Publications by authors named "Jean-Pierre Jacquot"

108 Publications

Thioredoxin and Glutaredoxin Systems Antioxidants Special Issue.

Antioxidants (Basel) 2019 Mar 18;8(3). Epub 2019 Mar 18.

Laboratory of Molecular Plant Physiology, Department of Pharmacy and Biotechnology, University of Bologna, via Irnerio 42, 40126 Bologna, Italy.

The special issue on Thioredoxin and Glutaredoxin systems (http://www [...].
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http://dx.doi.org/10.3390/antiox8030068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466572PMC
March 2019

Dark deactivation of chloroplast enzymes finally comes to light.

Proc Natl Acad Sci U S A 2018 09 30;115(38):9334-9335. Epub 2018 Aug 30.

UMR 1136 Interactions Arbres Microorganismes, Faculté des Sciences et Technologies, Institut National de la Recherche Agronomique , Université de Lorraine, 53506 Vandoeuvre-lès-Nancy, France

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http://dx.doi.org/10.1073/pnas.1814182115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156653PMC
September 2018

Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A.

PLoS One 2017 31;12(3):e0174753. Epub 2017 Mar 31.

UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.

Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174753PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375154PMC
August 2017

Dithiol disulphide exchange in redox regulation of chloroplast enzymes in response to evolutionary and structural constraints.

Plant Sci 2017 Feb 8;255:1-11. Epub 2016 Nov 8.

Université de Lorraine, UMR 1136 Interactions Arbres Microorganismes, F-54500 Vandœuvre-lès-Nancy, France; INRA, UMR 1136 Interactions Arbres Microorganismes, F-54280 Champenoux, France. Electronic address:

Redox regulation of chloroplast enzymes via disulphide reduction is believed to control the rates of CO fixation. The study of the thioredoxin reduction pathways and of various target enzymes lead to the following guidelines.
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http://dx.doi.org/10.1016/j.plantsci.2016.11.003DOI Listing
February 2017

Plastidic P2 glucose-6P dehydrogenase from poplar is modulated by thioredoxin m-type: Distinct roles of cysteine residues in redox regulation and NADPH inhibition.

Plant Sci 2016 Nov 8;252:257-266. Epub 2016 Aug 8.

Dipartimento di Biologia, Univ. di Napoli "Federico II", I-80126 Napoli, Italy. Electronic address:

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys and Cys (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C, C, C) are suggested to have a role in controlling the NADP access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys and Cys in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.
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http://dx.doi.org/10.1016/j.plantsci.2016.08.003DOI Listing
November 2016

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories.

Proc Natl Acad Sci U S A 2016 06 25;113(24):6779-84. Epub 2016 May 25.

Université de Lorraine, UMR 1136 Interactions Arbres Microorganismes, F-54500 Vandœuvre-les-Nancy, France; Institut national de la recherche agronomique (INRA), UMR 1136 Interactions Arbres Microorganismes, F-54280 Champenoux, France;

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.
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http://dx.doi.org/10.1073/pnas.1606241113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914176PMC
June 2016

Highly Efficient CYP167A1 (EpoK) dependent Epothilone B Formation and Production of 7-Ketone Epothilone D as a New Epothilone Derivative.

Sci Rep 2015 Oct 8;5:14881. Epub 2015 Oct 8.

Department of Biochemistry, Saarland University, 66123 Saarbrücken, Germany.

