18 results on '"Roberto A. Paggi"'
Search Results
2. In Vivo Protein Cross-Linking and Coimmunoprecipitation in Haloferax volcanii
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Roberto A, Paggi, Rosana E, De Castro, and Micaela, Cerletti
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Formaldehyde ,Sepharose ,Immunoprecipitation ,Proteins ,Propionates ,Peptides ,Haloferax volcanii ,Peptide Hydrolases - Abstract
Coimmunoprecipitation is a powerful and commonly used method to identify protein-protein interactions in a physiological context. Here, we report a coimmunoprecipitation protocol that was adapted and optimized for the haloarchaeon Haloferax volcanii to identify interacting partners to the LonB protease. This protocol includes the in vivo cross-linking of H. volcanii proteins using two different crosslinker agents, dithiobis(succinimidyl propionate) and formaldehyde, followed by immunoprecipitation with anti-LonB antibody conjugated to Protein A - Sepharose beads. Tryptic on-bead protein digestion was performed combined with Mass Spectrometry analysis of peptides for the identification and quantification of LonB ligands.
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- 2022
3. Proteome Turnover Analysis in Haloferax volcanii by a Heavy Isotope Multilabeling Approach
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Roberto A. Paggi, Stefan P. Albaum, Ansgar Poetsch, and Micaela Cerletti
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Proteomics ,Isotopes ,Proteome ,Isotope Labeling ,Haloferax volcanii - Abstract
The cellular protein repertoire is highly dynamic and responsive to internal or external stimuli. Its changes are largely the consequence of the combination of protein synthesis and degradation, referred collectively as protein turnover. Different proteomics techniques have been developed to determine the whole proteome turnover of a cell, but very few have been applied to archaea. In this chapter we describe a heavy isotope multilabeling method that allowed the successful analysis of relative protein synthesis and degradation rates on the proteome scale of the halophilic archaeon Haloferax volcanii. This method combines 15N and 13C isotope metabolic labeling with high-resolution mass spectrometry and data analysis tools (QuPE web-based platform) and could be applied to different archaea. © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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- 2022
4. The C-terminal region of phytoene synthase is a key element to control carotenoid biosynthesis in the haloarchaeon Haloferax volcanii
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Micaela Cerletti, Agustín Rabino, Roberto A. Paggi, Celeste Ferrari, Ansgar Poetsch, Harri Savilahti, Saija Kiljunen, and Rosana E. De Castro
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Glycogen Synthase ,Cell Biology ,Molecular Biology ,Biochemistry ,Haloferax volcanii ,Carotenoids - Abstract
Phytoene synthase (PSY) converts two molecules of geranyl-geranyl diphosphate to phytoene, the key regulatory step in carotenogenesis. However, post-translational mechanisms that control PSY expression are scarcely understood. Carotenoid biosynthesis (mainly bacterioruberin) is a distinctive feature of haloarchaea thriving in hypersaline environments. Carotenogenesis is negatively regulated by the AAA+ LonB protease in the haloarchaeon Haloferax volcanii as it controls PSY degradation. We investigated the relevance of the C-terminal portion of HvPSY as a regulatory element for carotenoid biosynthesis. H. volcanii mutants were constructed to express full-length HvPSY protein (strain HVPSYwt) and truncated HvPSY lacking 10 (HVPSY10), 20 (HVPSY20) or 34 amino acids (HVPSY34) at the C-terminus. Cells of HVPSY20 and HVPSY34 showed hyperpigmentation (bacterioruberin content 3-fold higher than HVPSYwt) which correlated with increased PSY protein abundance (2-fold in HVPSY34) while they contained less psy transcript level compared with HVPSYwt. In vivo degradation assays showed that HvPSY34 was more stable than HvPSYwt. Collectively, these results show that the C-terminal region of HvPSY contains a ‘recognition determinant’ for proteolysis in H. volcanii. Preliminary evidence suggests that LonB is involved in the recognition mechanism. This study provides the first identification of a regulatory sequence in an archaeal PSY for the post-translational control of carotenogenesis.
