Biomedical subjects

Valérie Labas

Publications and source records attributed to Valérie Labas.

At least 19 recordsLinked to original sources

PubMed2006

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins.

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Acute-Phase Proteins↗

PubMed2006

PDMS-based microfluidics for proteomic analysis.

A microfluidic poly(dimethylsiloxane) (PDMS) microdevice was realized, combining on-line protein electrophoretic separation, selection, and digestion of a protein of interest for identification by mass spectrometry. The system includes eight integrated valves and one micropump dedicated to control the flow operations. Myoglobin was successfully isolated from bovine serum albumin (BSA), then selected using integrated valves and digested in a rotary micromixer. Proteolytic peptides were recovered from the micromixer for protein identification. Total analysis from sample injection to protein identification is performed under 30 minutes, with samples of tens of nanolitres. The paper shows that PDMS technology can be successfully used for integrating complex preparation protocols of proteic samples prior to MS analysis.

Journal Article↗

PubMed2006

Polycalin (chlorophyllid A binding protein): a novel, very large fluorescent lipocalin from the midgut of the domestic silkworm Bombyx mori L.

We studied a protein from the midgut of the silkworm Bombyx mori characterized by its ability to bind the prosthetic group of chlorophyll, that confers fluorescent properties to this protein. Several techniques, 2D electrophoresis purification, MS-MS and Maldi-TOF peptide sequencing, RT-PCR and nucleotide sequencing were used to obtain the nucleotide sequence and the deduced amino acid sequence. The coding sequence was compared to the gene sequence to define the number and size of introns and exons. The gene spanned 45.5 kb of DNA and consisted of 46 exons. The cDNA encoded a protein of 2721 amino acids. The protein was identified as a lipocalin with novel features. Most lipocalins are proteins with high affinity to small lipophilic molecules, with a molecular size in the 25 kDa range and a well conserved tertiary structure. The apoprotein described here revealed 15 lipocalin like structures, in line. We called this protein a polycalin (pentadecacalin).

Amino Acid Sequence↗

PubMed2006

Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes.

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [(32)P]-orthophosphate, one-unit pH 2-DE, combined with MS. Highly reproducible 2-D profiles of phosphoproteins were obtained with early stationary-phase cells. The 2-D profiles contained at least 80 clearly labelled spots in the pH range 4-7. Forty-six spots were analysed by MS (confirmed in most cases by LC-MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary-phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.

Bacillus subtilis↗

PubMed2006

An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteins.

Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma (EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor (PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control.

Cell Line, Transformed↗

PubMed2005

3-phenylpropionate catabolism and the Escherichia coli oxidative stress response.

Cells have devised a variety of protection systems against the toxic effects of dioxygen. Dioxygenases are part of this defence mechanism. In Escherichia coli, the positive regulator HcaR, a member of the LysR family of regulators, controls expression of the neighbouring genes, hcaA1, hcaA2, hcaC, hcaB and hcaD, coding for the 3-phenylpropionate dioxygenase complex and 3-phenylpropionate-2',3'-dihydrodiol dehydrogenase, that oxidizes 3-phenylpropionate to 3-(2,3-dihydroxyphenyl) propionate. Differences between expression of hcaR and expression of its target, hcaA, suggest that HcaR is involved in control of other cellular processes or that other regulatory proteins modulate hcaA expression. Protein expression profiling was used to identify other HcaR targets. Two-dimensional gel electrophoresis was used to compare the proteomes of wild-type E. coli and strains in which hcaR was disrupted. Several polypeptides whose production was up- or downregulated in the hcaR mutant were involved in the oxidative stress response. Subsequent experiments demonstrated that hcaR disruption was involved in regulation of genes involved in the oxidative stress response. Modification of the stress response also occurred in an hcaA1A2CD mutant strain. Using gel retardation, the HcaR binding site was estimated to be located about -70 to -55 bp upstream of the hcaA transcription start site. The expression of hcaR was repressed in the absence of oxygen by the ArcA/ArcB two-component system.

Amino Acid Sequence↗

PubMed2005

Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

Apoptosis↗

PubMed2004

Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism.

We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).

Adenylyl Imidodiphosphate↗

PubMed2004

Tetraspanins connect several types of Ig proteins: IgM is a novel component of the tetraspanin web on B-lymphoid cells.

