Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition

Structural and functional properties of prefibrillar α-synuclein oligomers.

International audienceThe deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity

Structural mapping techniques distinguish the surfaces of fibrillar 1N3R and 1N4R human tau

International audienceThe rigid core of intracellular tau filaments from Alzheimer’s disease (AD), Pick’s disease (PiD), and Corticobasal disease (CBD) brains has been shown to differ in their cryo-EM atomic structure. Despite providing critical information on the intimate arrangement of a fraction of htau molecule within the fibrillar scaffold, the cryo-EM studies neither yield a complete picture of tau fibrillar assemblies structure nor contribute insights into the surfaces that define their interactions with numerous cellular components. Here, using proteomic approaches such as proteolysis and molecular covalent painting, we mapped the exposed amino acid stretches at the surface and those constituting the fibrillar core of in vitro-assembled fibrils of human htau containing one N-terminal domain and three (1N3R) or four (1N4R) C-terminal microtubule-binding repeat domains as a result of alternative splicing. Using limited proteolysis, we identified the proteolytic fragments composing the molecular “bar-code” for each type of fibril. Our results are in agreement with structural data reported for filamentous tau from AD, PiD, and CBD cases predigested with the protease pronase. Finally, we report two amino acid stretches, exposed to the solvent in 1N4R not in 1N3R htau, which distinguish the surfaces of these two kinds of fibrils. Our findings open new perspectives for the design of highly specific ligands with diagnostic and therapeutic potential

Atomic force microscopy imaging and nanomechanical properties of six Tau isoform assemblies

International audienceThe amyloid fibrillar form of the protein Tau is involved in a number of neurodegenerative diseases, also known as tauopathies. In this work, six different fibrillar Tau isoforms were assembled in vitro. The morphological and nanomechanical properties of these isoforms were studied using atomic force microscopy at high resolution in air and buffer. Our results demonstrate that all Tau isoform fibrils exhibit paired-helical-filament-like structures consisting of two protofibrils separated by a shallow groove. Interestingly, whereas the N-terminal inserts do not contribute to any morphological or mechanical difference between the isoforms with the same carboxyl-terminal microtubule-binding domain repeats, isoforms with four microtubule repeats (4R) exhibited a persistence length ranging from 2.0 to 2.8 mm, almost twofold higher than those with three repeats (3R). In addition, the axial Young's modulus values derived from the persistence lengths, as well as their radial ones determined via nanoindentation experiments, were very low compared to amyloid fibrils made of other proteins. This sheds light on the weak intermolecular interaction acting between the paired b-sheets within Tau fibrils. This may play an important role in their association into high molecular weight assemblies, their dynamics, their persistence, their clearance in cells, and their propagation

Hypopituitarism consecutive to the brain damage: a widely unexpected prevalence

Alors que la prévalence de l’hypopituitarisme est largement sous-estimée, son étiologie est souvent méconnue. Des données récentes indiquent que le traumatisme crânien constitue actuellement une cause importante d’hypopituitarisme acquis chez l’adulte. Les traumatismes crâniens représentent un problème majeur de santé publique, avec une incidence annuelle en Belgique de 30.000 patients par an. Plusieurs séries rétrospectives et prospectives ont identifié plus de 5.000 patients avec un traumatisme crânien et souffrant d’un déficit isolé ou combiné en hormone de croissance, gonadotrophines, adrénocorticotrophine, thyrotrophine et, occasionnellement, de prolactine et d’hormone antidiurétique. Nous faisons le point sur ces données récentes et discutons du diagnostic et de la prise en charge de l’hypopituitarisme secondaire au traumatisme crânien.While the prevalence of Hypopituitarism is widely underestimated, its etiology is often misunderstood. Recent data indicate that head trauma is currently a major cause of acquired Hypopituitarism in adulthood. Injuries are a major public health problem, with an annual incidence in Belgium of 30,000 patients per year. Several retrospective and prospective series have identified more than 5,000 patients with trauma injury and suffering from an isolated or in combination hormone growth deficiency, gonadotrophins, adrenocorticotrophin, thyrotrophin and, less frequently prolactin and antidiuretic hormone deficiency. In this presentation , we make the point on these recent data and discuss the diagnosis and management of the secondary Hypopituitarism injury, according to our experience and the review of the litterature

Abscisic acid-induced anion currents activation mediated by ADP-ribose/ryanodine receptor (RyR) in Arabidopsis thaliana suspensions cells

International audienc

Fibrillar Ure2p is not degraded by the 26S proteasome.

<p><b>(A)</b> Ure2p fibrils (2 μg) were incubated at 30°C under mild agitation (<300 rpm) in the presence of 2.5 mM ATP, with or without 26S proteasomes (1.6 μg), as indicated. Aliquots were removed at time intervals and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies. <b>(B)</b> Ure2p fibrils were incubated with or without 26S proteasomes and MG132 (100 μM), as indicated in (A). At the indicated time points, aliquots were diluted four-fold in proteasome assay buffer and then filtered through a cellulose acetate membrane (0.2 μm pore size) using a slot-blot vacuum manifold. Each well was then washed twice with 200 μL assay buffer and the membranes were immunostained with anti-Ure2p antibodies. <b>(C)</b> Ure2p fibrils were incubated with or without 26S proteasomes, as indicated in (A). Aliquots were withdrawn at time intervals and analyzed by SDD-AGE followed by immunoblotting with anti-Ure2p antibodies <b>(D)</b> Sup35p fibrils (1 μg) were mixed with purified 26S proteasomes (0.4 μg) in the presence of 2.5 mM ATP. The reactions mixes were incubated at 30°C under mild agitation (<300 rpm), and at the indicated time points, aliquots were removed and analyzed by SDS-PAGE followed by Western blotting using anti-Sup35p antibodies.</p

Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.

<p>Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131789#pone.0131789.s001" target="_blank">S1 Table</a>). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. <b>(Inset)</b> Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.</p

The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.

<p><b>(A)</b> Cartoon representation of the Ure2p variants used in this study. <b>(B)</b> Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE <b>(C)</b> Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. <b>(D)</b> Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.</p

Soluble Ure2p is a proteasomal substrate <i>in vitro</i>.

<p><b>(A)</b> Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. <b>(B)</b> Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). <b>(C)</b> Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).</p