Since their discovery in the soil bacterium Sorangium cellulosum, epothilones have emerged as a valuable substance class with promising anti-tumor activity. Because of their benefits in the treatment of cancer and neurodegenerative diseases, epothilones are targets for drug design and pharmaceutical research. The final step of their biosynthesis - a cytochrome P450 mediated epoxidation of epothilone C/D to A/B by CYP167A1 (EpoK) - needs significant improvement, in particular regarding the efficiency of its redox partners. Therefore, we have investigated the ability of various hetero- and homologous redox partners to transfer electrons to EpoK. Hereby, a new hybrid system was established with conversion rates eleven times higher and Vmax of more than seven orders of magnitudes higher as compared with the previously described spinach redox chain. This hybrid system is the most efficient redox chain for EpoK described to date. Furthermore, P450s from So ce56 were identified which are able to convert epothilone D to 14-OH, 21-OH, 26-OH epothilone D and 7-ketone epothilone D. The latter one represents a novel epothilone derivative and is a suitable candidate for pharmacological tests. The results revealed myxobacterial P450s from S. cellulosum So ce56 as promising candidates for protein engineering for biotechnological production of epothilone derivatives.
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http://dx.doi.org/10.1038/srep14881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4597204PMC
October 2015

Quinone- and nitroreductase reactions of Thermotoga maritima thioredoxin reductase.

Acta Biochim Pol 2015 22;62(2):303-9. Epub 2015 Jun 22.

Institute of Biochemistry of Vilnius University, LT-08662 Vilnius, Lithuania.

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E(1)7, -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E(1)7, thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31±0.03 V at pH 7.0, which may correspond to the FAD/FADH2 redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role.
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http://dx.doi.org/10.18388/abp.2015_1003DOI Listing
April 2016

Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

Appl Environ Microbiol 2014 Oct 8;80(20):6316-27. Epub 2014 Aug 8.

Université de Lorraine, IAM, UMR 1136, IFR 110 EFABA, Champenoux, France INRA, IAM, UMR 1136, Vandoeuvre-les-Nancy, France

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.
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http://dx.doi.org/10.1128/AEM.02103-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178660PMC
October 2014

Characterization of poplar GrxS14 in different structural forms.

Protein Cell 2014 May;5(5):329-33

Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing, 100871, China.

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http://dx.doi.org/10.1007/s13238-014-0042-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996159PMC
May 2014

Toward a refined classification of class I dithiol glutaredoxins from poplar: biochemical basis for the definition of two subclasses.

Front Plant Sci 2013 18;4:518. Epub 2013 Dec 18.

Interactions Arbres - Microorganismes, Université de Lorraine, UMR1136 Vandoeuvre-lès-Nancy, France ; Interactions Arbres - Microorganismes, Institut National de la Recherche Agronomique, UMR1136 Champenoux, France.

Glutaredoxins (Grxs) are small oxidoreductases particularly specialized in the reduction of protein-glutathione adducts. Compared to other eukaryotic organisms, higher plants present an increased diversity of Grxs which are organized into four classes. This work presents a thorough comparative analysis of the biochemical and catalytic properties of dithiol class I Grxs from poplar, namely GrxC1, GrxC2, GrxC3, and GrxC4. By evaluating the in vitro oxidoreductase activity of wild type and cysteine mutated variants and by determining their dithiol-disulfide redox potentials, pK a values of the catalytic cysteine, redox state changes in response to oxidative treatments, two subgroups can be distinguished. In accordance with their probable quite recent duplication, GrxC1 and GrxC2 are less efficient catalysts for the reduction of dehydroascorbate and hydroxyethyldisulfide compared to GrxC3 and GrxC4, and they can form covalent dimers owing to the presence of an additional C-terminal cysteine (Cys C ). Interestingly, the second active site cysteine (CysB) influences the reactivity of the catalytic cysteine (CysA) in GrxC1 and GrxC2 as already observed with GrxC5 (restricted to A. thaliana), but not in GrxC3 and C4. However, all proteins can form an intramolecular disulfide between the two active site cysteines (CysA-CysB) which could represent either a protective mechanism considering that this second cysteine is dispensable for deglutathionylation reaction or a true catalytic intermediate occurring during the reduction of particular disulfide substrates or in specific conditions or compartments where glutathione levels are insufficient to support Grx regeneration. Overall, in addition to their different sub-cellular localization and expression pattern, the duplication and maintenance along evolution of several class I Grxs in higher plants can be explained by the existence of differential biochemical and catalytic properties.
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http://dx.doi.org/10.3389/fpls.2013.00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3866529PMC
January 2014

Diversification of fungal specific class a glutathione transferases in saprotrophic fungi.

