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Alain Van Dorsselaer

Publications and source records attributed to Alain Van Dorsselaer.

At least 19 recordsLinked to original sources
PubMed2006

Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q).

An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].

Animals↗
PubMed2006

Proteome analysis of plant-virus interactome: comprehensive data for virus multiplication inside their hosts.

Known host-parasite molecular interactions are widespread among parasite families, but these interactions have to be particularly large considering that viruses generally encode few proteins. Although some particular virus-host interactions are well described, no global study has yet shown multiple and simultaneous interactions in a host-parasite biological system. To prove that these multiple interactions occur in biological conditions, the complexes formed by a plant virus (rice yellow mottle virus) and the proteins of its natural host (rice) were extracted and purified from infected tissue sample. Remarkably mass spectrometry permitted the identification of a large number of proteins from the complexes that are involved in different functions not encoded by the virus but probably essential for its biological life cycle. This recruiting of proteins was strongly confirmed by the repetition of experiments using different pairs of virus-host and the use of high salt concentration to extract the complexes. We mainly identified proteins involved in plant defense, metabolism, translation, and protein synthesis and some proteins involved in transport. This study demonstrates that viruses are able to recruit many proteins from their hosts to ensure their development. Among different pairs of virus-host, similar protein functions were identified suggesting a particular importance of these proteins for viruses. The identification of particular paralog proteins among multigenic families suggests the high specificity of the recruiting for some protein functions.

Dose-Response Relationship, Drug↗
PubMed2006

Oligomerization behavior of the archaeal Sm2-type protein from Archaeoglobus fulgidus.

As part of a functional analysis of archaeal Sm-related proteins, we have studied the oligomerization behavior of the Sm-2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA-bound heptamer increases at lower pH.

Archaeal Proteins↗
PubMed2006

Electrospray mass spectrometry analysis of dendritic branches bearing peripheral fullerene subunits.

The electrospray mass spectrometric characterization of neutral dendrons with a carboxylic acid function or a t-butyl ester moiety at the central point and up to eight peripheral C60 subunits has been performed and is described in detail. Molecules bearing a carboxylic acid group at the center turned out to be preferentially ionized by deprotonation, whereas those with a t-butyl ester head group were ionized by reduction of the C60 units in the infusion capillary of the electrospray source.

Carboxylic Acids↗
PubMed2006

Messages in molecules: ligand/cation coding and self-recognition in a constitutionally dynamic system of heterometallic double helicates.

Double helicates are known to exhibit self-recognition characteristics determined by the coordination geometry of the metal involved as well as by the topicity of the ligands. Combining tridentate (terpyridine, T) or bidentate (bipyridine, B) subunits in a tritopic strand affords a set of ligands able to assemble by pairs to form double helicates, homo- or heterostranded, homo- or heterotopic, depending on the coordination properties of the metals involved. The four ligand strands, BBB, TTT, BBT, and TBT form constitutionally dynamic sets of double helicates with the metal ions Cu(I), Cu(II), and Zn(II); these helicates correspond to the correct coding of the BB, BT, and TT pairs for tetra-, penta-, and hexacoordinate Cu(I), Cu(II), and Zn(II) cations, respectively.

Journal Article↗
PubMed2006

Synthesis of fullerodendrons with an ammonium unit at the focal point and their cooperative self-assembly on a fluorescent ditopic crown ether receptor.

Dendritic branches with 1, 2, or 4 peripheral fullerene subunits and an ammonium function at the focal point have been prepared. Their ability to form self-assembled dendritic structures with oligophenylenevinylene receptors bearing one or two crown ether moieties has been evidenced by ES-MS studies for the first time. These supramolecular complexes are multicomponent photoactive devices in which the emission of the central receptor is dramatically quenched by the fullerene units. This new property resulting from the association of the different molecular subunits allowed detailed investigations of the self-assembly process by means of fluorescence titrations. The binding studies have revealed positive cooperative effects for the assembly of the fullerodendrimers with the ditopic receptor. Interestingly, the stability of the supramolecular 2:1 structures increases as the size of the dendritic unit increases. This positive dendritic effect has been explained by the larger number of possible intramolecular fullerene-fullerene interactions between the two dendritic guests when the number of fullerene subunits is increased.

Crown Ethers↗
PubMed2006

Characterization of hemoglobin Würzburg (alpha2beta2 4(A1)Thr-->Asn), a new electrophoretically silent variant, by mass spectrometry and molecular modeling studies.