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- 2022
5. The Archaeal Proteome Project advances knowledge about archaeal cell biology through comprehensive proteomics
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Anita Marchfelder, Michael Hippler, Stefan Schulze, Micaela Cerletti, Friedhelm Pfeiffer, Sébastien Ferreira-Cerca, Christian Fufezan, Christof Lenz, Zivojin Jevtic, Rosana Esther de Castro, Henning Urlaub, Roberto A. Paggi, Julie A. Maupin-Furlow, Robert Knüppel, Zachary Adams, Ansgar Poetsch, Mechthild Pohlschroder, Maria Ines Gimenez, and Georgio Legerme
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0301 basic medicine ,Proteomics ,Glycosylation ,Proteome ,Systems biology ,Science ,Archaeal Proteins ,030106 microbiology ,General Physics and Astronomy ,Datasets as Topic ,Proteome informatics ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,Article ,purl.org/becyt/ford/1 [https] ,03 medical and health sciences ,Protein sequencing ,ARCHAEA ,Amino Acid Sequence ,lcsh:Science ,purl.org/becyt/ford/1.6 [https] ,Protein maturation ,Haloferax volcanii ,Multidisciplinary ,biology ,PROTEOME PROJECT ,General Chemistry ,biology.organism_classification ,Cell biology ,030104 developmental biology ,lcsh:Q ,ARCHAEAL BIOLOGY ,Archaeal biology ,Archaea - Abstract
While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as N-terminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics., While archaeal proteomics advanced rapidly, a comprehensive proteome database for archaea is lacking. Therefore, the authors here launch the Archaeal Proteome Project, a community-effort providing insights into archaeal cell biology via the combined reanalysis of Haloferax volcanii proteomics data.
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- 2020
6. The LonB protease modulates the degradation of CetZ1 involved in rod-shape determination in Haloferax volcanii
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Roberto A. Paggi, María Celeste Ferrari, Micaela Cerletti, Ansgar Poetsch, Christian Troetschel, and Rosana Esther de Castro
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Protease ,biology ,Archaeal Proteins ,medicine.medical_treatment ,Haloferax volcanii ,Biophysics ,biology.organism_classification ,Biochemistry ,Molecular biology ,medicine ,Gene Expression Regulation, Archaeal ,Cell shape ,Peptide Hydrolases - Abstract
Fil: Ferrari, Maria Celeste. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Mar del Plata. Instituto de Investigaciones Biologicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biologicas; Argentina
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- 2020
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7. Proteomic Study of the Exponential-Stationary Growth Phase Transition in the Haloarchaea Natrialba magadii and Haloferax volcanii
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Micaela Cerletti, Maria Ines Gimenez, Ansgar Poetsch, Christian Troetschel, Rosana Ester De Castro, Celeste D’ Alessandro, and Roberto A. Paggi
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0301 basic medicine ,Cell division ,Proteome ,Archaeal Proteins ,Bacterial growth ,Proteomics ,Biochemistry ,Mass Spectrometry ,purl.org/becyt/ford/1 [https] ,Ciencias Biológicas ,03 medical and health sciences ,Biología Celular, Microbiología ,Natrialba maagadii ,purl.org/becyt/ford/1.6 [https] ,Molecular Biology ,Haloferax volcanii ,Halobacteriaceae ,biology ,Chemistry ,Haloarchaea ,biology.organism_classification ,030104 developmental biology ,Comparative proteomics ,Bacteria ,Stationary phase ,CIENCIAS NATURALES Y EXACTAS ,Archaea - Abstract
The dynamic changes that take place along the phases of microbial growth (lag, exponential, stationary, and death) have been widely studied in bacteria at the molecular and cellular levels, but little is known for archaea. In this study, a high-throughput approach was used to analyze and compare the proteomes of two haloarchaea during exponential and stationary growth: the neutrophilic Haloferax volcanii and the alkaliphilic Natrialba magadii. Almost 2000 proteins were identified in each species (≈50% of the predicted proteome). Among them, 532 and 432 were found to be differential between growth phases in H. volcanii and N. magadii, respectively. Changes upon entrance into stationary phase included an overall increase in proteins involved in the transport of small molecules and ions, stress response, and fatty acid catabolism. Proteins related to genetic processes and cell division showed a notorious decrease in amount. The data reported in this study not only contributes to our understanding of the exponential–stationary growth phase transition in extremophilic archaea but also provides the first comprehensive analysis of the proteome composition of N. magadii. The MS proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier JPST000395. Fil: Cerletti, Micaela. 