PLoS One 2013 20;8(11):e80298. Epub 2013 Nov 20.

Université de Lorraine, IAM, UMR 1136, IFR 110 EFABA, Vandoeuvre-les-Nancy, France ; INRA, IAM, UMR 1136, Champenoux, France ; Laboratoire de biotechnologie, Pôle Biotechnologie et Sylviculture Avancée, FCBA, Campus Forêt-Bois de Pierroton, Cestas, France.

Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes and endogenous metabolism. The distribution of fungal-specific class A GSTs was investigated in saprotrophic fungi revealing a recent diversification within this class. Biochemical characterization of eight GSTFuA isoforms from Phanerochaete chrysosporium and Coprinus cinereus demonstrated functional diversity in saprotrophic fungi. The three-dimensional structures of three P. chrysosporium isoforms feature structural differences explaining the functional diversity of these enzymes. Competition experiments between fluorescent probes, and various molecules, showed that these GSTs function as ligandins with various small aromatic compounds, derived from lignin degradation or not, at a L-site overlapping the glutathione binding pocket. By combining genomic data with structural and biochemical determinations, we propose that this class of GST has evolved in response to environmental constraints induced by wood chemistry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080298PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835915PMC
September 2014

Monothiol glutaredoxin-BolA interactions: redox control of Arabidopsis thaliana BolA2 and SufE1.

Mol Plant 2014 Jan 7;7(1):187-205. Epub 2013 Nov 7.

a Université de Lorraine, Interactions Arbres-Microorganismes, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France.

A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluorescent protein (GFP) fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desulfurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionylated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.
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http://dx.doi.org/10.1093/mp/sst156DOI Listing
January 2014

Overexpression, purification and enzymatic characterization of a recombinant plastidial glucose-6-phosphate dehydrogenase from barley (Hordeum vulgare cv. Nure) roots.

Plant Physiol Biochem 2013 Dec 17;73:266-73. Epub 2013 Oct 17.

Dipartimento di Biologia, Università di Napoli "Federico II", Via Cinthia, 80126 Naples, Italy; Université de Lorraine, Unité Mixte de Recherches 1136 Interactions Arbres Microorganismes, F-54500 Vandoeuvre-lès-Nancy, France; INRA, Unité Mixte de Recherches 1136 Interactions Arbres Microorganismes, F-54280 Champenoux, France.

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coli, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies.
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http://dx.doi.org/10.1016/j.plaphy.2013.10.008DOI Listing
December 2013

Atypical features of a Ure2p glutathione transferase from Phanerochaete chrysosporium.

FEBS Lett 2013 Jul 24;587(14):2125-30. Epub 2013 May 24.

Université de Lorraine, IAM, UMR 1136, IFR 110 EFABA,Vandoeuvre-lès-Nancy F-54506, France.

Glutathione transferases (GSTs) are known to transfer glutathione onto small hydrophobic molecules in detoxification reactions. The GST Ure2pB1 from Phanerochaete chrysosporium exhibits atypical features, i.e. the presence of two glutathione binding sites and a high affinity towards oxidized glutathione. Moreover, PcUre2pB1 is able to efficiently deglutathionylate GS-phenacylacetophenone. Catalysis is not mediated by the cysteines of the protein but rather by the one of glutathione and an asparagine residue plays a key role in glutathione stabilization. Interestingly PcUre2pB1 interacts in vitro with a GST of the omega class. These properties are discussed in the physiological context of wood degrading fungi.
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http://dx.doi.org/10.1016/j.febslet.2013.05.031DOI Listing
July 2013

Cysteine-based redox regulation and signaling in plants.

