Caractérisations structurales et physico-chimiques de la protéine prion ovine

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

High-speed atomic force microscopy: Imaging and force spectroscopy

International audienceKeywords: High-speed atomic force microscopy High-speed force spectroscopy Membrane protein Membrane structure Titin Actin cortex a b s t r a c t Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding

Atomic Force Microscopy Mechanical Mapping of Micropatterned Cells Shows Adhesion Geometry-Dependent Mechanical Response on Local and Global Scales.

International audienceIn multicellular organisms, cell shape and organization are dictated by cell-cell or cell-extracellular matrix adhesion interactions. Adhesion complexes crosstalk with the cytoskeleton enabling cells to sense their mechanical environment. Unfortunately, most of cell biology studies, and cell mechanics studies in particular, are conducted on cultured cells adhering to a hard, homogeneous, and unconstrained substrate with nonspecific adhesion sites, thus far from physiological and reproducible conditions. Here, we grew cells on three different fibronectin patterns with identical overall dimensions but different geometries (▽, T, and Y), and investigated their topography and mechanics by atomic force microscopy (AFM). The obtained mechanical maps were reproducible for cells grown on patterns of the same geometry, revealing pattern-specific subcellular differences. We found that local Young's moduli variations are related to the cell adhesion geometry. Additionally, we detected local changes of cell mechanical properties induced by cytoskeletal drugs. We thus provide a method to quantitatively and systematically investigate cell mechanics and their variations, and present further evidence for a tight relation between cell adhesion and mechanics

Lien entre type génétique et résistance des ovins à la Tremblante : une approche structurale et physico-chimique

National audienceOvine prion protein genetic variants are associated with scrapie susceptibility or resistance : they pave the way to understanding the link between protein structural properties and pathogenesis. The studies developed by INRA teams, in association with other national groups, have brought unexpected informations on the stability and conversion of naturally occurring variants in European sheep breeds. The mechanisms underlying these characteristics were unveiled at the atomic level owing to the crystallographic determination of ovine 3D structure, providing the first experimental insight into the structure of the pathological protein. Unfolding intermediates revealed to be small soluble oligomers, which were purified and displayed neurotoxic effects on primary embryonic neurones. Mutational approaches are currently being developed to identify structural determinants of neuronal death on the one hand, and of prion replication on the other hand.Le polymorphisme génétique de la protéine prion ovine associé à des degrés divers de résistance ou de sensibilité à la tremblante ouvre une voie féconde pour comprendre le lien entre les propriétés structurales de la protéine et le mécanisme du développement de la pathologie. Les travaux menés par les équipes de l’INRA, en collaboration avec d’autres équipes nationales, ont apporté des renseignements inattendus sur la stabilité et la convertibilité des variants naturellement rencontrés dans les troupeaux européens. Les mécanismes, au niveau atomique, sous-tendant ces caractéristiques ont pu être explicités par la détermination cristallographique de la structure tri-dimensionnelle de la protéine ovine, apportant en même temps une première information expérimentale sur la structure de la protéine pathologique. Des formes intermédiaires de repliement, sous forme d’oligomères solubles, ont pu être isolées in vitro, et se sont révélées neurotoxiques, ouvrant de nouvelles pistes de recherche vers les déterminants respectifs de la mort neuronale et de la réplication du prion

Sequential generation of two structurally distinct ovine prion protein soluble oligomers displaying different biochemical reactivities