The first hemoglobin (Hb) variant carrying a mutation at beta4 was identified as beta4(A1)Thr-->Asn or Hb Würzburg and constituted 38% of the total hemoglobin. It showed a slightly elevated oxygen affinity and a slightly decreased cooperativity index (n50 = 2.3 versus n50 = 2.8). The analysis of the electrostatic potential showed an increased negative charge at the site of the mutation with a displacement of beta6(A3)Glu by 1.3A. The replacement of threonine by asparagine seems to stabilize the R conformation. This may explain partially both the high affinity and the reduction in cooperativity.

Amino Acid Substitution↗
PubMed2006

Proteomic analysis of brain tissue from an Alzheimer's disease mouse model by two-dimensional difference gel electrophoresis.

We used a beta-amyloid precursor protein (APP) transgenic (Tg) mouse model that displays some of the typical Alzheimer-associated pathological features to study the brain proteoma associated with amyloid plaque deposition. Two groups (male and female) of 14-month-old Tg mice were compared with their wild type littermates. We used differential 2D electrophoresis coupled with mass spectrometry to generate one of the first complete image of changes in brain protein expression occurring in this well-recognized model of Alzheimer's disease (AD). We identified 15 different proteins, which are significantly regulated in this pathology (p<0.05, > or =1.5-fold variation in expression comparing with the wild type samples). These comprise a number of proteins that were already known to be implicated in AD and neurodegeneration, as well as several proteins which relationship with AD had not been shown before. Identified proteins were grouped according to their biological key pathways. Results obtained are discussed in view of existing bibliographic data on human AD transcriptoma and proteoma.

Alzheimer Disease↗
PubMed2006

Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology.

In the horticulturally important ornamental species Cyclamen persicum Mill., somatic embryogenesis is an efficient vegetative propagation method and the development of artificial seeds is an ultimate aim. This study aims at a systematic comparison of the proteomes of zygotic embryos, somatic embryos grown in liquid medium containing 30 or 60 g l(-1) sucrose, germinating embryos of both types and endosperm in order to obtain novel insights into seed and germination physiology. Using high resolution two-dimensional isoelectric focussing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D IEF/SDS PAGE), 74% of the proteins expressed in zygotic embryos were found in similar abundance in somatic embryos grown in 60 g l(-1) sucrose. Somatic embryos grown in 30 g l(-1) sucrose accumulated fewer protein species than those grown in 60 g l(-1). Selected proteins were identified following mass spectrometry (nano-LC-MS/MS). Four enzymes involved in glycolysis (UDP-glucose pyrophosphorylase, fructose bisphosphate aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase GAPDH) were specifically induced in somatic embryos. 11S globulin proteins identified by MS were present in high levels in somatic embryos, zygotic embryos and endosperm, whereas 7S globulins were detected mainly in endosperm and zygotic embryos. These are the first storage proteins identified in C. persicum. Xyloglucans are known to be another group of seed storage compounds in C. persicum. Interestingly, xyloglucan endotransglycosylases were found to be highly expressed in endosperm tissue. We discuss the physiological implications of these observations.

Culture Media↗
PubMed2006

Identification of morphine-6-glucuronide in chromaffin cell secretory granules.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Alkaloids↗
PubMed2006

ICDS database: interrupted CoDing sequences in prokaryotic genomes.

Unrecognized frameshifts, in-frame stop codons and sequencing errors lead to Interrupted CoDing Sequence (ICDS) that can seriously affect all subsequent steps of functional characterization, from in silico analysis to high-throughput proteomic projects. Here, we describe the Interrupted CoDing Sequence database containing ICDS detected by a similarity-based approach in 80 complete prokaryotic genomes. ICDS can be retrieved by species browsing or similarity searches via a web interface (http://www-bio3d-igbmc.u-strasbg.fr/ICDS/). The definition of each interrupted gene is provided as well as the ICDS genomic localization with the surrounding sequence. Furthermore, to facilitate the experimental characterization of ICDS, we propose optimized primers for re-sequencing purposes. The database will be regularly updated with additional data from ongoing sequenced genomes. Our strategy has been validated by three independent tests: (i) ICDS prediction on a benchmark of artificially created frameshifts, (ii) comparison of predicted ICDS and results obtained from the comparison of the two genomic sequences of Bacillus licheniformis strain ATCC 14580 and (iii) re-sequencing of 25 predicted ICDS of the recently sequenced genome of Mycobacterium smegmatis. This allows us to estimate the specificity and sensitivity (95 and 82%, respectively) of our program and the efficiency of primer determination.

Bacillus↗
PubMed2006

Proteome analysis of Schizosaccharomyces pombe by two-dimensional gel electrophoresis and mass spectrometry.

