A partial metabolic pathway enables group b streptococcus to overcome quinone deficiency in a host bacterial community.

Aerobic respiration metabolism in Group B Streptococcus (GBS) is activated by exogenous heme and menaquinone. This capacity enhances resistance of GBS to acid and oxidative stress and improves its survival. In this work, we discovered that GBS is able to respire in the presence of heme and 1,4-dihydroxy-2-naphthoic acid (DHNA). DHNA is a biosynthetic precursor of demethylmenaquinone (DMK) in many bacterial species. A GBS gene (gbs1789) encodes a homolog of the MenA 1,4-dihydroxy-2-naphthoate prenyltransferase enzyme, involved in the synthesis of demethylmenaquinone. In this study, we showed that gbs1789 is involved in the biosynthesis of long-chain demethylmenaquinones (DMK-10). The Δgbs1789 mutant cannot respire in the presence of heme and DHNA, indicating that endogenously synthesized DMKs are cofactors of the GBS respiratory chain. We also found that isoprenoid side chains from GBS DMKs are produced by the protein encoded by the gbs1783 gene, since this gene can complement an Escherichia coli ispB mutant defective for isoprenoids chain synthesis. In the gut or vaginal microbiote, where interspecies metabolite exchanges occur, this partial DMK biosynthetic pathway can be important for GBS respiration and survival in different niches.

Dansyl-peptides matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI-MS/MS analysis of the proteome.

Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.

Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha.

A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile covalent attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The covalent attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the covalent attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).

The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling.

Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes.

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.

Synaptic multiprotein complexes associated with 5-HT(2C) receptors: a proteomic approach.

Membrane-bound receptors such as tyrosine kinases and ionotropic receptors are associated with large protein networks structured by protein-protein interactions involving multidomain proteins. Although these networks have emerged as a general mechanism of cellular signalling, much less is known about the protein complexes associated with G-protein-coupled receptors (GPCRs). Using a proteomic approach based on peptide affinity chromatography followed by mass spectrometry and immunoblotting, we have identified 15 proteins that interact with the C- terminal tail of the 5-hydroxytryptamine 2C (5-HT(2C)) receptor, a GPCR. These proteins include several synaptic multidomain proteins containing one or several PDZ domains (PSD95 and the proteins of the tripartite complex Veli3-CASK-Mint1), proteins of the actin/spectrin cytoskeleton and signalling proteins. Coimmunoprecipitation experiments showed that 5-HT(2C) receptors interact with PSD95 and the Veli3-CASK-Mint1 complex in vivo. Electron microscopy also indicated a synaptic enrichment of Veli3 and 5-HT(2C) receptors and their colocalization in microvilli of choroidal cells. These results indicate that the 5-HT(2C) receptor is associated with protein networks that are important for its synaptic localization and its coupling to the signalling machinery.