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

Interplay between ROS and autophagy in cancer cells, from tumor initiation to cancer therapy

Cancer formation is a complex and highly regulated multi-step process which is highly dependent of its environment, from the tissue to the patient. This complexity implies the development of specific treatments adapted to each type of tumor. The initial step of cancer formation requires the transformation of a healthy cell to a cancer cell, a process regulated by multiple intracellular and extracellular stimuli. The further steps, from the anarchic proliferation of cancer cells to form a primary tumor to the migration of cancer cells to distant organs to form metastasis, are also highly dependent of the tumor environment but of intracellular molecules and pathways as well. In this review, we will focus on the regulatory role of reactive oxygen species (ROS) and autophagy levels during the course of cancer development, from cellular transformation to the formation of metastasis. These data will allow us to discuss the potential of this molecule or pathway as putative future therapeutic targets

Nanobioengineering and Characterization of a Novel Estrogen Receptor Biosensor

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Specific or not specific recruitment of DNMTs for DNA methylation, an epigenetic dilemma

Abstract Our current view of DNA methylation processes is strongly moving: First, even if it was generally admitted that DNMT3A and DNMT3B are associated with de novo methylation and DNMT1 is associated with inheritance DNA methylation, these distinctions are now not so clear. Secondly, since one decade, many partners of DNMTs have been involved in both the regulation of DNA methylation activity and DNMT recruitment on DNA. The high diversity of interactions and the combination of these interactions let us to subclass the different DNMT-including complexes. For example, the DNMT3L/DNMT3A complex is mainly related to de novo DNA methylation in embryonic states, whereas the DNMT1/PCNA/UHRF1 complex is required for maintaining global DNA methylation following DNA replication. On the opposite to these unspecific DNA methylation machineries (no preferential DNA sequence), some recently identified DNMT-including complexes are recruited on specific DNA sequences. The coexistence of both types of DNA methylation (un/specific) suggests a close cooperation and an orchestration between these systems to maintain genome and epigenome integrities. Deregulation of these systems can lead to pathologic disorders

The ATG8 Family Proteins GABARAP and GABARAPL1 Target Antigen to Dendritic Cells to Prime CD4+ and CD8+ T Cells

Vaccine therapy is a promising method of research to promote T cell immune response and to develop novel antitumor immunotherapy protocols. Accumulating evidence has shown that autophagy is involved in antigen processing and presentation to T cells. In this work, we investigated the potential role of GABARAP and GABARAPL1, two members of the autophagic ATG8 family proteins, as surrogate tumor antigen delivery vectors to prime antitumor T cells. We showed that bone marrow-derived dendritic cells, expressing the antigen OVALBUMIN (OVA) fused with GABARAP or GABARAPL1, were able to prime OVA-specific CD4+ T cells in vitro. Interestingly, the fusion proteins were also degraded by the proteasome pathway and the resulting peptides were presented by the MHC class I system. We then asked if the aforementioned fusion proteins could improve tumor cell immunogenicity and T cell priming. The B16-F10 melanoma was chosen as the tumor cell line to express the fusion proteins. B16-F10 cells that expressed the OVA-ATG8 fused proteins stimulated OVA-specific CD8+ T cells, but demonstrated no CD4+ T cell response. In the future, these constructions may be used in vaccination trials as potential candidates to control tumor growth

Specific distribution of gabarap, gec1/gabarap Like 1, gate16/gabarap Like 2, lc3 messenger RNAs in rat brain areas by quantitative real-time PCR.

International audienceGABARAP and GEC1/GABARAPL1 interact with tubulin and GABA(A) receptor and belong to a new protein family. This family includes GATE 16 and LC3, potentially involved in intracellular transport processes. In this study, we combined brain dissection and quantitative real-time reverse transcription polymerase chain reaction to study discriminatively gabarap, gec1/gabarapL1, gate16/gabarapL2, lc3 mRNA distribution in multiple rat brain areas

Effect of 7-O-beta-D-glucopyranosylchrysin and its aglycone chrysin isolated from Podocytisus caramanicus on estrogen receptor alpha transcriptional activity.

International audienceA screening of Greek Fabaceae extracts identified the methanolic extract of Podocytisus caramanicus Boiss. & Heldr. as having proliferative activity on human breast cancer cells (MCF-7). Using transient transfection experiments, we have first used three compounds described for their estrogen-like properties, E (2), genistein (Gen) and biochanin A (Bch), as controls to evaluate our cellular model. Secondly, we have demonstrated that the 7- O-beta- D-glucopyranosylchrysin (Glc-chr), the most abundant flavone of the extract, and its aglycone chrysin were able to increase estrogen receptor alpha transcriptional activity in MCF-7 cells. We have also shown that the estrogenic activity of Glc-chr could be completely suppressed by the pure estrogen antagonist ICI 182,780 suggesting that the effect of Glc-chr is mediated by ERalpha

Carbon nanotubes as gene carriers: Focus on internalization pathways related to functionalization and properties

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