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; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Gimenez, Maria Ines. Universidad Nacional de Mar del Plata; Argentina. 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; Argentina Fil: Tröetschel, Christian. Ruhr Universität Bochum; Alemania Fil: D'alessandro, Celeste Paola. Universidade de Sao Paulo; Brasil Fil: Poetsch, Ansgar. University Of Plymouth; . Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. 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; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina. 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; Argentina
- Published
- 2018
8. LonB Protease Is a Novel Regulator of Carotenogenesis Controlling Degradation of Phytoene Synthase in Haloferax volcanii
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Christian Troetschel, Roberto A. Paggi, Micaela Cerletti, Stefan P. Albaum, Rosana Esther de Castro, Carina Ramallo Guevara, María Celeste Ferrari, and Ansgar Poetsch
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0301 basic medicine ,Protease La ,Proteome ,membrane protease ,medicine.medical_treatment ,Mutant ,Biochemistry ,Substrate Specificity ,Tandem Mass Spectrometry ,Proteome turnover ,Membrane protease ,Haloferax volcanii ,chemistry.chemical_classification ,Phytoene synthase ,biology ,medicine.diagnostic_test ,Chemistry ,phytoene synthase ,Geranylgeranyl-Diphosphate Geranylgeranyltransferase ,Isotope Labeling ,proteome turnover ,volcanii ,CIENCIAS NATURALES Y EXACTAS ,Proteolysis ,Archaeal Proteins ,LonB substrates ,Ciencias Biológicas ,03 medical and health sciences ,Biología Celular, Microbiología ,medicine ,Haloferax ,Protease ,Molecular Sequence Annotation ,General Chemistry ,biology.organism_classification ,Archaea ,Carotenoids ,030104 developmental biology ,Enzyme ,Gene Ontology ,Cytoplasm ,Protein Biosynthesis ,Mutation ,biology.protein ,bacteria ,Gene Expression Regulation, Archaeal ,Lonb substrates ,Chromatography, Liquid - Abstract
The membrane protease LonB is an essential protein in the archaeon Haloferax volcanii and globally impacts its physiology. However, natural substrates of the archaeal Lon protease have not been identified. The whole proteome turnover was examined in a H. volcanii LonB mutant under reduced and physiological protease levels. LC-MS/MS combined with stable isotope labeling was applied for the identification/quantitation of membrane and cytoplasm proteins. Differential synthesis and degradation rates were evidenced for 414 proteins in response to Lon expression. A total of 58 proteins involved in diverse cellular processes showed a degradation pattern (none/very little degradation in the absence of Lon and increased degradation in the presence of Lon) consistent with a LonB substrate, which was further substantiated for several of these candidates by pull-down assays. The most notable was phytoene synthase (PSY), the rate-limiting enzyme in carotenoid biosynthesis. The rapid degradation of PSY upon LonB induction in addition to the remarkable stabilization of this protein and hyperpigmentation phenotype in the Lon mutant strongly suggest that PSY is a LonB substrate. This work identifies for the first time candidate targets of the archaeal Lon protease and establishes proteolysis by Lon as a novel post-translational regulatory mechanism of carotenogenesis. Fil: Cerletti, Micaela. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina Fil: Troetschel, Christian. Ruhr Universität Bochum; Alemania Fil: Ferrari, María Celeste. Universidad Nacional de Mar del Plata; Argentina Fil: Guevara, Carina Ramallo. Ruhr Universität Bochum; Alemania Fil: Albaum, Stefan. Universitat Bielefeld; Alemania Fil: Poetsch, Ansgar. Plant Biochemistry, Ruhr University Bochum; Alemania Fil: de Castro, Rosana Esther. Universidad Nacional de Mar del Plata; Argentina
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- 2018
9. Haloferax volcanii Proteome Response to Deletion of a Rhomboid Protease Gene
