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Biomedical subjects

Jean-Pierre Jacquot

Publications and source records attributed to Jean-Pierre Jacquot.

At least 37 records · Page 2Linked to original sources
PubMed2004

Isolation of Pea Thioredoxin f Precursor Protein and Characterization of its Biochemical Properties.

Like many other soluble chloroplastic enzymes, thioredoxin f is nuclear-encoded and expressed as a precursor protein. After synthesis in the cytosol, it is imported into the chloroplast with subsequent cleavage of the transit sequence in the stroma. We report the expression and the partial purification of the recombinant precursor thioredoxin f protein. The prethioredoxin f was found to be located essentially in the insoluble Echerichia coli fraction, but could be renatured after urea treatment followed by dialysis. The renatured protein was active in the dithiothreitol- and thioredoxin-dependent activation of NADP malate dehydrogenase and also of fructose bisphosphatase and in the ferredoxin-thioredoxin-dependent fructose bisphosphatase activation. These data are discussed in relation with the known properties of mature thioredoxin f.

Journal Article↗
PubMed2004

Generation of polyclonal antibodies against multiple proteins in a single rabbit and subsequent isolation of the specific immunoglobulins as a tool for proteomics research.

A single rabbit has been immunized with a mixture of six different proteins: green fluorescent protein, ferredoxin:NADP oxidoreductase, NADPH:thioredoxin reductase, thioredoxin f, fructose bisphosphatase and cyclophilin. Individual IgGs were re-purified from the serum through specific interaction on small columns generated by grafting the antigen on a CNBr-Sepharose matrix. The specificity of the isolated IgG was checked by Western-blot analysis. Purified and specific IgG fractions were obtained with all antigens except thioredoxin f.

Animals↗
PubMed2004

Thioredoxin Ch1 of Chlamydomonas reinhardtii displays an unusual resistance toward one-electron oxidation.

To test thioredoxin resistance to oxidizing free radicals, we have studied the one-electron oxidation of wild-type thioredoxin and of two forms with the point mutations D30A and W35A, using azide radicals generated by gamma-ray or pulse radiolysis. The oxidation patterns of wild-type thioredoxin and D30A are similar. In these forms, Trp35 is the primary target and is 'repaired' by one-electron reduction; first by intramolecular electron transfer from tyrosine, and then from other residues. Conversely, during oxidation of W35A, Trp13 is poorly reactive. For all proteins, activity is conserved showing an unusual resistance toward oxidation.

Algal Proteins↗
PubMed2003

Effect of pH on the oxidation-reduction properties of thioredoxins.

Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.

Alanine↗
PubMed2003

Crystallization and preliminary X-ray studies of the glutaredoxin from poplar in complex with glutathione.

A monocysteinic mutant of poplar glutaredoxin (C30S) has been overproduced and purified. The protein has been crystallized in complex with glutathione using the hanging-drop vapour-diffusion technique in the presence of PEG 4000 as a precipitating agent. A native data set was collected at 1.55 A resolution. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.7, b = 49.1, c = 104.8 A. Isomorphous crystals of a selenomethionine derivative were grown under the same conditions. Three data sets were collected at 1.73 A using the FIP synchrotron beamline at the ESRF. The positions of the Se atoms were determined and model rebuilding and refinement are in progress.

Cloning, Molecular↗
PubMed2003

Characterization of the redox properties of poplar glutaredoxin.

The presence of glutaredoxins in plants is now well recognized, but their functions and natural substrates remain largely unknown. Recently, a poplar glutaredoxin has been biochemically characterized and several mutants have been engineered in order to explore its reactivity. This work focuses on some physiological functions of the enzyme. According to our findings, the poplar glutaredoxin can serve as an electron donor to the bacterial 3'-phosphoadenylylsulfate reductase as it supports both the catalysis by the enzyme in vitro and complements a methionine auxotroph strain of Escherichia coli. In addition, poplar glutaredoxin is able to reduce the Escherichia coli ribonucleotide reductase 1a (in vitro reduction of cytidine diphosphate). Although this glutaredoxin is described as an electron donor to a phloem-located peroxiredoxin, whose function is to detoxify hydroperoxides, we found that it does not directly reduce hydrogen peroxide or other alkyl hydroperoxides as described for yeast and rice glutaredoxins. However, the poplar glutaredoxin may be involved in the response to oxidative stress as its overexpression in Escherichia coli resulted in a higher resistance toward hydrogen peroxide, menadione, and tert-butyl hydroperoxide.

Amino Acid Sequence↗
PubMed2002

Exploring the active site of plant glutaredoxin by site-directed mutagenesis.

Six mutants (Y26A, C27S, Y29F, Y29P, C30S and Y26W/Y29P) have been engineered in order to explore the active site of poplar glutaredoxin (Grx) (Y26CPYC30). The cysteinic mutants indicate that Cys 27 is the primary nucleophile. Phe is a good substitute for Tyr 29, but the Y29P mutant was inactive. The Y26A mutation caused a moderate loss of activity. The YCPPC and WCPPC mutations did not improve the reactivity of Grx with the chloroplastic NADP-malate dehydrogenase, a well known target of thioredoxins (Trxs). The results are discussed in relation with the known biochemical properties of Grx and Trx.

Amino Acid Sequence↗
PubMed2002

Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence.

Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells. Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.

