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Jean-Marc Lancelin

Publications and source records attributed to Jean-Marc Lancelin.

At least 19 recordsLinked to original sources
PubMed2006

Protein-protein interactions within peroxiredoxin systems.

Peroxiredoxin systems in plants were demonstrated involved in crucial roles related to reactive oxygenated species (ROS) metabolism and the linked cell signalling to ROS. Peroxiredoxins function as peroxidasic systems that combine at least a reactivating reductant agent like thioredoxins, and sometimes glutaredoxins and glutathion. In the past three years a number of peroxiredoxin structures were solved by crystallography in different experimental crystallisation conditions. The structures have revealed a significant propensity of peroxiredoxins for oligomerism that was confirmed by biophysical studies in solution using NMR and other methods as analytical ultra-centrifugation. These studies showed that quaternary structures of peroxiredoxins involve specific protein-protein interaction interfaces that rely upon the peroxiredoxin types and/or their redox conditions. The protein-protein interactions with the reactivating redoxins essentially lead to transient unstable complexes. We review herein the different protein-protein interactions characterized or deduced from those reports.

Models, Molecular↗
PubMed2006

NMR structure determination of a synthetic analogue of bacillomycin Lc reveals the strategic role of L-Asn1 in the natural iturinic antibiotics.

Iturins are a group of antifungal produced by Bacillus subtilis. All are cyclic lipopeptides with seven alpha-amino acids of configuration LDDLLDL and one beta-amino fatty acid. The bacillomycin L is a member of this family and its NMR structure was previously resolved using the sequence Asp-Tyr-Asn-Ser-Gln-Ser-Thr. In this work, we carefully examined the NMR spectra of this compound and detected an error in the sequence. In fact, Asp1 and Gln5 need to be changed into Asn1 and Glu5, which therefore makes it identical to bacillomycin Lc. As a consequence, it now appears that all iturinic peptides with antibiotic activity share the common beta-amino fatty acid 8-L-Asn1-D-Tyr2-D-Asn3 sequence. To better understand the conformational influence of the acidic residue L-Asp1, present, for example in the inactive iturin C, the NMR structure of the synthetic analogue SCP [cyclo (L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6-L-Thr7-beta-Ala8)] was determined and compared with bacillomycin Lc recalculated with the corrected sequence. In both cases, the conformers obtained were separated into two families of similar energy which essentially differ in the number and type of turns. A detailed analysis of both cyclopeptide structures is presented here. In addition, CD and FTIR spectra were performed and confirmed the conformational differences observed by NMR between both cyclopeptides.

Amino Acid Sequence↗
PubMed2006

Interfacial behaviour of bovine testis hyaluronidase.

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.

Adsorption↗
PubMed2006

Glutathionylation induces the dissociation of 1-Cys D-peroxiredoxin non-covalent homodimer.

1-Cys peroxiredoxins (1-Cys Prxs) are antioxidant enzymes that catalyze the reduction of hydroperoxides into alcohols using a strictly conserved cysteine. 1-Cys B-Prxs, homologous to human PrxVI, were recently shown to be reactivated by glutathione S-transferase (GST) pi via the formation of a GST-Prx heterodimer and Prx glutathionylation. In contrast, 1-Cys D-Prxs, homologous to human PrxV, are reactivated by the glutaredoxin-glutathione system through an unknown mechanism. To investigate the mechanistic events that mediate the 1-Cys D-Prx regeneration, interaction of the Prx with glutathione was studied by mass spectrometry and NMR. This work reveals that the Prx can be glutathionylated on its active site cysteine. Evidences are reported that the glutathionylation of 1-Cys D-Prx induces the dissociation of the Prx non-covalent homodimer, which can be recovered by reduction with dithiothreitol. This work demonstrates for the first time the existence of a redox-dependent dimer-monomer switch in the Prx family, similar to the decamer-dimer switch for the 2-Cys Prxs.

Binding Sites↗
PubMed2006

Probing the structure-function relationship of polyene macrolides: engineered biosynthesis of soluble nystatin analogues.

Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin. However, both analogues were shown to have lost antifungal activity, implying that the integrity of the hydrophobic polyene region of the nystatin molecule is crucial for the fungicidal action. NMR data and computer modeling performed for the new analogues suggested conformational changes together with a significantly increased structural disorder, which may account for both increased solubility and the loss of activity.

Antifungal Agents↗
PubMed2006

F-ara-AMP is a substrate of cytoplasmic 5'-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation.

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

5'-Nucleotidase↗
PubMed2005

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants.

Thioredoxin f (TRXf) is a key factor in the redox regulation of chloroplastic carbon fixation enzymes, whereas glutathione is an important thiol buffer whose status is modulated by stress conditions. Here, we report specific glutathionylation of TRXf. A conserved cysteine is present in the TRXf primary sequence, in addition to its two active-site cysteines. The additional cysteine becomes glutathionylated when TRXf is exposed to oxidized glutathione or to reduced glutathione plus oxidants. No other chloroplastic TRX, from either Arabidopsis or Chlamydomonas, is glutathionylated under these conditions. Glutathionylation decreases the ability of TRXf to be reduced by ferredoxin-thioredoxin reductase and results in impaired light activation of target enzymes in a reconstituted thylakoid system. Although several mammalian proteins undergoing glutathionylation have already been identified, TRXf is among the first plant proteins found to undergo this posttranslational modification. This report suggests that a crosstalk between the TRX and glutathione systems mediates a previously uncharacterized form of redox signaling in plants in stress conditions.

Animals↗
PubMed2005

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface.

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.

