Study on the internalization mechanism of the ZEBRA cell-penetrating peptide

Cell-penetrating peptides (CPPs) represent a noninvasive method for delivering functional biomolecules into living cells. We have recently shown that the Epstein-Barr virus transcriptional factor ZEBRA contains a protein transduction domain, named Z9 or minimal domain (MD). Only few of currently identified CPPs including MD are able to rapidly cross the mammalian cell membrane without being entrapped into endosomal compartments, even when fused to cargo macromolecules. In this work, a series of MD deletion mutants has been engineered and their cellular uptake has been analyzed by confocal microscopy and FACS. We identified a domain MD11 (8 amino acids shorter than MD) able to enter mammalian cells via a mainly endocytosis-independent mechanism. All the other generated truncated forms exhibited reduced cellular uptake and penetrated into cells through endocytic mechanisms. These results have highlighted the role of the MD11 C-terminal region as essential for efficient cellular entry and endosomal escape  and open new perspectives for the use of this CPP as carrier for delivering biologically active macromolecules with therapeutic potential

Human Speedy: a novel cell cycle regulator that enhances proliferation through activation of Cdk2

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition

Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

Expression and Purification of ZEBRA Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells

International audienceThe recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution

Expression and purification of ZEBRA fusion proteins and applications for the delivery of macromolecules into mammalian cells.

International audienceThe recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution

Production of recombinant proteoliposomes for therapeutic uses.

International audienceOne of the major challenges in human therapy is to develop delivery systems that are convenient and effective for tackling problems in disease treatments. In the past 20 years, liposomes have represented promising pharmaceutical carriers for drug delivery. Due to their biophysical properties, liposomes can deliver and specifically target a large set of bioactive molecules, they can protect molecules from degradation, and their composition is easily modifiable. The use of recombinant proteoliposomes containing therapeutic membrane proteins is a recently developed technology that allows biologically active proteins to penetrate across the plasma membrane of eukaryotic cells. One of the bottlenecks in this powerful delivery system lies in the production of functional therapeutic membrane proteins mainly due to their biophysical characteristics. Membrane proteins represent about 30% of the total proteins from an organism, and play a central role in drug discovery as potential pharmaceutical targets. This chapter describes the methodology for the production of bioactive proteoliposomes containing therapeutic, proapoptotic membrane proteins synthesized with an optimized cell-free expression system. We will examine (1) the design of the expression vectors and the liposome compositions compatible with the cell-free expression system; (2) the production of membrane proteins using a cell-free expression system in combination with liposomes, to obtain in a one-step reaction functional therapeutic proteoliposomes; (3) proteoliposome purification for further use in the treatment of cancer cells; and (4) the methodology for detecting apoptosis in cells after treatment. Furthermore, this system can be easily adapted for producing "difficult to express proteins" compared with the classical overexpression (bacterial or eukaryotic) systems

Les capteurs biomimétiques en recherche biomédicale

International audienceThe recent research on both the synthesis of membrane proteins by cell-free systems and the reconstruction of planar lipid membranes, has led to the development of a cross-technology to produce biosensors or filters. Numerous biomimetic membranes are currently being standardized and used by the industry, such as filters containing aquaporin for water desalination, or used in routine at the laboratory scale, for example the bacteriorhodopsin as a light sensor. In the medical area, several fields of application of these biomimetic membranes are under consideration today, particularly for the screening of therapeutic molecules and for the developing of new tools in diagnosis, patient monitoring and personalized medicine.Le récent développement de la synthèse de protéines membranaires par des systèmes dits acellulaires et la reconstitution des membranes lipidiques planes ont permis l’émergence d’une technologie transversale pour la production de biocapteurs ou de filtres. Un certain nombre de membranes biomimétiques sont actuellement normalisées et utilisées par l’industrie (comme par exemple, un filtre contenant l’aquaporine pour la désalinisation de l’eau), ou en routine à l’échelle du laboratoire (comme la bactériorhodopsine en tant que capteur de lumière). Dans le domaine médical, plusieurs champs d’applications pour ces membranes biomimétiques sont à l’étude pour du criblage de molécules thérapeutiques et pour répondre concrètement aux besoins de développement de nouveaux outils pour le diagnostic, le suivi du patient et la médecine personnalisée

Nouvelles approches thérapeutiques pour le traitement du glioblastome (exemple du développement d'une protéine pro-apoptotique)

Le glioblastome est la tumeur du cerveau la plus fréquente et la plus agressive. Il n existe actuellement pas de traitement curatif de ce cancer. Les thérapies standards utilisées actuellement sont la chirurgie de résection macroscopique et la radiothérapie externe en association avec une chimiothérapie concomitante et adjuvante, le plus souvent par témozolomide. Grâce à la découverte du témozolomide et des progrès accomplis au niveau de la chirurgie et de la radiothérapie, la durée de vie des patients a été augmentée de façon significative mais pas suffisamment. La compréhension des dérèglements, notamment de l apoptose et des voies de signalisation, conduisant au développement des glioblastomes est corrélée avec l essor de nouvelles molécules. Il s agit de thérapies ciblées agissant, entre autre, sur les effecteurs ou les récepteurs de ces voies de signalisation. Leur nombre est en constante augmentation et les résultats obtenus par certaines de ces molécules sont très prometteurs. Cependant le coût d un traitement de thérapie ciblée reste élevé et peut-être un frein pour l accès au traitement des patients. Quoi qu il en soit, ces potentiels médicaments, en combinaison avec les traitements standards, représentent un espoir dans la prise en charge des glioblastomes.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

The translational factor eIF3f: the ambivalent eIF3 subunit.

International audienceThe regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth. Subtle changes of proteins involved in the translation process may alter the rate of the protein synthesis and modify the cell fate by shifting the balance from normal status into a tumoral or apoptotic one. The largest eukaryotic initiation factor involved in translation regulation is eIF3. Amongst the 13 factors constituting eIF3, the f subunit finely regulates this balance in a cell-type-specific manner. Loss of this factor causes malignancy in several cells, and atrophy in normal muscle cells. The intracellular interacting partners which influence its physiological significance in both cancer and muscle cells are detailed in this review. By delineating the global interaction network of this factor and by clarifying its intracellular role, it becomes apparent that the f subunit represents a promising candidate molecule to use for biotherapeutic applications