The tetraspanins form a family of about 30 molecules mainly expressed on the cell surface. They have been reported to be involved in many physiological or pathological processes, such as fertilization, immune response, development of the nervous system, and metastasis, as well as in infectious diseases (HCV, malaria, etc.). The tetraspanins may play a role as "organizers" of multimolecular complexes on the cell surface associating numerous proteins, the "tetraspanin web." To better define the composition of the tetraspanin web, its characterization has been recently performed using mass spectrometry and proteomics. We report the proteomic analysis of tetraspanin complexes on B-lymphoid cells. Immunoprecipitation experiments were performed using mAbs directed against the tetraspanin CD9, and associated molecules were identified by MALDI-TOF (matrix assisted laser desorption ionization time of flight) mass spectrometry. This led to the identification of IgM as a novel component of the complexes. Thus, tetraspanins may connect several types of proteins with Ig domains, including HLA-DR, EWI-2, and IgM, that may play a role in immune responses.

Antigen Presentation↗

PubMed2004

Use of deoxyribose by intestinal and extraintestinal pathogenic Escherichia coli strains: a metabolic adaptation involved in competitiveness.

We showed that the deoK operon, which confers the ability to use deoxyribose as a carbon source, is more common among pathogenic than commensal Escherichia coli strains. The expression of the deoK operon increases the competitiveness of clinical isolates, suggesting that this biochemical characteristic plays a role in host infectivity.

Conserved Sequence↗

PubMed2003

EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.

Animals↗

PubMed2003

Proteomic analysis of a lymphoma-derived cell line (DG75) following treatment with a demethylating drug: modification of membrane-associated proteins.

5'-azacytidine (AZC) is a potent DNA demethylating agent used clinically for treatment of patients with malignant hemopathies. We have previously shown that AZC induces a halt in cell growth and a decrease of cell activity, without affecting cell viability. We have also shown using proteomics, that 35 polypeptides were differentially expressed in a cytoplasmic fraction. The aim of this study was to provide a more complete picture of modifications in AZC-treated cells using cell membrane preparations. Therefore the protein pattern changes following AZC treatment of the cell line DG75 were studied on a detergent-solubilized fraction obtained from these membranes. Results showed that 49 proteins were differentially expressed in the membrane fraction. Seven polypeptides were down-regulated, while 42 were up-regulated. The identity of most of these differentially expressed proteins was determined by mass spectrometry (liquid chromatography-tandem mass spectrometry or matrix-assisted laser desorption/ionization-time of flight), and the identified proteins were grouped based on cellular function and participation in biochemical and signaling pathways.

Antimetabolites, Antineoplastic↗

PubMed2003

Proteomic study of human umbilical vein endothelial cells in culture.

The endothelium is a single layer of cells lining the inside face of all blood vessels. It constitutes a major metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological processes such as coagulation, hemostasis, inflammation, atherosclerosis, angiogenesis and cancerous metastasis dissemination. In order to increase our knowledge about the protein content and the main biological pathways of human vascular endothelial cells, we have undertaken the proteomic analysis of the most explored present endothelial cell model, i.e. primocultures of human umbilical vein endothelial cells (HUVECs). Using low levels of protein loads (~ 30 nug), the association of two-dimensional electrophoresis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, liquid chromatography-tandem mass spectrometry and database interrogations allowed us to identify 53 proteins of suspected endothelial origin in quiescent HUVECs. Beside cytoskeletal proteins such as actin, tubulin, tropomyosin and vimentin, we identified various proteins more especially implicated in cellular motility and plasticity (e.g. cofilin, F-actin capping protein and prefoldin), in regulation of apoptosis and senescence (protease inhibitor 9, glucose related proteins, heat shock proteins, thioredoxin peroxidase, nucleophosmin) as well as other proteins implicated in coagulation (annexin V, high mobility group protein), antigen presentation (valosin containing protein and ubiquitin carboxyl terminal hydrolase isozyme L1) and enzymatic capabilities (glutathione-S-transferase, protein disulfide isomerases, lactate deshydrogenase). The presented annotated 2-D maps of HUVECs will be soon available on the web at http://www. huvec.com.

Cells, Cultured↗

PubMed2003

Drug induced proteome changes in Candida albicans: comparison of the effect of beta(1,3) glucan synthase inhibitors and two triazoles, fluconazole and itraconazole.