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Ansgar Poetsch, Maria Ines Gimenez, Roberto A. Paggi, Rosana Esther de Castro, Christian Trötschel, Micaela Cerletti, and Mariana Inés Costa
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0301 basic medicine ,Proteases ,Spectrometry, Mass, Electrospray Ionization ,Glycosylation ,Protease substrate ,Proteome ,medicine.medical_treatment ,Archaeal Proteins ,Mutant ,Biochemistry ,Substrate Specificity ,Ciencias Biológicas ,03 medical and health sciences ,chemistry.chemical_compound ,Biología Celular, Microbiología ,Endopeptidases ,Metalloproteins ,medicine ,Shotgun proteomics ,Cell Adhesion ,Integral membrane protein ,Rhomboid protease ,Haloferax volcanii ,Protease ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Halorerax volcanii ,Membrane Proteins ,Molecular Sequence Annotation ,General Chemistry ,biology.organism_classification ,Archea ,Cell biology ,DNA-Binding Proteins ,030104 developmental biology ,Gene Ontology ,Gene Expression Regulation, Archaeal ,Protein Processing, Post-Translational ,CIENCIAS NATURALES Y EXACTAS ,Gene Deletion ,Bacterial Outer Membrane Proteins - Abstract
Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO-1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism. Fil: Costa, Mariana Inés. Universidad Nacional de Mar del Plata; Argentina Fil: Cerletti, Micaela. Universidad Nacional de Mar del Plata; Argentina Fil: Paggi, Roberto Alejandro. Universidad Nacional de Mar del Plata; Argentina Fil: Trötschel, Christian. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. Universidad Nacional de Mar del Plata; Argentina. 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; Argentina Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: Gimenez, Maria Ines. Universidad Nacional de Mar del Plata; Argentina
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- 2018
10. Data in support of global role of the membrane protease LonB in Archaea : Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii
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Micaela Cerletti, Carina Ramallo Guevara, Roberto A. Paggi, Rosana Esther de Castro, and Ansgar Poetsch
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Phytoene synthase ,Otras Ciencias Biológicas ,medicine.medical_treatment ,Mutant ,Cell ,LonB protease ,Shotgun ,Biology ,lcsh:Computer applications to medicine. Medical informatics ,Microbiology ,purl.org/becyt/ford/1 [https] ,Ciencias Biológicas ,medicine ,purl.org/becyt/ford/1.6 [https] ,lcsh:Science (General) ,Haloferax volcanii ,Data Article ,Multidisciplinary ,Protease ,biology.organism_classification ,Quantitative Shotgun Proteomics ,Archaea ,Protein substrates ,Cytosol ,medicine.anatomical_structure ,Biochemistry ,Proteome ,lcsh:R858-859.7 ,CIENCIAS NATURALES Y EXACTAS ,lcsh:Q1-390 - Abstract
This data article provides information in support of the research article "Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii" [1]. The proteome composition of a wt and a LonB protease mutant strain (suboptimal expression) in the archaeon Haloferax volcanii was assessed by a quantitative shotgun proteomic approach. Membrane and cytosol fractions of H. volcanii strains were examined at two different growth stages (exponential and stationary phase). Data is supplied in the present article. This study represents the first proteome examination of a Lon-deficient cell of the Archaea Domain. Fil: Cerletti, Micaela. 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; Argentina Fil: Paggi, Roberto Alejandro. 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; Argentina Fil: Ramallo Guevara, Carina. Ruhr Universität Bochum; Alemania Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. 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; Argentina
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- 2015
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11. A simple technique to improve the resolution of membrane acidic proteins of the haloarchaeonHaloferax volcaniiby 2D electrophoresis
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Roberto A. Paggi, María Inés Giménez, Andreina Cesari, and Rosana Esther de Castro
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chemistry.chemical_classification ,Two-dimensional gel electrophoresis ,biology ,Clinical Biochemistry ,Haloferax volcanii ,biology.organism_classification ,Biochemistry ,Thermosome ,Analytical Chemistry ,Membrane ,Membrane protein ,chemistry ,Cell envelope ,Glycoprotein ,Integral membrane protein - Abstract
Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.