Front Plant Sci 2013 29;4:105. Epub 2013 Apr 29.

UMR1136 Université de Lorraine-INRA, Interactions Arbres/Micro-organismes, IFR110, Faculté des Sciences Vandoeuvre, France.

Living organisms are subjected to oxidative stress conditions which are characterized by the production of reactive oxygen, nitrogen, and sulfur species. In plants as in other organisms, many of these compounds have a dual function as they damage different types of macromolecules but they also likely fulfil an important role as secondary messengers. Owing to the reactivity of their thiol groups, some protein cysteine residues are particularly prone to oxidation by these molecules. In the past years, besides their recognized catalytic and regulatory functions, the modification of cysteine thiol group was increasingly viewed as either protective or redox signaling mechanisms. The most physiologically relevant reversible redox post-translational modifications (PTMs) are disulfide bonds, sulfenic acids, S-glutathione adducts, S-nitrosothiols and to a lesser extent S-sulfenyl-amides, thiosulfinates and S-persulfides. These redox PTMs are mostly controlled by two oxidoreductase families, thioredoxins and glutaredoxins. This review focuses on recent advances highlighting the variety and physiological roles of these PTMs and the proteomic strategies used for their detection.
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http://dx.doi.org/10.3389/fpls.2013.00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638127PMC
May 2013

Putative involvement of Thioredoxin h in early response to gravitropic stimulation of poplar stems.

J Plant Physiol 2013 May 5;170(7):707-11. Epub 2013 Mar 5.

Laboratoire de Biologie et Physiologie Végétales, Département de Biologie, Faculté des Sciences de Tunis, campus universitaire, 1060, Tunis, Tunisia.

Gravity perception and gravitropic response are essential for plant development. In herbaceous species, it is widely accepted that one of the primary events in gravity perception involves the displacement of amyloplasts within specialized cells. However, the early signaling events leading to stem reorientation are not fully known, especially in woody species in which primary and secondary growth occur. Thirty-six percent of the identified proteins that were differentially expressed after gravistimulation were established as potential Thioredoxin targets. In addition, Thioredoxin h expression was induced following gravistimulation. In situ immunolocalization indicated that Thioredoxin h protein co-localized with the amyloplasts located in the endodermal cells. These investigations suggest the involvement of Thioredoxin h in the first events of signal transduction in inclined poplar stems, leading to reaction wood formation.
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http://dx.doi.org/10.1016/j.jplph.2012.12.017DOI Listing
May 2013

Xenomic networks variability and adaptation traits in wood decaying fungi.

Microb Biotechnol 2013 May 2;6(3):248-63. Epub 2013 Jan 2.

Université de Lorraine, IAM, UMR 1136, IFR 110 EFABA, Vandoeuvre-lès-Nancy, F-54506, France.

Fungal degradation of wood is mainly restricted to basidiomycetes, these organisms having developed complex oxidative and hydrolytic enzymatic systems. Besides these systems, wood-decaying fungi possess intracellular networks allowing them to deal with the myriad of potential toxic compounds resulting at least in part from wood degradation but also more generally from recalcitrant organic matter degradation. The members of the detoxification pathways constitute the xenome. Generally, they belong to multigenic families such as the cytochrome P450 monooxygenases and the glutathione transferases. Taking advantage of the recent release of numerous genomes of basidiomycetes, we show here that these multigenic families are extended and functionally related in wood-decaying fungi. Furthermore, we postulate that these rapidly evolving multigenic families could reflect the adaptation of these fungi to the diversity of their substrate and provide keys to understand their ecology. This is of particular importance for white biotechnology, this xenome being a putative target for improving degradation properties of these fungi in biomass valorization purposes.
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http://dx.doi.org/10.1111/1751-7915.12015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815920PMC
May 2013

New substrates and activity of Phanerochaete chrysosporium Omega glutathione transferases.

