X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins.

Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization. Following this line, we show here that crystals of membrane proteins display systematically higher diffraction anisotropy than those of soluble proteins. This phenomenon dramatically hampers structure solution and refinement, and has a strong impact on the quality of electron-density maps. A farther search for origins of this phenomenon showed that the type of crystallization, and thus the crystal packing, has no impact on anisotropy, nor does the nature or function of the membrane protein. Membrane proteins fully embedded within the membrane display equal anisotropy compared to the ones with extra membranous domains or fusions with soluble proteins. Overall, these results overturn common beliefs and call for a specific handling of their diffraction data

Polymer "ruthenium-cyclopentadienyl" conjugates - New emerging anti-cancer drugs

In this work, we aimed to understand the biological activity and the mechanism of action of three polymer-'ruthenium-cyclopentadienyl' conjugates (RuPMC) and a low molecular weight parental compound (Ru1) in cancer cells. Several biological assays were performed in ovarian (A2780) and breast (MCF7, MDA-MB-231) human cancer derived cell lines as well as in A2780cis, a cisplatin resistant cancer cell line. Our results show that all compounds have high activity towards cancer cells with low IC50 values in the micromolar range. We observed that all Ru-PMC compounds are mainly found inside the cells, in contrast with the parental low molecular weight compound Ru1 that was mainly found at the membrane. All compounds induced mitochondrial alterations. PMC3 and Ru1 caused F-actin cytoskeleton morphology changes and reduced the clonogenic ability of the cells. The conjugate PMC3 induced apoptosis at low concentrations comparing to cisplatin and could overcame the platinum resistance of A2780cis cancer cells. A proteomic analysis showed that these compounds induce alterations in several cellular proteins which are related to the phenotypic disorders induced by them.Our results suggest that PMC3 is foreseen as a lead candidate to future studies and acting through a different mechanism of action than cisplatin. Here we established the potential of these Ru compounds as new metallodrugs for cancer chemotherapy.This work was financed by the Portuguese Foundation for Science and Technology (Fundacao para a Ciencia e a Tecnologia, FCT) within the scope of projects UID/QUI/00100/2013 and PTDC/QUI-QIN/28662/2017. This work was supported by the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI). Andreia Valente acknowledges the COST action CM1302 (SIPs), the Investigator FCT2013 Initiative for the project IF/01302/2013 (acknowledging FCT, as well as POPH and FSE - European Social Fund) and the Royal Society of Chemistry's Research Fund. Pierre Falson and Elisabeta Comsa warmly acknowledge Thibault Andrieu from the cytometry plateau of SFR bioscience -UMS 3444- at Lyon-Gerland, France for assistance on CytoF. This work was also supported by the Marie Curie Initial Training Network: FP7-PEOPLE-2012-ITN proposal no 317297 - acronym GLYCOPHARM and PITN-GA-2012-317297. The high resolution mass spectrometer at CIRE-PAIB was financed (SMHART project no3069) by the European Regional Development Fund (ERDF), the Conseil Regional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm)

Au courant computation of the PDB to audit diffraction anisotropy of soluble and membrane proteins.

This data article makes available the informed computation of the whole Protein Data Bank (PDB) to investigate diffraction anisotropy on a large scale and to perform statistics. This data has been investigated in detail in "X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins" [1]. Diffraction anisotropy is traditionally associated with absence of contacts in-between macromolecules within the crystals in a given direction of space. There are however many case that do not follow this empirical rule. To investigate and sort out this discrepancy, we computed diffraction anisotropy for every entry of the PDB, and put them in context of relevant metrics to compare X-ray diffraction in reciprocal space to the crystal packing in real space. These metrics were either extracted from PDB files when available (resolution, space groups, cell parameters, solvent content), or calculated using standard procedures (anisotropy, crystal contacts, presence of ligands). More specifically, we separated entries to compare soluble vs membrane proteins, and further separated the later in subcategories according to their insertion in the membrane, function, or type of crystallization (Type I vs Type II crystal packing). This informed database is being made available to investigators in the raw and curated formats that can be re-used for further downstream studies. This dataset is useful to test ideas and to ascertain hypothesis based on statistical analysis

Etude de mutants de la levure Schizosaccharomyces pombe affectes au niveau d'acides amines impliques dans la catalyse et la regulation de l'ATPase mitochondriale : modifications genetiques et chimiques