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- 2014
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12. The LonB protease controls membrane lipids composition and is essential for viability in the extremophilic haloarchaeonHaloferax volcanii
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María Julia Martinez, Maria Ines Gimenez, Rosana Esther de Castro, Micaela Cerletti, Roberto A. Paggi, and Diego E. Sastre
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Protease ,biology ,Biochemistry ,medicine.medical_treatment ,Membrane lipids ,Haloferax volcanii ,Haloarchaea ,medicine ,biology.organism_classification ,Microbiology ,Ecology, Evolution, Behavior and Systematics - Abstract
Fil: Cerletti, Micaela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Mar del Plata. Instituto de Investigaciones Biologicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina
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- 2014
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13. Global role of the membrane protease LonB in Archaea: Potential protease targets revealed by quantitative proteome analysis of a lonB mutant in Haloferax volcanii
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Rosana Esther de Castro, Roberto A. Paggi, Micaela Cerletti, Ansgar Poetsch, and Carina Ramallo Guevara
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Proteomics ,Proteases ,Cytoplasm ,Spectrometry, Mass, Electrospray Ionization ,Proteome ,Otras Ciencias Biológicas ,medicine.medical_treatment ,Archaeal Proteins ,Mutant ,Biophysics ,Biochemistry ,Ciencias Biológicas ,PHYTOENE SYNTHASE ,medicine ,ARCHAEA ,Amino Acids ,Shotgun proteomics ,Haloferax volcanii ,Protease ,biology ,Cell Membrane ,Membrane Proteins ,HALOFERAX VOLCANII ,biology.organism_classification ,Lipid Metabolism ,Carotenoids ,Membrane protein ,PROTEIN SUBSTRATES ,Mutation ,QUANTITATIVE SHOTGUN PROTEOMICS ,LONB PROTEASE ,bacteria ,Electrophoresis, Polyacrylamide Gel ,CIENCIAS NATURALES Y EXACTAS ,Peptide Hydrolases - Abstract
The membrane-associated LonB protease is essential for viability in Haloferax volcanii, however, the cellular processes affected by this protease in archaea are unknown. In this study, the impact of a lon conditional mutation (down-regulation) on H. volcanii physiology was examined by comparing proteomes of parental and mutant cells using shotgun proteomics. A total of 1778 proteins were identified (44% of H. volcanii predicted proteome) and 142 changed significantly in amount (≥. 2 fold). Of these, 66 were augmented in response to Lon deficiency suggesting they could be Lon substrates. The "Lon subproteome" included soluble and predicted membrane proteins expected to participate in diverse cellular processes. The dramatic stabilization of phytoene synthase (57 fold) in concert with overpigmentation of lon mutant cells suggests that Lon controls carotenogenesis in H. volcanii. Several hypothetical proteins, which may reveal novel functions and/or be involved in adaptation to extreme environments, were notably increased (300 fold). This study, which represents the first proteome examination of a Lon deficient archaeal cell, shows that Lon has a strong impact on H. volcanii physiology evidencing the cellular processes controlled by this protease in Archaea. Additionally, this work provides a platform for the discovery of novel targets of Lon proteases. Biological Significance: The proteome of a Lon-deficient archaeal cell was examined for the first time showing that Lon has a strong impact on H. volcanii physiology and evidencing the proteins and cellular processes controlled by this protease in Archaea. This work will facilitate future investigations aiming to address Lon function in archaea and provides a platform for the discovery of endogenous targets of the archaeal-type Lon as well as novel targets/processes regulated by Lon proteases. This knowledge will advance the understanding on archaeal physiology and the biological function of membrane proteases in microorganisms. Fil: Cerletti, Micaela. 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; Argentina Fil: Paggi, Roberto Alejandro. 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; Argentina Fil: Ramallo Guevara, Carina. Ruhr Universität Bochum; Alemania Fil: Poetsch, Ansgar. Ruhr Universität Bochum; Alemania Fil: de Castro, Rosana Esther. 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; Argentina
- Published
- 2015
14. A rhomboid protease gene deletion affects a novel oligosaccharide N-linked to the S-layer glycoprotein of Haloferax volcanii
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María Celeste Ferrari, Alicia S. Couto, Adriana C. Casabuono, Rosana Esther de Castro, Maria Ines Gimenez, Juliana Elena Parente, and Roberto A. Paggi
- Subjects