Biochimie 2013 Feb 11;95(2):336-46. Epub 2012 Oct 11.

UMR 1136 INRA-UHP Interactions Arbres/Micro-Organismes, IFR110 Ecosystèmes Forestiers, Agroressources, Bioprocédés et Alimentation, Université de Lorraine, Faculté des Sciences et Technologies, BP 70239, 54506 Vandoeuvre-les-Nancy, France.

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chrysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions.
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http://dx.doi.org/10.1016/j.biochi.2012.10.003DOI Listing
February 2013

Glutathione- and glutaredoxin-dependent reduction of methionine sulfoxide reductase A.

FEBS Lett 2012 Nov 25;586(21):3894-9. Epub 2012 Sep 25.

UMR1136 Université de Lorraine-INRA, Interactions Arbres-Microorganismes, IFR 110, Faculté des Sciences, 54500 Vandoeuvre, France.

A natural fusion occurring between two tandemly repeated glutaredoxin (Grx) modules and a methionine sulfoxide reductase A (MsrA) has been detected in Gracilaria gracilis. Using an in vivo yeast complementation assay and in vitro activity measurements, we demonstrated that this fusion enzyme was able to reduce methionine sulfoxide into methionine using glutathione as a reductant. Consistently, a poplar cytosolic MsrA can be regenerated in vitro by glutaredoxins with an efficiency comparable to that of thioredoxins, but using a different mechanism. We hypothesize that the glutathione/glutaredoxin system could constitute an evolutionary conserved alternative regeneration system for MsrA.
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http://dx.doi.org/10.1016/j.febslet.2012.09.020DOI Listing
November 2012

Characterization of a Phanerochaete chrysosporium glutathione transferase reveals a novel structural and functional class with ligandin properties.

J Biol Chem 2012 Nov 24;287(46):39001-11. Epub 2012 Sep 24.

Université de Lorraine, Interactions Arbre-Microorganismes, UMR 1136, Institut Fédératif de Recherche 110 EFABA, Vandoeuvre-lès-Nancy F-54506, France.

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional β-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix α4' in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.
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http://dx.doi.org/10.1074/jbc.M112.402776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493941PMC
November 2012

Quinone- and nitroreductase reactions of Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme.

Arch Biochem Biophys 2012 Dec 12;528(1):50-6. Epub 2012 Sep 12.

Institute of Biochemistry of Vilnius University, Mokslininkų 12, LT-08662 Vilnius, Lithuania.

Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme (Prx-NR) consists of a FMN-containing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromatics by NR proceeded in a two-electron manner, and follows a 'ping-pong' scheme with sometimes pronounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx-NR was characterized by the linear dependence of their reactivity (logk(cat)/K(m)) on their single-electron reduction potentials E(7)(1), while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the specificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx-NR, and to a significant diversity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics.
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http://dx.doi.org/10.1016/j.abb.2012.08.014DOI Listing
December 2012

Two Sinorhizobium meliloti glutaredoxins regulate iron metabolism and symbiotic bacteroid differentiation.

Environ Microbiol 2013 Mar 14;15(3):795-810. Epub 2012 Aug 14.

UMR 'Institut Sophia Agrobiotech' INRA 1355-CNRS 7254 - Université de Nice-Sophia Antipolis, 400 routes des Chappes, 06903 Sophia-Antipolis cedex, France.