International audienceIn pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. In prion diseases the lethal evolution is associated with formation of PrPSc, a misfolded and amyloid form of the normal cellular prion protein PrP. Although several molecular mechanisms were proposed to account for the propagation of the infectious agent, the events responsible for cell death are still unclear. The correlation between PrPC expression level and the rate of disease evolution on one side, and the fact that PrPSc deposition in brain did not strictly correlate with the apparition of clinical symptoms on the other side, suggested a potential role for diffusible oligomers in neuronal death. To get better insight into the molecular mechanisms of PrPC oligomerization, we studied the heat-induced oligomerization pathway of the fulllength recombinant ovine PrP at acidic pH. This led to the irreversible formation of two well-identified soluble oligomers that could be recovered by size-exclusion chromatography. Both oligomers displayed higher b-sheet content when compared to the monomer. A sequential two-step multimolecular process accounted for the rate of their formation and their ratio partition, both depending on the initial protein concentration. Smallangle X-ray scattering allowed the determination of the molecular masses for each oligomer, 12mer and 36mer, as well as their distinct oblate shapes. The two species differed in accessibility of polypeptide chain epitopes and of pepsin-sensitive bonds, in a way suggesting distinct conformations for their monomeric unit. The conversion pathway leading to these novel oligomers, displaying contrasted biochemical reactivities, might be a clue to unravel their biological roles

Conformational dynamics and oligomerization pathways of prion protein investigated by rational mutagenesis

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Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

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Insight into the PrPC -> PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants

Prion diseases are associated with the conversion of the_-helix rich prion protein (PrPC) into a _-structure-rich insoluble conformer (PrPSc) that is thought to be infectious. The mechanism for the PrPC 3 PrPSc conversion and its relationship with the pathological effects of prion diseases are poorly understood, partly because of our limited knowledge of the structure of PrPSc. In particular, the way in which mutations in the PRNP gene yield variants that confer different susceptibilities to disease needs to be clarified. We report here the 2.5-Å-resolution crystal structures of three scrapie-susceptibility ovine PrP variants complexed with an antibody that binds to PrPC and to PrPSc; they identify two important features of the PrPC 3 PrPSc conversion. First, the epitope of the antibody mainly consists of the last two turns of ovine PrP second _-helix. We show that this is a structural invariant in the PrPC 3 PrPSc conversion; taken together with biochemical data, this leads to a model of the conformational change in which the two PrPC Cterminal _-helices are conserved in PrPSc, whereas secondary structure changes are located in the N-terminal _-helix. Second, comparison of the structures of scrapie-sensitivity variants defines local changes in distant parts of the protein that account for the observed differences of PrPC stability, resistant variants being destabilized compared with sensitive ones. Additive contributions of these sensitivity-modulating mutations to resistance suggest a possible causal relationship between scrapie resistance and lowered stability of the PrP protein

The detrimental invasiveness of glioma cells controlled by gadolinium chelate-coated gold nanoparticles

International audienceGlioblastoma are characterized by an invasive phenotype, which is thought to be responsible for recurrences and the short overall survival of patients. In last decade, the promising potential of ultrasmall gadolinium chelate-coated gold nanoparticles (namely Au@DTDTPA(Gd)) was evidenced for image-guided radiotherapy in brain tumors. Considering the threat posed by invasiveness properties of glioma cells, we were interested to further investigate the biological effects of Au@DTDTPA(Gd) by examining their impact on GBM cell migration and invasion. In our work, exposure of U251 glioma cells to Au@DTDTPA(Gd) led into high accumulation of gold nanoparticles, that were mainly diffusely distributed in the cytoplasm of the tumor cells. Experiments pointed out a significant decrease in glioma cells invasiveness when exposed to nanoparticles. As the proteolysis activities were not directly affected by the intracytoplasmic accumulation of Au@DTDTPA(Gd), the anti-invasive effect cannot be attributed to a matrix remodeling impairment. Rather, Au@DTDTPA(Gd) nanoparticles affected the intrinsic biomechanical properties of U251 glioma cells, such as cell stiffness, adhesion and generated traction forces, and significantly reduced the formation of protrusions, thus exerting an inhibitory effect on their migration capacities. These results highlight the interest of Au@DTDTPA(Gd) nanoparticles for the therapeutic management of astrocytic tumors, not only as radio-enhancing agent but also by reducing invasive potential of glioma cells