Deletion of UCP2 in iNOS Deficient Mice Reduces the Severity of the Disease during Experimental Autoimmune Encephalomyelitis

Uncoupling protein 2 is a member of the mitochondrial anion carrier family that is widely expressed in neurons and the immune cells of humans. Deletion of Ucp2 gene in mice pre-activates the immune system leading to higher resistance toward infection and to an increased susceptibility to develop chronic inflammatory diseases as previously exemplified with the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for multiple sclerosis. Given that oxidative stress is enhanced in Ucp2−/− mice and that nitric oxide (NO) also plays a critical function in redox balance and in chronic inflammation, we generated mice deficient for both Ucp2 and iNos genes and submitted them to EAE. Mice lacking iNos gene exhibited the highest clinical score (3.4+/−0.5 p<0.05). Surprisingly, mice deficient for both genes developed milder disease with reduced immune cell infiltration, cytokines and ROS production as compared to iNos−/− mice

Bacteria‐Based Production of Thiol‐Clickable, Genetically Encoded Lipid Nanovesicles

International audienceDespite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Here we present a microbial platform for high yield production of lipidic nanovesicles, with clickable thiol moieties in their outer corona. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented and customizable corona of transmembrane polypeptides. In addition, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients or detergents) and their straightforward surface functionalization taking advantage of the diversity of commercially-available maleimide derivatives, engineering bacterial-based proteoliposomes are an attractive eco-friendly alternative that can outperform current liposome preparation methods

Microbial expression systems for membrane proteins

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture

Etude de la protéine mitochondriale UCP2 au cours de l'encéphalite auto-immune expérimentale

La protéine découplante UCP2 fait partie de la famille des transporteurs mitochondriaux. Son expression prédomine dans les cellules immunitaires. Chez la souris, le gène est situé dans une région chromosomique fortement associée à l Encéphalite Auto-immune Expérimentale (EAE), un modèle murin de sclérose en plaques (SEP). En absence d UCP2, la réponse auto-immune est amplifiée. Sur le plan clinique, cela se traduit par des troubles moteurs beaucoup plus sévères. Le but de ce travail était, dans un premier temps, de comprendre les relations entre UCP2 et la NO synthase inductible (iNOS), une enzyme surexprimée chez les souris Ucp2-/- pendant l EAE. Chez les souris inactivées pour ces deux gènes, la maladie apparaît plus tardivement, l inflammation et le stress oxydant sont diminués. Le gène Ucp2 s exprime également dans diverses régions du système nerveux central. Ces observations nous ont conduit à étudier la réactivité des souris Ucp2-/- en réponse à un stress environnemental, reproduit expérimentalement par la disruption sociale. Les résultats montrent un état d anxiété plus grand chez la souris Ucp2-/-. Enfin, l étude d UCP2 au cours de l anesthésie montre que son expression est augmentée par différents produits anesthésiques. Par ailleurs, la délétion du gène entraîne un phénotype d hyperactivité locomotrice.PARIS-BIUP (751062107) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Editorial overview: Membranes.

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Escherichia Coli as Host for Membrane Protein Structure Determination: A Global Analysis

Hattab G, Moncoq K, Warschawski D, Miroux B. Escherichia Coli as Host for Membrane Protein Structure Determination: A Global Analysis. Biophysical Journal. 2015;106(2, Suppl. 1): 46a

Escherichia coli as host for membrane protein structure determination: a global analysis

Hattab G, Warschawski DE, Moncoq K, Miroux B. Escherichia coli as host for membrane protein structure determination: a global analysis. Scientific Reports. 2015;5(1): 12097.The structural biology of membrane proteins (MP) is hampered by the difficulty in producing and purifying them. A comprehensive analysis of protein databases revealed that 213 unique membrane protein structures have been obtained after production of the target protein in E. coli. The primary expression system used was the one based on the T7 RNA polymerase, followed by the arabinose and T5 promoter based expression systems. The C41λ(DE3) and C43λ(DE3) bacterial mutant hosts have contributed to 28% of non E. coli membrane protein structures. A large scale analysis of expression protocols demonstrated a preference for a combination of bacterial host-vector together with a bimodal distribution of induction temperature and of inducer concentration. Altogether our analysis provides a set of rules for the optimal use of bacterial expression systems in membrane protein production

Inducible intracellular membranes: molecular aspects and emerging applications

International audienceMembrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering an abnormal accumulation of membrane structures inside the cells (or membrane proliferation) share two major common features: (1) they promote the formation of highly curved membrane domains and (2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, and inspire new applications, either for academia (high-yield membrane protein production and nanovesicle production) or industry (biofuel production and vaccine preparation)