Legumes interact symbiotically with bacteria of the Rhizobiaceae to form nitrogen-fixing root nodules. We investigated the contribution of the three glutaredoxin (Grx)-encoding genes present in the Sinorhizobium meliloti genome to this symbiosis. SmGRX1 (CGYC active site) and SmGRX3 (CPYG) recombinant proteins displayed deglutathionylation activity in the 2-hydroethyldisulfide assay, whereas SmGRX2 (CGFS) did not. Mutation of SmGRX3 did not affect S. meliloti growth or symbiotic capacities. In contrast, SmGRX1 and SmGRX2 mutations decreased the growth of free-living bacteria and the nitrogen fixation capacity of bacteroids. Mutation of SmGRX1 led to nodule abortion and an absence of bacteroid differentiation, whereas SmGRX2 mutation decreased nodule development without modifying bacteroid development. The higher sensitivity of the Smgrx1 mutant strain as compared with wild-type strain to oxidative stress was associated with larger amounts of glutathionylated proteins. The Smgrx2 mutant strain displayed significantly lower levels of activity than the wild type for two iron-sulfur-containing enzymes, aconitase and succinate dehydrogenase. This lower level of activity could be associated with deregulation of the transcriptional activity of the RirA iron regulator and higher intracellular iron content. Thus, two S. meliloti Grx proteins are essential for symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and the regulation of iron metabolism.
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http://dx.doi.org/10.1111/j.1462-2920.2012.02835.xDOI Listing
March 2013

Glutathione regulates the transfer of iron-sulfur cluster from monothiol and dithiol glutaredoxins to apo ferredoxin.

Protein Cell 2012 Sep 12;3(9):714-21. Epub 2012 Aug 12.

Beijing Nuclear Magnetic Resonance Center, College of Chemistry and Molecular Engineering, School of Life Sciences, Peking University, Beijing, 100871, China.

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.
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http://dx.doi.org/10.1007/s13238-012-2051-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4875373PMC
September 2012

In the absence of thioredoxins, what are the reductants for peroxiredoxins in Thermotoga maritima?

Antioxid Redox Signal 2013 May 24;18(13):1613-22. Epub 2012 Sep 24.

Unité Mixte de Recherches 1136 INRA-Lorraine Université, Interactions Arbres-Microorganismes, IFR 110, Faculté des Sciences, Vandoeuvre Cedex, France.

Three peroxiredoxins (Prxs) were identified in Thermotoga maritima, which possesses neither glutathione nor typical thioredoxins: one of the Prx6 class; one 2-Cys PrxBCP; and a unique hybrid protein containing an N-terminal 1-Cys PrxBCP domain fused to a flavin mononucleotide-containing nitroreductase (Ntr) domain. No peroxidase activity was detected for Prx6, whereas both bacterioferritin comigratory proteins (BCPs) were regenerated by a NADH/thioredoxin reductase/glutaredoxin (Grx)-like system, constituting a unique peroxide removal system. Only two of the three Grx-like proteins were able to support peroxidase activity. The inability of TmGrx1 to regenerate oxidized 2-Cys PrxBCP probably results from the thermodynamically unfavorable difference in their disulfide/dithiol E(m) values, -150 and -315 mV, respectively. Mutagenesis of the Prx-Ntr fusion, combined with kinetic and structural analyses, indicated that electrons are not transferred between its two domains. However, their separate activities could function in a complementary manner, with peroxide originating from the chromate reductase activity of the Ntr domain reduced by the Prx domain.
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http://dx.doi.org/10.1089/ars.2012.4739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613187PMC
May 2013

Fifty years in the thioredoxin field and a bountiful harvest.

Biochim Biophys Acta 2012 Nov 27;1820(11):1822-9. Epub 2012 Jul 27.

Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.

Discovered 50 years ago as a hydrogen donor for the reduction of ribonucleotides, thioredoxin is currently recognized as a protein central to the regulation of multiple processes in the cell. Two meetings separated by a period of 30 years serve as benchmarks for assessing this transition-the first held in Berkeley (California) in 1981 and the other convened in 2011 in Sant Feliu de Guixols (Spain). The four of us contributing this article attended both meetings and thus have witnessed the development of the thioredoxin field and its notable extension in unanticipated new directions. In this Perspective we briefly recount the unfolding of this remarkable story.
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http://dx.doi.org/10.1016/j.bbagen.2012.07.006DOI Listing
November 2012

Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine.