glycoprotein ,Proteases ,Glycosylation ,glycosylation ,Glycobiology and Extracellular Matrices ,Oligosaccharides ,Biology ,S- layerglycoprotein ,Biochemistry ,purl.org/becyt/ford/1 [https] ,Ciencias Biológicas ,Biología Celular, Microbiología ,Mutant protein ,halophiles ,Endopeptidases ,purl.org/becyt/ford/1.6 [https] ,Haloferax volcanii ,Molecular Biology ,mass spectrometry ,chemistry.chemical_classification ,Membrane Glycoproteins ,Rhomboid protease ,Rhomboid ,Ciencias Químicas ,Wild type ,Cell Biology ,biology.organism_classification ,Archaea ,carbohydrates (lipids) ,chemistry ,rhomboid Protease ,Gene Knockdown Techniques ,Rhomboid proteases ,Glycoprotein ,S-layer ,CIENCIAS NATURALES Y EXACTAS - Abstract
Rhomboid proteases occur in all domains of life; however, their physiological role is not completely understood, and nothing is known of the biology of these enzymes in Archaea. One of the two rhomboid homologs of Haloferax volcanii (RhoII) is fused to a zinc finger domain. Chromosomal deletion of rhoII was successful, indicating that this gene is not essential for this organism; however, the mutant strain (MIG1) showed reduced motility and increased sensitivity to novobiocin. Membrane preparations of MIG1 were enriched in two glycoproteins, identified as the S-layer glycoprotein and an ABC transporter component. The H. volcanii S-layer glycoprotein has been extensively used as a model to study haloarchaeal protein N-glycosylation. HPLC analysis of oligosaccharides released from the S-layer glycoprotein after PNGase treatment revealed that MIG1 was enriched in species with lower retention times than those derived from the parent strain. Mass spectrometry analysis showed that the wild type glycoprotein released a novel oligosaccharide species corresponding to GlcNAc-GlcNAc(Hex)2-(SQ-Hex)6 in contrast to the mutant protein, which contained the shorter form GlcNAc2(Hex)2-SQ-Hex-SQ. A glycoproteomics approach of the wild type glycopeptide fraction revealed Asn-732 peptide fragments linked to the sulfoquinovose-containing oligosaccharide. This work describes a novel N-linked oligosaccharide containing a repeating SQ-Hex unit bound to Asn-732 of the H. volcanii S-layer glycoprotein, a position that had not been reported as glycosylated. Furthermore, this study provides the first insight on the biological role of rhomboid proteases in Archaea, suggesting a link between protein glycosylation and this protease family. Fil: Parente, Juliana Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Casabuono, Adriana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Ferrari, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Paggi, Roberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina Fil: Couto, Alicia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina Fil: Gimenez, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina
- Published
- 2014
15. A simple technique to improve the resolution of membrane acidic proteins of the haloarchaeon Haloferax volcanii by 2D electrophoresis
- Author
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Roberto A, Paggi, María Inés, Giménez, Rosana E, De Castro, and Andreina, Cesari
- Subjects
Archaeal Proteins ,Membrane Proteins ,Electrophoresis, Gel, Two-Dimensional ,Hydrogen-Ion Concentration ,Haloferax volcanii - Abstract
Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the "omics" level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9-5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.
- Published
- 2014
16. The LonB protease controls membrane lipids composition and is essential for viability in the extremophilic haloarchaeon Haloferax volcanii
- Author
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Micaela, Cerletti, María J, Martínez, María I, Giménez, Diego E, Sastre, Roberto A, Paggi, and Rosana E, De Castro
- Subjects
Microbial Viability ,Base Sequence ,Pigmentation ,Archaeal Proteins ,Gene Expression ,Microbial Sensitivity Tests ,Anti-Bacterial Agents ,Membrane Lipids ,Bacitracin ,Puromycin ,Lovastatin ,Gene Expression Regulation, Archaeal ,Haloferax volcanii ,Novobiocin ,Peptide Hydrolases ,Protein Binding - Abstract
Although homologs of the ATP-dependent Lon protease exist in all domains of life, the relevance of this protease in archaeal physiology remains a mystery. In this study, we have constructed and phenotypically characterized deletion and conditional lon mutants in the model haloarchaeon Haloferax volcanii to elucidate the role of the unusual membrane-bound LonB protease in archaea. Hvlon could be deleted from the chromosome only when a copy of the wild type gene was provided in trans suggesting that Lon is essential for survival in this archaeon. Successful complementation of the lethal phenotype of ΔHvlon was attained by expression of the heterologous protease gene Nmlon from the haloalkaliphilic archaeon Natrialba magadii, meaning that the biological function of Lon is conserved in these organisms. Suboptimal cellular levels of Lon protein affected growth rate, cell shape, cell pigmentation, lipid composition and sensitivity to various antibiotics. The contents of bacterioruberins and some polar lipids were increased in the lon mutants suggesting that Lon is linked to maintenance of membrane lipid balance which likely affects cell viability in this archaeon. The phenotypes associated to a membrane-bound LonB protease mutant were examined for the first time providing insight on the relevance of this protease in archaeal physiology.