Amino Acid Sequence↗
PubMed2005

Crystal structure and solution NMR dynamics of a D (type II) peroxiredoxin glutaredoxin and thioredoxin dependent: a new insight into the peroxiredoxin oligomerism.

Peroxiredoxins (Prxs) constitute a family of thiol peroxidases that reduce hydrogen peroxide, peroxinitrite, and hydroperoxides using a strictly conserved cysteine. Very abundant in all organisms, Prxs are produced as diverse isoforms characterized by different catalytic mechanisms and various thiol-containing reducing agents. The oligomeric state of Prxs and the link with their functionality is a subject of intensive research. We present here a combined X-ray and nuclear magnetic resonance (NMR) study of a plant Prx that belongs to the D-Prx (type II) subfamily. The Populus trichocarpa Prx is the first Prx shown to be regenerated in vitro by both the glutaredoxin and thioredoxin systems. The crystal structure and solution NMR provide evidence that the reduced protein is a specific noncovalent homodimer both in the crystal and in solution. The dimer interface is roughly perpendicular to the plane of the central beta sheet and differs from the interface of A- and B-Prx dimers, where proteins associate in the plane parallel to the beta sheet. The homodimer interface involves residues strongly conserved in the D (type II) Prxs, suggesting that all Prxs of this family can homodimerize. The study provides a new insight into the Prx oligomerism and the basis for protein-protein and enzyme-substrate interaction studies by NMR.

Amino Acid Sequence↗
PubMed2004

NMR solution structures of delta-conotoxin EVIA from Conus ermineus that selectively acts on vertebrate neuronal Na+ channels.

Delta-conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. delta-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNa(v)1.2a, rNa(v)1.3, and rNa(v)1.6), without affecting rat skeletal muscle (subtype rNa(v)1.4) and human cardiac muscle (subtype hNa(v)1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu(12)-Pro(13) peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu(12)-Pro(13) cis/trans isomerism, a Pro(13) --> Ala delta-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A delta-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.

Amino Acid Sequence↗
PubMed2004

NMR of redox proteins of plants, yeasts and photosynthetic bacteria.

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.

Journal Article↗
PubMed2004

Chemical diversity of polyene macrolides produced by Streptomyces noursei ATCC 11455 and recombinant strain ERD44 with genetically altered polyketide synthase NysC.

The gram-positive bacterium Streptomyces noursei ATCC 11455 produces a complex mixture of polyene macrolides generally termed nystatins. Although the structures for nystatins A(1) and A(3) have been reported, the identities of other components of the nystatin complex remain obscure. Analyses of the culture extract from the S. noursei wild type revealed the presence of several nystatin-related compounds for which chemical structures could be suggested on the basis of their molecular weights, their UV spectra, and knowledge of the nystatin biosynthetic pathway. Nuclear magnetic resonance (NMR) studies with one of these polyene macrolides identified it as a nystatin analogue containing a mycarose moiety at C-35. A similar investigation was performed with the culture extract of the ERD44 mutant, which has a genetically altered polyketide synthase (PKS) NysC and which was previously shown to produce a heptaene nystatin analogue. The latter compound, tentatively named S44HP, and its derivative, which contains two deoxysugar moieties, were purified; and their structures were confirmed by NMR analysis. Nystatin analogues with an expanded macrolactone ring were also observed in the extract of the ERD44 mutant, suggesting that the altered PKS can "stutter" during the polyketide chain assembly. These data provide new insights into the biosynthesis of polyene macrolide antibiotics and the functionalities of PKSs and post-PKS modification enzymes.

Anti-Bacterial Agents↗
PubMed2003

Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR.

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.

Amino Acid Sequence↗
PubMed2003

A putative consensus sequence for the nucleotide-binding site of annexin A6.

Reaction-induced infrared difference spectroscopy (RIDS) has been used to investigate the nature of interactions of human annexin A6 (ANXA6) with nucleotides. RIDS results for ANXA6, obtained after the photorelease of GTP-gamma-S, ATP, or P(i) from the respective caged compounds, were identical, suggesting that the interactions between the nucleotide and ANXA6 were dominated by the phosphate groups. Phosphate-induced structural changes in ANXA6 were small and affected only seven or eight amino acid residues. The GTP fluorescent analogue, 2'(3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP), quenched tryptophan fluorescence of ANXA6 when bound to the protein. A binding stoichiometry of 1 mol of nucleotide/mol ANXA6 was established with a K(D) value of 2.8 microM for TNP-GTP. The bands observed on RIDS of ANXA6 halves (e.g., N-terminal half, ANXA6a, and C-terminal half, ANXA6b) were similar to those of the whole molecule. However, their amplitudes were smaller by a factor of 2 compared to those of whole ANXA6. TNP-GTP bound to both fragments of ANXA6 with a stoichiometry of 0.5 mol/mol. However, the binding affinities of ANXA6a and ANXA6b differed from that of ANXA6. Simulated molecular modeling revealed a nucleotide-binding site which was distributed in two distinct domains. Residues K296, Y297, K598, and K644 of ANXA6 were less than 3 A from the bound phosphate groups of either GTP or ATP. The presence of two identical sequences in ANXA6 with the F-X-X-K-Y-D/E-K-S-L motif, located in the middle of ANXA6, at residues 293-301 (within ANXA6a) and at 641-649 (within ANXA6b), suggested that the F-X-X-K-Y-D/E-K-S-L motif was the putative sequence in ANXA6 for nucleotide binding.

Adenosine Triphosphate↗
PubMed2003

The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity.

The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.

Amino Acid Sequence↗