Caracterización de S-glutationilación de proteínas a nivel de proteoma

Comunicaciones a congreso

S-Nitrosation of E3 Ubiquitin Ligase Complex Components Regulates Hormonal Signalings in Arabidopsis

E3 ubiquitin ligases mediate the last step of the ubiquitination pathway in the ubiquitin-proteasome system (UPS). By targeting transcriptional regulators for their turnover, E3s play a crucial role in every aspect of plant biology. In plants, SKP1/CULLIN1/F-BOX PROTEIN (SCF)-type E3 ubiquitin ligases are essential for the perception and signaling of several key hormones including auxins and jasmonates (JAs). F-box proteins, TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and CORONATINE INSENSITIVE 1 (COI1), bind directly transcriptional repressors AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) and JASMONATE ZIM-DOMAIN (JAZ) in auxin- and JAs-depending manner, respectively, which permits the perception of the hormones and transcriptional activation of signaling pathways. Redox modification of proteins mainly by S-nitrosation of cysteines (Cys) residues via nitric oxide (NO) has emerged as a valued regulatory mechanism in physiological processes requiring its rapid and versatile integration. Previously, we demonstrated that TIR1 and Arabidopsis thaliana SKP1 (ASK1) are targets of S-nitrosation, and these NO-dependent posttranslational modifications enhance protein-protein interactions and positively regulate SCFTIR1 complex assembly and expression of auxin response genes. In this work, we confirmed S-nitrosation of Cys140 in TIR1, which was associated in planta to auxin-dependent developmental and stress-associated responses. In addition, we provide evidence on the modulation of the SCFCOI1 complex by different S-nitrosation events. We demonstrated that S-nitrosation of ASK1 Cys118 enhanced ASK1-COI1 protein-protein interaction. Overexpression of non-nitrosable ask1 mutant protein impaired the activation of JA-responsive genes mediated by SCFCOI1 illustrating the functional relevance of this redox-mediated regulation in planta. In silico analysis positions COI1 as a promising S-nitrosation target, and demonstrated that plants treated with methyl JA (MeJA) or S-nitrosocysteine (NO-Cys, S-nitrosation agent) develop shared responses at a genome-wide level. The regulation of SCF components involved in hormonal perception by S-nitrosation may represent a key strategy to determine the precise time and site-dependent activation of each hormonal signaling pathway and highlights NO as a pivotal molecular player in these scenarios.Fil: Terrile, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Tebez, Nuria Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Colman, Silvana Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Mateos, Julieta Lisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Morato López, Esperanza. CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA (CBMSO) ; UNIVERSIDAD AUTONOMA DE MADRID;Fil: Sánchez López, Nuria. CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA (CBMSO) ; UNIVERSIDAD AUTONOMA DE MADRID;Fil: Izquierdo Álvarez, Alicia. No especifíca;Fil: Marina, Anabel. CENTRO DE BIOLOGIA MOLECULAR SEVERO OCHOA (CBMSO) ; UNIVERSIDAD AUTONOMA DE MADRID;Fil: Calderón Villalobos, Luz Irina A.. Donald Danforth Plant Science Center; Estados UnidosFil: Estelle, Mark. No especifíca;Fil: Martínez Ruiz, Antonio. No especifíca;Fil: Fiol, Diego Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Casalongue, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Iglesias, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Procesamiento y localización subcelular del precursor de la proteina B-amiloide en células de glia

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 4-11-199

The Experimental Proteome of Leishmania infantum Promastigote and Its Usefulness for Improving Gene Annotations

© 2020 by the authors.Leishmania infantum causes visceral leishmaniasis (kala-azar), the most severe form of leishmaniasis, which is lethal if untreated. A few years ago, the re-sequencing and de novo assembling of the L. infantum (JPCM5 strain) genome was accomplished, and now we aimed to describe and characterize the experimental proteome of this species. In this work, we performed a proteomic analysis from axenic cultured promastigotes and carried out a detailed comparison with other Leishmania experimental proteomes published to date. We identified 2352 proteins based on a search of mass spectrometry data against a database built from the six-frame translated genome sequence of L. infantum. We detected many proteins belonging to organelles such as glycosomes, mitochondria, or flagellum, as well as many metabolic enzymes and many putative RNA binding proteins and molecular chaperones. Moreover, we listed some proteins presenting post-translational modifications, such as phosphorylations, acetylations, and methylations. On the other hand, the identification of peptides mapping to genomic regions previously annotated as non-coding allowed for the correction of annotations, leading to the N-terminal extension of protein sequences and the uncovering of eight novel protein-coding genes. The alliance of proteomics, genomics, and transcriptomics has resulted in a powerful combination for improving the annotation of the L. infantum reference genome.This research was funded by grants (to B.A. and J.M.R.) from Proyecto del Ministerio de Economía, Industria y Competitividad SAF2017-86965-R, and by the Network of Tropical Diseases Research RICET (RD16/0027/0008); both grants are co-funded with FEDER funds. A.S. was funded by a postdoctoral contract from the “Programa de Empleo Juvenil” of the Community of Madrid, Spain, within the European Youth Employment Initiative (YEI). A.M. was funded by project PRB3-ISCIII (supported by grant PT17/0019) of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santander.Peer reviewe

Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression

Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy.Instituto de Salud Carlos III (co-funded by Fondo Europeo de Desarrollo Regional (FEDER)) (PIE13/00041); BFU2014-55478-R from Ministerio de Economía y Competitividad (BFU2014-55478-R); and from Fundación Ramón Areces to MY-M. Z.A. was supported by Juan de la Cierv

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.Ministerio Espanol de Economia y Competitividad (MINECO), grant from the Fundacion Ramon Areces and Leonardo Grant from fBBVA to Maria Yanez-M

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.Ministerio Espanol de Economia y Competitividad (MINECO), grant from the Fundacion Ramon Areces and Leonardo Grant from fBBVA to Maria Yanez-M

New palaeoecological approaches to interpret climatic fluctuations in Holocenic sites of the Pampean Region of Argentina

The apparently regular and favourable climate that characterizes the Holocene as an interglacial period shows, however, important climatic instability well documented in the Northern Hemisphere. These fluctuations from colder to warmer or wetter to drier affected both biodiversity and human societies in the last 12,000 years, although the impact in Southern America is still poorly known. We are here investigating the biodiversity of small mammal faunas, more sensitive to climatic changes than large mammals, combining taphonomic and palaeoecological data in the Argentine Pampas to better understand the global nature and effect of these Holocene climatic fluctuations. This paper is pioneering applying in this region palaeoecological methodologies practised in European sites, such as the chorotype classification and biomes overlap analyses. The Pampean Region is an ecotone with a confluence of three climatic regions where any change in climatic conditions should be easily detected. Our results, based on the palaeoecological requirements of small mammals, do not indicate severe changes, and most of the sites show climatic stability except for one of them, in which a possible trend towards present conditions (temperate/humid) can be inferred.Fil: García Morato, Sara. Universidad Complutense de Madrid; España. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Fernández Jalvo, Yolanda. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Montalvo, Claudia Inés. Universidad Nacional de La Pampa; ArgentinaFil: Andrews, Peter. Natural History Museum; Reino UnidoFil: Marin Monfort, María Dolores. Universidad de Valencia; España. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Fagoaga, Ana. Universidad de Valencia; EspañaFil: Domínguez García, Ángel C.. Universidad Complutense de Madrid; EspañaFil: Alberdi, María Teresa. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Bonini, Ricardo Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano. Universidad Nacional del Centro de la Provincia de Buenos Aires. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano; ArgentinaFil: Cerdeño Serrano, Maria Esperanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Provincia de Mendoza. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales. Universidad Nacional de Cuyo. Instituto Argentino de Nivología, Glaciología y Ciencias Ambientales; ArgentinaFil: Denys, Christiane. Sorbonne University; FranciaFil: Domingo, Laura. Universidad Complutense de Madrid; España. University of California at Santa Cruz; Estados UnidosFil: Domingo, Soledad. Universidad Complutense de Madrid; EspañaFil: Gutierrez, Maria Amelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano. Universidad Nacional del Centro de la Provincia de Buenos Aires. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano; ArgentinaFil: López Cantalapiedra, Juan. Universidad de Alcalá; EspañaFil: Pesquero Fernández, María Dolores. Consejo Superior de Investigaciones Científicas. Museo Nacional de Ciencias Naturales; EspañaFil: Prado, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano. Universidad Nacional del Centro de la Provincia de Buenos Aires. Investigaciones Arqueológicas y Paleontológicas del Cuaternario Pampeano; ArgentinaFil: Sevilla, Paloma. Universidad Complutense de Madrid; EspañaFil: Stoetzel, Emmanuelle. Sorbonne University; FranciaFil: Tomassini, Rodrigo Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Geológico del Sur. Universidad Nacional del Sur. Departamento de Geología. Instituto Geológico del Sur; ArgentinaFil: Fernández, Fernando Julián. Universidad de Buenos Aires. Facultad de Ingeniería; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

A multicentric study to evaluate the use of relative retention times in targeted proteomics

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.All laboratories from Spain are members of ProteoRed (Plataforma de Recursos Biomoleculares y Bioinformáticos) and are supported by grant PT13/0001 funded by Instituto de Salud Carlos III (ISCIII) and FEDER.S