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Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol...

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Autores principales: Damgaard, Dres, Bjørn, Mads Emil, Steffensen, Maria A., Pruijn, Ger J. M., Nielsen, Claus H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858833/
https://www.ncbi.nlm.nih.gov/pubmed/27149996
http://dx.doi.org/10.1186/s13075-016-1000-7
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author Damgaard, Dres
Bjørn, Mads Emil
Steffensen, Maria A.
Pruijn, Ger J. M.
Nielsen, Claus H.
author_facet Damgaard, Dres
Bjørn, Mads Emil
Steffensen, Maria A.
Pruijn, Ger J. M.
Nielsen, Claus H.
author_sort Damgaard, Dres
collection PubMed
description BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.
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spelling pubmed-48588332016-05-07 Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase Damgaard, Dres Bjørn, Mads Emil Steffensen, Maria A. Pruijn, Ger J. M. Nielsen, Claus H. Arthritis Res Ther Research Article BACKGROUND: Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. METHODS: Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. RESULTS: No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. CONCLUSIONS: Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space. BioMed Central 2016-05-05 2016 /pmc/articles/PMC4858833/ /pubmed/27149996 http://dx.doi.org/10.1186/s13075-016-1000-7 Text en © Damgaard et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Damgaard, Dres
Bjørn, Mads Emil
Steffensen, Maria A.
Pruijn, Ger J. M.
Nielsen, Claus H.
Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title_full Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title_fullStr Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title_full_unstemmed Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title_short Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
title_sort reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858833/
https://www.ncbi.nlm.nih.gov/pubmed/27149996
http://dx.doi.org/10.1186/s13075-016-1000-7
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