Biomedical subjects

Emmanuelle Leize-Wagner

Publications and source records attributed to Emmanuelle Leize-Wagner.

15 recordsLinked to original sources

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

Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view.

The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e., devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e., including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria.

DNA, Mitochondrial↗

PubMed2006

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining.

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.

Ammonia↗

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↗

PubMed2005

Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall.

The exoproteome of the fungus Fusarium graminearum grown on glucose and on hop (Humulus lupulus, L.) cell wall has been investigated. The culture medium was found to contain a higher quantity of proteins and the proteins are more diverse when the fungus is grown on cell wall. Using both 1D and 2D electrophoresis followed by mass spectrometry analysis and protein identification based on similarity searches, 84 unique proteins were identified in the cell wall-grown fungal exoproteome. Many are putatively implicated in carbohydrate metabolism, mainly in cell wall polysaccharide degradation. The predicted carbohydrate-active enzymes fell into 24 different enzymes classes, and up to eight different proteins within a same class are secreted. This indicates that fungal metabolism becomes oriented towards synthesis and secretion of a whole arsenal of enzymes able to digest almost the complete plant cell wall. Cellobiohydrolase is one of the only four proteins found both after growth on glucose and on plant cell wall and we propose that this enzyme could act as a sensor of the extracellular environment. Extensive knowledge of this very diverse F. graminearum exoproteome is an important step towards the full understanding of Fusarium/plants interactions.

Cell Wall↗

PubMed2005

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases.

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K (m) (I-) = 2.5 mM; k (cat) (I-) = 462 s(-1)) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K (m) (I-) =18.1 mM; k (cat) (I-) = 38 s(-1)). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.

Amino Acid Sequence↗

PubMed2005

Proteomic analysis identifies novel proteins of the Maurer's clefts, a secretory compartment delivering Plasmodium falciparum proteins to the surface of its host cell.

A novel method was validated for the efficient distinction between malaria parasite-derived and host cell proteins in mass spectrometry analyses. This method was applied to a ghost fraction from Plasmodium falciparum-infected erythrocytes containing the red blood cell plasma membrane, the erythrocyte submembrane skeleton, and the Maurer's clefts, a Golgi-like apparatus linked to and addressing parasite proteins to the host cell surface. This method allowed the identification of 78 parasite proteins. Among these we identified seven novel proteins of the Maurer's clefts based on immunofluorescence studies and proteinase K digestion assays. The products of six contiguous genes located on chromosome 5 were identified, and the location within the Maurer's clefts was established for two of them. This suggests a clustering of genes encoding Maurer's cleft proteins. Our study sheds new light on the biological function of the Maurer's clefts, which are central to the pathogenesis and to the intraerythrocytic development of P. falciparum.

Animals↗

PubMed2005

Oxidative stress response: a proteomic view.

The oxidative stress response is characterized by various effects on a range of biologic molecules. When examined at the protein level, both expression levels and protein modifications are altered by oxidative stress. While these effects have been studied in the past by classic biochemical methods, the recent onset of proteomics methods has allowed the oxidative stress response to be studied on a much wider scale. The input of proteomics in the study of oxidative stress response and in the evidence of an oxidative stress component in biologic phenomena is reviewed in this paper.

Animals↗

PubMed2004

Carcinogenic properties of proteins with pro-inflammatory activity from Streptococcus infantarius (formerly S.bovis).

Several studies reported linkage between bacterial infections and carcinogenesis. Streptococcus bovis was traditionally considered as a lower grade pathogen frequently involved in bacteremia and endocarditis. This bacterium became important in human health as it was shown that 25-80% of patients who presented a S.bovis bacteremia had also a colorectal tumor. Moreover, in previous experiments, we demonstrated that S.bovis or S.bovis wall extracted antigens (WEA) were able to promote carcinogenesis in rats. The aim of the present study was: (i) to identify the S.bovis proteins responsible for in vitro pro-inflammatory properties; (ii) to purify them; (iii) to examine their ability to stimulate in vitro IL-8 and COX-2 expression by human colon cancer cells; and (iv) to assess in vivo their pro-carcinogenic potential in a rat model of colon carcinogenesis. The purified S300 fraction, as determined by proteomic analysis, contained 72 protein spots in two-dimensional gel electrophoresis representing 12 different proteins able to trigger human epithelial colonic Caco-2 cells and rat colonic mucosa to release CXC chemokines (human IL-8 or rat CINC/GRO) and prostaglandins E2, correlated with an in vitro over-expression of COX-2. Moreover, these proteins were highly effective in the promotion of pre-neoplastic lesions in azoxymethane-treated rats. In the presence of these proteins, Caco-2 cells exhibited enhanced phosphorylation of the three classes of MAP kinases. Our results show a relationship between the pro-inflammatory potential of S.bovis proteins and their pro-carcinogenic properties, confirming the linkage between inflammation and colon carcinogenesis. These data support the hypothesis that colonic bacteria can contribute to cancer development particularly in chronic infection/inflammation diseases where bacterial components may interfere with cell function.

Animals↗

PubMed2004

About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis.