Plant Physiol 2012 Jun 20;159(2):592-605. Epub 2012 Apr 20.

UMR 1136 Lorraine University-INRA, Interactions Arbres-Microorganismes, Institut Fédératif de Recherche 110 Ecosystèmes Forestiers, Agroressources, Bioprocédés, et Alimentation, Faculté des Sciences, Vandoeuvre-lès-Nancy, France.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.
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http://dx.doi.org/10.1104/pp.112.197723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375927PMC
June 2012

Functional diversification of fungal glutathione transferases from the ure2p class.

Int J Evol Biol 2011 22;2011:938308. Epub 2011 Nov 22.

Unité Mixte de Recherches INRA UHP 1136 Interaction Arbres Microorganismes, IFR 110 Ecosystèmes Forestiers, Agroressources, Bioprocédés et Alimentation, Faculté des Sciences et Technologies, Nancy Université BP 70239, 54506 Vandoeuvre-lès-Nancy Cedex, France.

The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints.
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http://dx.doi.org/10.4061/2011/938308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227518PMC
August 2012

Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5: kinetics, catalytic mechanism and oxidative inactivation.

Biochem J 2012 Mar;442(2):369-80

UMR1136 Nancy University-INRA, Interactions Arbres-Microorganismes, IFR 110, EFABA 54506 Vandoeuvre-lès-Nancy cedex, France.

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1·s-1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.
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http://dx.doi.org/10.1042/BJ20111378DOI Listing
March 2012

RETRACTED: Redox regulation of the glutathione reductase/iso-glutaredoxin system in germinating pea seed exposed to cadmium.

Plant Sci 2010 Nov 6;179(5):423-36. Epub 2010 Jul 6.

Bio-Physiologie Cellulaires, Faculté des Sciences de Bizerte, 7021 Zarzouna, Tunisia; Unité Mixte de Recherches, 1136 Interaction arbres-microorganismes INRA-Université Henri-Poincaré, IFR110, Faculté des Sciences, BP 239, 54506 Vandoeuvre cedex, France.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The editors would like to confirm the retraction of this paper at the request of the co-authors who had no prior knowledge on the actions of the lead author. This article contains data that was duplicated in: Smiri M, Chaoui A, Rouhier N, Gelhaye E, Jacquot JP, El Ferjani E. Cadmium Affects the Glutathione/Glutaredoxin System in Germinating Pea Seeds. Biol. Trace Elem. Res., 142 (2010) 93-105, doi:10.1007/s12011-010-8749-3; Smiri M, Chaoui A, Rouhier N, Gelhaye E, Jacquot JP, El Ferjani E. Effect of cadmium on resumption of respiration in cotyledons of germinating pea seeds. Ecotox. Environ. Safe., 73 (2010) 1246-1254, doi:10.1016/j.ecoenv.2010.05.015; Smiri M, Chaoui A, Rouhier N, Gelhaye E, Jacquot JP, El Ferjani E. NAD pattern and NADH oxidase activity in pea (Pisum sativum L.) under cadmium toxicity. Physiol. Mol. Biol. Plants, 16 (2010) 305-315, doi:10.1007/s12298-010-0033-7; Smiri M, Chaoui A, Rouhier N, Gelhaye E, Jacquot JP, El Ferjani E. Oxidative damage and redox change in pea seeds treated with cadmium. C. R. Biol., 333 (2010) 801-807, doi:10.1016/j.crvi.2010.09.002; Smiri M, Chaoui A, Rouhier N, Kamel C, Gelhaye E, Jacquot JP, El Ferjani E. Cadmium induced mitochondrial redox changes in germinating pea seed. BioMetals, 23 (2010) 973-984, doi:10.1007/s10534-010-9344-y. The co-authors apologize for this unfortunate incident.
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http://dx.doi.org/10.1016/j.plantsci.2010.06.015DOI Listing
November 2010