SIGLECNRS T 64208 MIC / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Inhibition différentielle par des pseudopeptides des transporteurs de résistance aux agents chimiothérapeutiques, ABCB1 et ABCG2 (développement d un système d expression dans les cellules humaines du transporteur ABCG2 couplé à la GFP)

La thérapie la plus courante pour un cancer est la chimiothérapie. Malheureusement, son efficacité est réduite par notamment le phénotype MDR (MultiDrug Resistance) ; il est dû à la surexpression de protéines membranaires, les transporteurs ABC, provoquant l expulsion des agents anticancéreux hors de la cellule. L étude du rôle physiologique de ces protéines et des mécanismes conduisant au phénotype MDR est entreprise afin de développer par la suite, des inhibiteurs. Le test d une série de composés peptidomimétiques dérivant de la réversine 121 a été entreprise sur ABCB1 et ABCG2. L étude s est effectuée par cytométrie en flux en mesurant l accumulation de mitoxantrone. La relation structure-activité de ces composés a permis d identifier 2 groupements indispensables à l efficacité des composés : le carbonyle de la liaison peptidique et l encombrement stérique de la région A pouvant être modulé, soit par la présence ou non d une lysine dans la région B. L étude structure-fonction d ABCG2 se fait fréquemment par mutagenèse dirigée. J ai développé un nouveau système d expression conduisant à une protéine de fusion ABCG2-GFP, permettant ainsi la détection plus rapide des mutants directement in cellulo par microscopie à fluorescence et sans destruction des cellules.GRENOBLE1-BU Médecine pharm. (385162101) / SudocSudocFranceF

Expression et caractérisation biochimique d'une boucle extracellulaire de la Breast Cancer Resistance Protein (BCRP)

LYON1-BU Santé (693882101) / SudocSudocFranceF

Drug candidates targeting multidrug resistance in cancer and infections

International audienceMultidrug resistance (MDR) is the phenomenon by which prokaryotic and eukaryotic cells have developed resistance to drugs. The SARSCov-19 pandemic has spotlighted the lethal effect of MDR in the context of nosocomial diseases. In this context, the MDR against anticancer drugs and antibiotics are by far the most investigated areas. MDR finds multiple origins including intracellular drug inactivation, biological target alteration, activation of DNA repair mechanisms (in the case of cancer) and drug expulsion from inside to outside the cells leading to drug concentration below the active threshold. In the latter case, the high expression of efflux proteins at the cell membrane plays a critical role in the transport and pumping out of drugs

PDR-like ABC systems in pathogenic fungi

International audienc

Overcoming the toxicity of membrane peptide expression in bacteria by upstream insertion of Asp-Pro sequence

AbstractTransmembrane (TM) peptides often induce toxic effects when expressed in bacteria, probably due to membrane destabilization. We report here that in the case of the TM domains of hepatitis C virus (HCV) E1 and E2 envelope proteins, which are both particularly toxic for the bacteria, the insertion of the Asp-Pro (DP) sequence dramatically reduced their toxicities and promoted their expressions when produced as glutathione S-transferase (GST) GST-DP-TM chimeras. Subcellular fractionation showed that these chimeras co-sediment with the membrane fraction and contain active GST that could be solubilized with a mild detergent. Surprisingly, immuno-gold electron microscopy clearly showed that such chimeras are not localized in the membrane but in the cytosol. We thus postulate that they likely form proteo-lipidic aggregates, which prevent the bacteria from toxicity by sequestering the TM part of the chimeras. The reduction of toxicity in the presence of the Asp-Pro sequence is possibly due to Asp's negative charge that probably disadvantages the binding of the TM peptides to the membrane. In addition, the structural features of Pro residue could promote the formation of chimera aggregates

Contribution of lipid components to the permeability barrier of oral mucosa

The aim of this paper is to review the lipid composition of different regions of the oral mucosa, bearing in mind that, in general, molecular diffusion occurs through the intercellular lipids. Lipid content and organization is described to explain the permeability differences between keratinized and nonkeratinized tissues. Some of the thermotropic and lyotropic transitions observed in simple lipid model systems, such as liposomes, are also taken into consideration, in an attempt to explain the contribution of each lipid class to the permeability barrier