The dimorphic fungus Candida albicans is an opportunistic human pathogen. Candidiasis is usually treated with azole antifungal agents. However clinical treatments may fail due to the appearance of resistance to this class of antifungal agents in Candida. Echinocandin derivatives are an alternative for the treatment of these fungal infections and are active against azole resistant isolates of C. albicans. Azoles inhibit the lanosterol 14 alpha demethylase which is a key enzyme in the synthesis of ergosterol. In contrast, the echinocandin class of antibiotics inhibit noncompetitively beta-(1,3)-D-glucan synthesis in vitro. We have investigated the impact of mulundocandin on the proteome of C. albicans and compared it to those of a mulundocandin derivative, as well as to two azoles of different structure, fluconazole and itraconazole. The changes in gene expression underlying the antifungal responses were analyzed by comparative 2-D PAGE. Dose dependant responses were kinetically studied on C. albicans grown at 25 degrees C (yeast form) in synthetic dextrose medium. This study shows that antifungals with a common mechanism of action lead to comparable effects at the proteome level and that a proteomics approach can be used to distinguish different antifungals, with the promise to become a useful tool to study drugs of unknown mechanism of action.

Antifungal Agents↗

PubMed2003

MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner.

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclin-dependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system.

Amino Acid Sequence↗

PubMed2003

Identification of rat targets of anti-soluble liver antigen autoantibodies by serologic proteome analysis.

BACKGROUND: Anti-soluble liver antigen (SLA) autoantibodies are specific for autoimmune hepatitis type 1 and are the only immunologic marker found in 15-20% of hepatitis cases previously considered cryptogenic. Anti-SLA antibodies react with the 100 000g supernatant from rat liver homogenate, but the molecular targets remain controversial. METHODS: We characterized anti-SLA targets by one- and two-dimensional immunoblotting analysis. The recognized proteins were identified by peptide mass fingerprint analysis after matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. RESULTS: Three proteins of 35 kDa and pI 6.0, 50 kDa and pI between 6.0 and 6.5, and 58 kDa and pI between 6.5 and 7.0 were stained more intensely by anti-SLA positive-sera than by control sera. After in-gel tryptic digestion, MALDI-TOF analysis of the generated peptides enabled the clear identification of N-hydroxyarylamine sulfotransferase, isoforms of alpha-enolase, and isoforms of catalase. CONCLUSIONS: Possible antigens for anti-SLA antibodies include a sulfotransferase, alpha-enolase(s), and catalase(s). Two-dimensional electrophoresis combined with mass spectrometry offers a versatile tool to identify molecular targets of autoantibodies and thus to improve diagnostic tools and the understanding of the immune process.

Animals↗

PubMed2002

Novel target sequences for Pax-6 in the brain-specific activating regions of the rat aldolase C gene.

Upstream activating sequences of the rat aldolase C gene are shown here to confer brain-specific expression in transgenic mice. In addition to binding sites described previously for the brain-expressed POU proteins Brn-1 and Brn-2 (Skala, H., Porteu, A., Thomas, M., Szajnert, M. F., Okazawa, H., Kahn, A., and Phan-Dinh-Tuy, F. (1998) J. Biol. Chem. 273, 31806-31814), we have identified two novel DNA elements critical for an interaction with a brain-specific, high affinity DNA-binding protein. Characterization of this binding protein showed it to be sensitive to thiol oxidation and stable to heat at 100 degrees C. This protein was purified on the basis of its thermostability and its selective adsorption to streptavidin magnetic particles via a biotinylated multimer of its target DNA binding site. Liquid chromatography coupled to tandem mass spectrometry analysis, binding competition with consensus oligonucleotides, and antibody supershift assays led to its identification as the homeodomain paired protein Pax-6. This result suggests that the brain-specific aldolase C gene could constitute a new target for the transcription factor Pax-6, which is implicated increasingly in neurogenesis.

Amino Acid Motifs↗

PubMed2002

Effect of mild acid pH on the functioning of bacterial membranes in Vibrio cholerae.

In this paper, we initiated the first two-dimensional electrophoresis map of Vibrio cholerae, the aetiological agent of cholera disease. In this pathogen the efficient adaptation to detrimental conditions plays an important role in its survival in both the aquatic reservoir and human intestine. By proteome analysis we investigated the effect of mild acid treatment on the physiology of V. cholerae. More than 50 proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and database searching. Amongst them, pH regulated proteins belong to various functional classes such as intermediary metabolism and bacterial envelope. Several proteins whose accumulation level was decreased in response to acidic pH are known to be involved in the organization and the functioning of membranes, including lipopolysaccharide. Consistent with this, we observed an increased susceptibility to hydrophobic drugs, a loss of motility and a reduction in the ability to form a biofilm in cells grown at pH 6. Our results suggest that V. cholerae is able to sense a moderate decrease in pH and to modify accordingly its structure and physiology.

Bacterial Proteins↗