The fission yeast Schizosaccharomyces pombe (S. pombe) is a unicellular eukaryote and contains many genes and regulatory mechanisms that are close to those of mammals. In this study, we performed a global proteomic analysis of the fission yeast S. pombe wild type h(-S) L 972 proteome. More than 1,500 protein spots were visualized on silver stained 2-D gels in the 3-10 pI range with a high resolution and high reproducibility. Protein identification was carried out by MALDI-TOF-MS and/or nanoLC-MS/MS. Advantage of the complementarity of these two MS approaches was used to enhance the identification quality. So far, 364 proteins (representing 157 different proteins) have been identified. We report here the identification of 117 new proteins on our 2-D reference map of this yeast compared to the first reference map. Of these identified proteins, 40.1% were involved in metabolism. The present work provides a very useful tool for all studies relying on S. pombe as a model organism and is a considerable complement to the first reference map of S. pombe published recently by Sun and coworkers (Sun, N., Jang, J., Lee, S., Kim, S. et al.., Proteomics 2005, 5, 1574-1579).

Amino Acid Sequence↗
PubMed2006

Discovery and identification of potential biomarkers in a prospective study of chronic lymphoid malignancies using SELDI-TOF-MS.

The accurate diagnosis of the different forms of chronic mature B-cell lymphocytic malignancies is of primary importance to determine an appropriate and efficient treatment. Usually, the diagnosis is achieved by morphology and immunophenotyping. Nevertheless, the diagnostic tools available are not able to discriminate pathologies with variable evolution, or to classify some of them. To discover new biomarkers, we used peptide and protein profiling SELDI-TOF-MS, to analyze 39 chronic B-cell malignancies and 20 control serum samples. Markers of interest were subsequently identified and characterized. In the obtained SELDI-MS profiles, most of the differences were observed in three mass ranges (m/z = 13 000; m/z = 9000; m/z < 2000). Identification of these biomarkers was achieved either by direct enrichment on the ProteinChip arrays followed by on-chip-MS/MS or by chromatographic fractionation, 1D-gel followed by nanoLC-MS/MS analysis. An increase of a sulfite form of transthyretin (13,841 Da) was observed in the patient group. A second set of markers at 8.6 and 8.9 kDa was identified as complement related fragment proteins, the C3a and C4a anaphylatoxins. In the low mass range, several peptides originating from N-terminal and C-terminal processing of the C3 alpha and C4 alpha chains were specifically observed in 38% of the patient sera, but in none of the control sera. This study emphasizes the usefulness of mass spectrometry studies in such malignancies.

Amino Acid Sequence↗
PubMed2006

Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment.

Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.

Bryopsida↗
PubMed2006

Global comparison of the membrane subproteomes between a multidrug-resistant Acinetobacter baumannii strain and a reference strain.

Acinetobacter baumannii causes severe infections in compromised patients. We combined SDS-PAGE, two-dimensional gel electrophoresis and mass spectrometry (LC-MS/MS and MALDI-TOF) to separate and characterize the proteins of the cell envelope of this bacterium. In total, 135 proteins (inner and outer membrane proteins) were identified. In this analysis, we described the expression by this bacterium of RND-type efflux systems and some potential virulence factors. We then compared the membrane subproteome of a clinical multidrug-resistant (MDR) isolate with that of a reference strain. We found that the MDR strain expressed lower levels of the penicillin-binding-protein 1b, produced a CarO protein having different primary and quaternary structures to that of the reference strain, and expressed OmpW isoforms. We also showed that the clinical strain has a high ability to form biofilms consistent with the accumulation of some outer membrane proteins (OMPs) such as NlpE or CsuD that have already been described as involved in bacterial adhesion. These features may partly explain the MDR emergence of the clinical isolate.

Acinetobacter baumannii↗
PubMed2006

Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry.

In the human body the mineralocorticoid aldosterone is responsible for maintaining water and electrolyte homeostasis and therefore controlling blood pressure. In addition, aldosterone has recently been associated with severe heart failure. Besides receptor-dependent action, the damaging effects of aldosterone may also be partly mediated through non-genomic mechanisms. The present study focuses on the mineralocorticoid receptor-independent action of aldosterone at the protein level. We chose the fission yeast Schizosaccharomyces pombe as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Using 2D-electrophoresis we identified for the first time protein spots affected by aldosterone in a nuclear receptor-free system. Mass spectrometry analysis using MALDI-TOF MS and nanoLC-MS/MS approaches allowed the unambiguous identification of 11 proteins that showed increased or decreased levels, which may represent newly identified players and pathways of aldosterone-induced action. Two proteins with a connection to osmotic regulation (NAD-dependent malic enzyme and glycerol-3-phosphate-dehydrogenase), as well as two proteins involved in the overall organization of the cytoskeleton, vip1 and glyceraldehyde-3-phosphate dehydrogenase, which was also found to be specifically affected by aldosterone in human HCT116 cells, are discussed.

Aldosterone↗
PubMed2005

Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome.

The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.

Amino Acid Sequence↗