The influence of thiol blocking on the resolution of basic proteins by two-dimensional electrophoresis was investigated. Cysteine blocking greatly increased resolution and decreased streaking, especially in the basic region of the gels. Two strategies for cysteine blocking were found to be efficient: classical alkylation with maleimide derivatives and mixed disulfide exchange with an excess of a low molecular weight disulfide. The effect on resolution was significant enough to allow correct resolution of basic proteins with in-gel rehydration on wide gradients (e.g. 3-10 and 4-12), but anodic cup-loading was still required for basic gradients (e.g. 6-12 or 8-12). These results demonstrate that thiol-related problems are not solely responsible for streaking of basic proteins on two-dimensional gels.

Alkylation↗

PubMed2004

About the mechanism of interference of silver staining with peptide mass spectrometry.

The mechanism by which silver staining of proteins in polyacrylamide gels interferes with mass spectrometry of peptides produced by proteolysis has been investigated. It was demonstrated that this interference increases with time between silver staining and gel processing, although the silver image is constant. This suggested an important role of the formaldehyde used in silver staining development in this interference process. Consequently, a formaldehyde-free staining protocol has been devised, using carbohydrazide as the developing agent. This protocol showed much increased peptide coverage and retained the sensitivity of silver staining. These results were however obtained at the expense of an increased background in the stained gels and of a reduced staining homogeneity.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Regeneration of peroxiredoxins during recovery after oxidative stress: only some overoxidized peroxiredoxins can be reduced during recovery after oxidative stress.

Peroxiredoxins (prx) are redox enzymes using an activated cysteine as their active site. This activated cysteine can be easily overoxidized to cysteine sulfinic acid or cysteine sulfonic acid, especially under oxidative stress conditions. The regeneration of peroxiredoxins after a short, intense oxidative stress was studied, using a proteomics approach. Important differences in regeneration speed were found, prx2 being the fastest regenerated protein, followed by prx1, whereas prx3 and prx6 were regenerated very slowly. Further study of the mechanism of this regeneration by pulse-chase experiments using stable isotope labeling and cycloheximide demonstrated that the fast-regenerating peroxiredoxins are regenerated at least in part by a retroreduction mechanism. This demonstrates that the overoxidation can be reversible under certain conditions. The pathway of this retroreduction and the reasons explaining the various regeneration speeds of the peroxiredoxins remain to be elucidated.

Binding Sites↗

PubMed2003

The brown algal kelp Laminaria digitata features distinct bromoperoxidase and iodoperoxidase activities.

Different haloperoxidases, one specific for the oxidation of iodide and another that can oxidize both iodide and bromide, were separated from the sporophytes of the brown alga Laminaria digitata and purified to electrophoretic homogeneity. The iodoperoxidase activity was approximately seven times more efficient than the bromoperoxidase fraction in the oxidation of iodide. The two enzymes were markedly different in their molecular masses, trypsin digestion profiles, and immunological characteristics. Also, in contrast to the iodoperoxidase, bromoperoxidases were present in the form of multimeric aggregates of near-identical proteins. Two full-length haloperoxidase cDNAs were isolated from L. digitata, using haloperoxidase partial cDNAs that had been identified previously in an Expressed Sequence Tag analysis of the life cycle of this species (1). Sequence comparisons, mass spectrometry, and immunological analyses of the purified bromoperoxidase, as well as the activity of the protein expressed in Escherichia coli, all indicate that these almost identical cDNAs encode bromoperoxidases. Haloperoxidases form a large multigenic family in L. digitata, and the potential functions of haloperoxidases in this kelp are discussed.

Amino Acid Sequence↗

PubMed2002

Properties and regulation of the bifunctional enzyme HPr kinase/phosphatase in Bacillus subtilis.

The bifunctional allosteric enzyme HPr kinase/phosphatase (HPrK/P) from Bacillus subtilis is a key enzyme in the main mechanism of carbon catabolite repression/activation (i.e. a means for the bacteria to adapt rapidly to environmental changes in carbon sources). In this regulation system, the enzyme can phosphorylate and dephosphorylate two proteins, HPr/HPr(Ser(P)) and Crh/Crh(Ser(P)), sensing the metabolic state of the cell. To acquire further insight into the properties of HPrK/P, electrospray ionization mass spectrometry, dynamic light scattering, and BIACORE were used to determine the oligomeric state of the protein under native conditions, revealing that the enzyme exists as a hexamer at pH 6.8 and as a monomer and dimer at pH 9.5. Using an in vitro radioactive assay, the influence of divalent cations, pH, temperature, and different glycolytic intermediates on the activity as well as kinetic parameters were investigated. The presence of divalent cations was found to be essential for both opposing activities of the enzyme. Furthermore, pH values equal to the internal pH of vegetative cells seem to favor the kinase activity, whereas lower pH values increased the phosphatase activity. Among the glycolytic intermediates evaluated, fructose 1,6-diphosphate and fructose 2,6-diphosphate were found to be allosteric activators in the kinase assay, whereas high concentrations inhibited the phosphatase activity, except for fructose 1,6-diphosphate in the case of HPr(Ser(P)). Phosphatase activity was induced by inorganic phosphate as well as acetyl phosphate and glyceraldehyde 3-phosphate. Kinetic parameters indicate a preference for binding of HPr compared with Crh to the enzyme and supported a strong positive cooperativity. This work suggests that the oligomeric state of the enzyme is influenced by several effectors and is correlated to the kinase or phosphatase activity. The phosphatase activity is mainly supported by the hexameric form.

Adenosine Triphosphate↗

PubMed2002

A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress.

Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.

Amino Acid Sequence↗