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JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62 JOURNAL OF INTEGRATED OMICS Journal of Integrated Omics A METHODOLOGICAL JOURNAL HTTP://WWW.JIOMICS.COM Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015) Effects of heavy metals on Cyanothece sp. CCY 0110 growth, extracellular polymeric substances (EPS) production, ultrastructure and protein profiles R. Mota 1,2,3 , S. B. Pereira 1,2 , M. Meazzini 4 , R. Fernandes 1,2 , A. Santos 1,2,3 , C. A. Evans 5 , R. De Philippis 4,6 , P. C. Wright 5 , and P. Tamagnini* 1,2,3 1 i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. 2 IBMC - Instituto de Biologia Molecular e Celular, Univ Porto. Rua do Campo Alegre, 823. 4150-180. Porto Portugal. 3 Faculdade de Ciências, Dept. Biologia, Univ. Porto, Rua do Campo Alegre, Edifício FC4, 4169-007. Porto. Portugal. 4 Dept. Agrifood Production and Environmental Sciences, Univ. Florence. Piazzale delle Cascine 24. I- 50144. Florence. Italy. 5 ChELSI Institute, Dept. Chemical and Biological Engineering, Univ. Sheffield. Hadfield building, Mappin Street. S1 3J. Sheffield. U. K. 6 Institute of Ecosystem Study, National Research Council (CNR). Via Madonna del Piano, 10. 50019. Sesto Fiorentino. Italy. *Corresponding author: pmtamagn@ibmc.up.pt Available Online: 31 December 2015 Abstract Purpose: The aim of this study was to evaluate the effects of several heavy metals on the growth/survival, EPS production, ultrastructure and protein profiles of the highly efficient extracellular polymeric substances (EPS)-producer cyanobacterium Cyanothece sp. CCY 0110. Experimental description: After exposure to different concentrations of metals (Cu 2+ , Pb 2+ , Cd 2+ , or Li + ), cell growth, production of total carbohydrates and RPS (released polysaccharides) and ultrastructural changes were investigated. Proteomes were compared by two 8-plex iTRAQ studies and results were validated with data from Western blots, O 2 evolution and ROS measurements and enzymatic assays. Results: The heavy metals affected the cells differently, triggering distinctive responses. Concerning chronic exposure to the metals, the cells were more affected by Cu 2+ followed by Pb 2+ , Cd 2+ , and Li + . The presence of metals led to remarkable ultrastructural changes, mainly at the thylakoid level. The comparison of the proteomes allowed to follow the stress responses and to distinguish specific effects related to the time of exposure and/or the concentration of an essential (Cu 2+ ) and a non-essential (Cd 2+ ) metal. The majority of the proteins identified with significant fold changes were associated with photosynthesis, CO 2 fixation and carbohydrate metabolism, translation, and nitrogen and amino acid metabolism. Unexpectedly, the amount of released polysaccharides (RPS) was not enhanced by the presence of heavy metals. Conclusions: This work shows the holistic effects of different heavy metals on the cyanobacterium Cyanothece sp. CCY 0110. Overall, the results suggest that during Cu 2+ chronic exposure the cells adjust their metabolism to invest the spare energy in the activation of metal detoxification mechanisms. In contrast, the toxic effects of Cd 2+ accumulate over time suggesting that cells might not have the same capacity to deal with this non-essential metal. Keywords: Cyanobacteria; Cyanothece; extracellular polymeric substances (EPS); heavy metals; iTRAQ; proteome. Acknowledgements: The work was funded by FEDER Funds through the Operational Competitiveness Programme – COMPETE and national funds through FCT – Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-028314 (PTDC/BIA- MIC/2889/2012) and the scholarships SFRH/BD/84914/2012 and SFRH/BDP/72400/2010. We thank Professor Lucas Stal for providing Cyanothece sp. CCY 0110. Sheffield acknowledges the EPSRC (EP/E036252/1). 1-62: 41
JIOMICS | VOL 5 | ISSUE 2 | DECEMBER 2015 | 1-62 JOURNAL OF INTEGRATED OMICS Journal of Integrated Omics A METHODOLOGICAL JOURNAL HTTP://WWW.JIOMICS.COM Special Issue: Proceeding Abstracts of the 4 th International Congress on Analytical Proteomics (ICAP 2015) Brain ubiquitin mitochondrial subproteome and its changes induced by neurotoxins and neuroprotectors A.E. Medvedev* 1 , O.A. Buneeva 1 , A.T. Kopylov 1 , M.V.Medvedeva 2 , O.V.Tikhonova 1 , V.G. Zgoda 1 1 Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry, 10 Pogodinskaya Street, Moscow, 119121, Russia; 2 Moscow State University, Vorobievy Gori 1, Bld.12, Moscow, Russia 119234 Russia. *Corresponding author: professor57@yandex.ru Available Online: 31 December 2015 Abstract Ubiquitin is a 76-residue protein that tags proteins and thus regulates numerous intracellular processes. Incubation of the rat brain mitochondrial fraction with biotinylated ubiquitin in vitro followed by avidin-agarose chromatography and mass spectrometry (MS) detection revealed direct ubiquitination of extra- and intramitochondrial proteins. However, the proportion of directly ubiquitinated proteins represented not more than 20% of the total number of identified proteins that specifically bound to avidin-agarose. Profiling of endogenously ubiquitinated proteins of the mitochondrial compartment by means of affinity chromatography on the proteasome subunit S5a conjugated with agarose followed by their MS detection revealed 58 individual proteins identified by a characteristic diglycine fragment of their tryptic peptides. Administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inducing symptoms of parkinsonism in mice had a minor effect on the total number of identified proteins (59) but caused qualitative changes in the proteomic profile. Pretreatment with a nonpeptide neuroprotector, isatin, decreased MPTP-induced neurotoxicity and significantly reduced the total number of individual ubiquitinated proteins (to 36). Isatin (interacting with a wide range of isatin-binding proteins in the brain and regulating expression of isatin-sensitive genes) administered to control mice also decreased the total number of ubiquitinated proteins (to 30). Conclusions: The qualitative changes in the ubiquitin mitochondrial subproteome suggest that the neuroprotector effect of isatin is associated with “metabolic immobilization of the brain” induced by interaction of isatin with isatin binding proteins and altered patterns of ubiquitinated proteins of the mitochondrial compartments and inhibition of monoamine oxidase (B) as well. Keywords: mitochondrial subproteome, affinity-based profiling, ubiquitin, isatin, isatin-binding proteins, MPTP Acknowledgements: The work was done in the framework of the State Academies of Sciences Fundamenal Research Program for 2013- 2020 and the Russian Foundation for Basic Research (project nos. 13-04-00161 and 15-04-01545). 1-62: 42
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Volume 5 | Issue 2 | December 2015
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Randen Patterson Center for Computa
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Biodiversity Research Center, Acade
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Belgium Bart De Spiegeleer Ghent Un
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