Amino Acid Sequence↗
PubMed2002

Thioredoxins and related proteins in photosynthetic organisms: molecular basis for thiol dependent regulation.

Thioredoxins are small molecular weight disulfide oxidoreductases specialized in the reduction of disulfide bonds on other proteins. Generally, the enzymes which are selectively and reversibly reduced by these proteins oscillate between an oxidized and inactive conformation and a reduced and active conformation. Thioredoxin constitutes the archetype of a family of protein disulfide oxidoreductases which comprises glutaredoxin and protein disulfide isomerase. Thioredoxin and glutaredoxin serve many roles in the cell, including the redox regulation of target enzymes and transcription factors. They can also serve as hydrogen donors to peroxiredoxins, recently discovered heme free peroxidases, the function of which is to get rid of hydroperoxides in the cell. This review describes the molecular basis for the functioning and interaction between these enzymes in photosynthetic organisms.

Animals↗
PubMed2002

The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.

Experiments initiated in the early 1960s on fermentative bacteria led to the discovery of ferredoxin-dependent alpha-ketocarboxylation reactions that were later found to be key to a new cycle for the assimilation of carbon dioxide in photosynthetic bacteria (the reductive carboxylic acid or reverse citric cycle). The latter finding set the stage for the discovery of a regulatory system, the ferredoxin/thioredoxin system, functional in photosynthesis in chloroplasts and oxygen-evolving photosynthetic prokaryotes. The chloroplast research led to a description of the extraplastidic NADP/thioredoxin system that is now known to function in heterotrophic plant processes such as seed germination and self-incompatibility. Extensions of the fundamental research have begun to open doors to the broad application of thioredoxin in technology and medicine.

Journal Article↗
PubMed2002

Plant peroxiredoxins: alternative hydroperoxide scavenging enzymes.

The role of plant peroxiredoxins in the detoxification systems is discussed in relation with the existence of many isoforms of this protein in distinct plant compartments. Phylogenetic analyses indicate that plant peroxiredoxins can be divided into four classes. Two of these classes correspond to chloroplastic enzymes. All isoforms contain at least one conserved catalytic cysteine. The enzymes belonging to the 1-Cys Prx class seem to be seed restricted and to play a role of detoxification during the germination process. At least one putative cytosolic isoform can use both thioredoxin and glutaredoxin as an electron donor, but the chloroplastic isoforms characterized depend on reduced thioredoxin. Mutagenesis and plant transformation studies support the proposal that the chloroplastic peroxiredoxins play an important role in combating the ROS species generated at the level of the chloroplastic electron transfer chain.

Journal Article↗
PubMed2002

Isolation and characterization of an extended thioredoxin h from poplar.

A cDNA coding for a thioredoxin h has been isolated from a xylem/phloem poplar cDNA library by RACE-PCR. The nucleotide sequence called popTrx-h2 is homologous to other thioredoxins h isolated from plants but differs from the other thioredoxins h by presenting a 30 amino acid long N-terminus extension. A variant of this cDNA lacking the N-terminal extension was also generated by PCR. Both cDNAs have been introduced into an expression plasmid (pET-3d) and the recombinant proteins have been expressed to a high level and purified from Escherichia coli cells. Protein sequencing showed that a part of the N-terminal extension was cleaved in the E. coli cells, with the first 19 amino acids missing, suggesting the presence of a putative cleavage site in the N-terminal extension of popTrx-h2. Both recombinant proteins display unusual catalytic properties compared to other thioredoxins h characterized so far, i.e. a weak reduction by Arabidopsis thaliana NADPH-dependent thioredoxin reductase, and a weak activation of the chloroplastic NADP-malate dehydrogenase, a non-physiological target enzyme. Northern blot experiments indicate that the transcripts of popTrx-h2 are present in leaves and roots, albeit at a lower level compared to the earlier characterized popTrx-h1.

Journal Article↗
PubMed2002

Isolation and characterization of a thioredoxin-dependent peroxidase from Chlamydomonas reinhardtii.

All living organisms contain redox systems involving thioredoxins (Trx), proteins featuring an extremely conserved and reactive active site that perform thiol-disulfide interchanges with disulfide bridges of target proteins. In photosynthetic organisms, numerous isoforms of Trx coexist, as revealed by sequencing of Arabidopsis genome. The specific functions of many of them are still unknown. In an attempt to find new molecular targets of Trx in Chlamydomonas reinhardtii, an affinity column carrying a cytosolic Trx h mutated at the less reactive cysteine of its active site was used to trap Chlamydomonas proteins that form mixed disulfides with Trx. The major protein bound to the column was identified by amino-acid sequencing and mass spectrometry as a thioredoxin-dependent 2Cys peroxidase. Isolation and sequencing of its gene revealed that this peroxidase is most likely a chloroplast protein with a high homology to plant 2Cys peroxiredoxins. It is shown that the Chlamydomonas peroxiredoxin (Ch-Prx1) is active with various thioredoxin isoforms, functions as an antioxidant toward reactive oxygen species (ROS), and protects DNA against ROS-induced degradation. Expression of the peroxidase gene in Chlamydomonas was found to be regulated by light, oxygen concentration, and redox state. The data suggest a role for the Chlamydomonas Prx in ROS detoxification in the chloroplast.

Amino Acid Sequence↗