Strategy of macromolecular grafting onto a gold substrate dedicated to protein-protein interaction measurements.

International audienceMany biotechnology applications use proteins immobilized on surface. For biosensor, the sensing layer is a key component interfacing the transducer and the sample. Strategies employed to activate the bidimensional surface act directly on the performance of the biosensor. In this paper we propose a novel strategy for engineered proteins self-assembly. Our original supramolecular structure allows a direct and fast covalent attachment of proteins onto bare gold substrate through a homobifunctional cross-linker, 1,4-di-([2'-pyridyldithio]propionamido)butane (DPDPB). In this work, engineered proteins and linker-protein complexes were synthesized and characterized by gel electrophoresis, chromatography and spectroscopy experiments. Macromolecular construction "DPDPB-GST tag-GEC1 protein" was conceived in order to guarantee a 2D architecture enhancing the capabilities of the target (tubulin) to recognize its partner (GEC1). Surface plasmon resonance measurements clearly showed potential of this particular self-assembled protein layer compared to a commercial immunosensor interface. At the concentrations tested, the recognition process occurs between tubulin and the immobilized GEC1; moreover enhanced binding was obtained with the home-made 2D sensing layer more than with 3D carboxymethyl dextran matrix

Estrogènes et prolifération : identification du gène SOx-3 codant une sulfhydryl-oxydase et étude de son expression dans des cellules endométriales

La prolifération cellulaire est un processus complexe qui requiert des mécanismes de contrôle spécifiques positifs et négatifs, impliquant notamment les hormones stéroïdes et en particulier les estrogènes. Au laboratoire, un nouveau gène gec-3/S0x-3 potentiellement régulé négativement par les estrogènes a été identifié par hybridation différentielle dans des cellules épithéliales glandulaires d'endomètre de Cobaye (CEGE). Cependant la séquence 5' de l'ADNc gec-3/S0x-3, ne comportant pas l'ATG d'initiation de la traduction, était incomplète. L'amplification et le clonage de la séquence codante complète de l'ADNc gec-3/S0x-3 a permis de confirmer la séquence 5' et de mettre en évidence le codon A TG. Cet ADNc présente de fortes homologies avec le gène humain quiescin 06 et le gène sulfhydryl oxydase 2 de rat, et nous l'avons renommé SOx-3. L'expression du messager SOx-3 est induite par la déplétion sérique dans les cellules épithéliales glandulaires et stromales d'endomètre de Cobaye normales en culture, puis décroît lors d'une stimulation par le sérum. Ces effets ne sont pas observés dans des CEGE immortalisées par l'antigène Grand Tsv4o, ni dans des cellules d'adénocarcinome du sein humain (MCF-7). Des protéines SOx-3 recombinantes, puis des anticorps ont été produits et nous avons montré que la protéine SOx-3 est fortement exprimée dans les CEGE, mais pas dans les cellules stromales. De plus l'expression de la protéine est plus importante au 10ème qu'au 1ier jour du cycle estrien, suggérant une régulation par les estrogènes et/ou la progestérone. De plus, la surexpression de la protéine SOx-3 dans des cellules MCF-7 a un effet inhibiteur sur la prolifération de ces cellules, résultant probablement d'une modification de l'adhérence. La protéine SOx-3 possède une activité sulfhydryl-oxydasique et est sécrétée. Ainsi, elle pourrait être impliquée dans la modification de l'état d'oxydo-réduction des protéines de la matrice extracellulaire et/ou des protéines de la membrane plasmique et par conséquent dans les interactions cellules/cellules et/ou cellules/matrice extracellulaire.BESANCON-BU Sciences Staps (250562103) / SudocSudocFranceF

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC50 of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.This work was supported by the Ministe©re del'Education Nationale, de la Recherche et de la TechnologiePeer reviewe

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines

AbstractPokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC50 of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID50 of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect

Involvement of sulfhydryl oxidase QSOX1 in the protection of cells against oxidative stress-induced apoptosis.

International audienceThe QSOX1 protein, belonging to a new class of FAD-linked Quiescin/Sulfhydryl oxidase, catalyzes disulfide bond formation. To give new insight into the biological function of QSOX1, we studied its involvement in oxidative stress-induced apoptosis and cell recovery of PC12 cells. By real time RT-PCR and flow cytometric analysis, we show that the QSOX1 mRNA and protein levels increased late after the beginning of oxidative treatment and were sustained for 72 h. These levels were still high when the PC12 cells were not dying but had resumed proliferation. The kinetics of QSOX1 expression suggest a more protective effect of QSOX1 rather than an involvement of this protein in apoptosis. Human breast cancer MCF-7 cell lines overexpressing the guinea pig QSOX1 protein submitted to the same treatments appeared less sensitive to cell death than the MCF-7 control cells. The protective effect is partly due to a preservation of the mitochondrial polarization generally lost after an oxidative stress. These results strengthen our hypothesis of a protective role of QSOX1 against apoptosis

A Bregman-proximal point algorithm for robust non-negative matrix factorization with possible missing values and outliers - application to gene expression analysis

BackgroundNon-Negative Matrix factorization has become an essential tool for feature extraction in a wide spectrum of applications. In the present work, our objective is to extend the applicability of the method to the case of missing and/or corrupted data due to outliers.ResultsAn essential property for missing data imputation and detection of outliers is that the uncorrupted data matrix is low rank, i.e. has only a small number of degrees of freedom. We devise a new version of the Bregman proximal idea which preserves nonnegativity and mix it with the Augmented Lagrangian approach for simultaneous reconstruction of the features of interest and detection of the outliers using a sparsity promoting ℓ 1 penality.ConclusionsAn application to the analysis of gene expression data of patients with bladder cancer is finally proposed.KeywordsFeature extraction Non-negative matrix factorization Gene expression analysis Outliers and missing dat