- Published
- 2013
17. Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii
- Author
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Diego Manuel Ruiz, Maria Ines Gimenez, Roberto A. Paggi, and Rosana Esther de Castro
- Subjects
Signal peptide ,Proteases ,archaea ,medicine.medical_treatment ,Archaeal Proteins ,Protein domain ,Biology ,tat system ,Microbiology ,Subtilase ,Gene Expression Regulation, Enzymologic ,purl.org/becyt/ford/1 [https] ,Ciencias Biológicas ,Serine ,Biología Celular, Microbiología ,medicine ,purl.org/becyt/ford/1.6 [https] ,Protein precursor ,Molecular Biology ,Protease ,Haloferax volcanii ,fungi ,Serine Endopeptidases ,protease ,Articles ,biology.organism_classification ,Archaea ,Protein Transport ,Biochemistry ,Gene Expression Regulation, Archaeal ,CIENCIAS NATURALES Y EXACTAS - Abstract
Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and are salt dependent for structural integrity and functionality. In contrast to bacterial subtilases, thematurationmechanismof halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus motif (GRRSVL), an N-terminal propeptide, the protease domain, and a C-terminal domain. In this study, we used Nep as amodel protease to examine the secretion andmaturation of halolysins by using genetic and biochemical approaches.Mutant variants of Nep were constructed by site-directedmutagenesis and expressed in H. volcanii, which were then analyzed by protease activity andWestern blotting. The Tat dependence of Nep secretion was demonstrated in Nep RR/KK variants containing double lysine (KK) in place of the twin arginines (RR), in which Nep remained cell associated and the extracellular activity was undetectable. High-molecular-mass Nep polypeptides without protease activity were detected as cell associated and extracellularly in the Nep S/A variant, in which the catalytic serine 352 had been changed by alanine, indicating that Nep protease activity was needed for precursor processing and activation. Nep NSN 1-2 containing amodification in two potential cleavage sites for signal peptidase I (ASA) was not efficiently processed and activated. This study examined for the first time the secretion and maturation of a Tat-dependent halophilic subtilase. Fil: Ruiz, Diego M,. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Paggi, Roberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Gimenez, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina
- Published
- 2012
18. Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep)
- Author
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Julie A. Maupin-Furlow, Maria Ines Gimenez, Rosana Esther de Castro, María Ximena Silveyra, Diego Manuel Ruiz, and Roberto A. Paggi
- Subjects
Proteases ,Archaeal Proteins ,medicine.medical_treatment ,Molecular Sequence Data ,Restriction Mapping ,NATRIALBA MAGADII ,SOLVENT TOLERANCE ,Biology ,Molecular cloning ,medicine.disease_cause ,Microbiology ,Article ,purl.org/becyt/ford/1 [https] ,Sequence Analysis, Protein ,Natrialba ,TAT PATHWAY ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,purl.org/becyt/ford/1.6 [https] ,Escherichia coli ,Conserved Sequence ,DNA Primers ,Halobacteriaceae ,Protease ,Base Sequence ,Serine Endopeptidases ,Haloferax volcanii ,fungi ,GENE CLONING AND EXPRESSION ,Subtilisin ,Sequence Analysis, DNA ,General Medicine ,biology.organism_classification ,Molecular biology ,HALOALKALIPHILIC PROTEASE ,DNA, Archaeal ,Biochemistry ,Haloarchaea ,Molecular Medicine ,Sequence Alignment ,Peptide Hydrolases ,Plasmids - Abstract
The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease. © 2008 Springer., This work was also supported in part by research grants from ANPCyT (PICT 15-25456), CONICET (PIP-6522), UNMDP (Argentina) awarded to De Castro and grants from NIH (R01 GM057498) and DOE (DE-FG02-05ER15650) awarded to Maupin-Furlow.
- Published
- 2008
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