(Public Library of Science (PLOS), 2016-03-24) Fuentealba, Matías; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana R.; Cabrera, RicardoGlucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.Ítem Acceso Abierto A long-chain flavodoxin protects pseudomonas aeruginosa from oxidative stress and host bacterial clearance(Public Library of Science (PLOS), 2014-02-13) Moyano, Alejandro José; Tobares, Romina Alín; Rizzi, Yanina S.; Krapp, Adriana R.; Mondotte, Juan Alberto; Bocco, José Luis; Saleh, Maria-Carla; Carrillo, Néstor; Smania, Andrea M.Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.Ítem Acceso Abierto Transcriptional and metabolic changes associated to the infection by fusarium verticillioides in maize inbreds with contrasting ear rot resistance(Public Library of Science (PLOS), 2013-04-18) Campos Bermúdez, Valeria Alina; Fauguel, Carolina M.; Tronconi, Marcos A.; Casati, Paula; Presello, Daniel A.; Andreo, Carlos SantiagoFusarium verticillioides causes ear rot and grain mycotoxins in maize (Zea mays L.), which are harmful to human and animal health. Breeding and growing less susceptible plant genotypes is one alternative to reduce these detrimental effects. A better understanding of the resistance mechanisms would facilitate the implementation of strategic molecular agriculture to breeding of resistant germplasm. Our aim was to identify genes and metabolites that may be related to the Fusarium reaction in a resistant (L4637) and a susceptible (L4674) inbred. Gene expression data were obtained from microarray hybridizations in inoculated and non-inoculated kernels from both inbreds. Fungal inoculation did not produce considerable changes in gene expression and metabolites in L4637. Defense-related genes changed in L4674 kernels, responding specifically to the pathogen infection. These results indicate that L4637 resistance may be mainly due to constitutive defense mechanisms preventing fungal infection. These mechanisms seem to be poorly expressed in L4674; and despite the inoculation activate a defense response; this is not enough to prevent the disease progress in this susceptible line. Through this study, a global view of differential genes expressed and metabolites accumulated during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize.Ítem Acceso Abierto Engineering Climate-Change-Resilient Crops: New Tools and Approaches(MDPI, 2021-07-23) Shahinnia, Fahimeh; Carrillo, Néstor; Hajirezaei, Mohammad-RezaÍtem Acceso Abierto CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential(Royal Society of Chemistry, 2021-02-04) Szuster, Jonathan; Zitare, Ulises A.; Castro, María A.; Leguto, Alcides J.; Morgada, Marcos Nicolás; Vila, Alejandro J.; Murgida, Daniel H.Ítem Acceso Abierto Environmental selection pressures related to iron utilization are involved in the loss of the flavodoxin gene from the plant genome(Oxford University Press, 2015-02-16) Pierella Karlusich, Juan J.; Ceccoli, Romina Denis; Graña, Martín; Romero, Héctor; Carrillo, NéstorÍtem Acceso Abierto The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters(Elsevier, 2011) Cupello Peixoto, Mauricio; Fernandes de Souza, Cíntia; Buchensky, Celeste; Rocha Corrêa Soares, Juliana Baptista; Travassos Laranja, Gustavo Augusto; Garcia Pinto Coelho, Marsen; Cricco, Julia Alejandra; Paes, Marcia CristinaÍtem Acceso Abierto Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication(Portland Press, 2017-06-27) Merli, Marcelo Luciano; Cirulli, Brenda Analía; Menendez Bravo, Simón M. ; Cricco, Julia AlejandraÍtem Acceso Abierto Biosynthesis of heme O in intraerythrocytic stages of Plasmodium falciparum and potential inhibitors of this pathway(Springer Nature, 2019-12-17) Simão-Gurge, Raquel M.; Wunderlich, Gerhard; Cricco, Julia Alejandra; Galindo Cubillos, Eliana F.; Domenech-Carbó, Antonio; Cebrián-Torrejón, Gerardo; Almeida, Fernando G.; Cirulli, Brenda Analía; Katzin, Alejandro M.Ítem Acceso Abierto The Trypanosoma cruzi Protein TcHTE Is Critical for Heme Uptake(Public Library of Science PLOS, 2016-01) Merli, Marcelo Luciano; Pagura, Lucas; Hernández, Josefina; Barisón, María Julia; Pral, Elizabeth M. F.; Silber, Ariel M.; Cricco, Julia AlejandraÍtem Acceso Abierto Role of Heme and Heme-Proteins in Trypanosomatid essential metabolic pathways(Hindawi, 2011) Tripodi, Karina E. J.; Menendez Bravo, Simón M.; Cricco, Julia AlejandraÍtem Acceso Abierto The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae(Oxford University Press, 2010-11) Buchensky, Celeste; Almirón, Paula; Suarez Mantilla, Brian; Silber, Ariel M.; Cricco, Julia Alejandra