Varied Probability of Staying Collapsed/Extended at the Conformational Equilibrium of Monomeric Aβ40 and Aβ42.

International audienceIn present study, we set out to investigate the conformation dynamics of Aβ40 and Aβ42 through exploring the impact of intra-molecular interactions on conformation dynamics using equilibrium molecular dynamics simulations. Our 40 microsecond-scale simulations reveal heterogeneous conformation ensembles of Aβ40 and Aβ42 that encompass ~35% β-strand and ~60% unstructured coils. Two conformational states were identified in both alloforms: a collapsed state (CS) that resembles the structural motif of face-to-face hydrophobic clustering in amyloid fibrils, and an extended state (ES) that features the structural characteristics of anti-parallel β-sheets in amyloid oligomers. In Aβ40, the C-terminus remains unstructured and rarely interacts with other parts, thereof the hydrophobic clustering is in loose contact and the peptide assumes ES with high probability. In contrast, the C-terminus of Aβ42 adopts a β-strand structure that strongly interacts with segments E3-R5 and V18-A21. The active association leads to a more compact hydrophobic collapse and refrain the alloform from ES. Based on the structural characterization, we propose that the fibril and oligomer assembly pathways could respectively take off from CS and ES, and their aggregation propensity may be governed by the probability of visiting the corresponding conformational states at the equilibrium

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level.

International audienceBacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis

PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway

PCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 M) and analyzed for cell proliferation by the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway

Gating process in Omp-Pst2 monomer B at negative voltage.

<p>(A) Snapshots of L3 tip. In each snapshot, residues W104 to D117 are shown in cartoon; W111, D114, E258 and D312 in sticks; and K⁺ within 3.5 Å of the L3 tip in blue spheres. The acidic niche I (E258) and niche II (D312) are highlighted in gray. (B) W111 movements in every 50 ns. The RMSD of 112-AG-113 (C), the phi/psi angle of W111 (D) and the RMSD of W111 (E) are laid out to demonstrate the sequential events leading to niches exposure and subsequent binding of cations within them (F). G) The electrostatic potential surface at 0 ns and 500 ns. The extent of color scale is ± 100 kT/e. Y95, Y99, A103 and W111 are shown in sticks.</p

The calculated current, conductance and ion selectivity from MD simulations.

<p>(A) The currents are calculated every 50 ns monomer-wise and the total channel currents are shown in in-set panels in comparison to the experimentally determined values (dash lines). (B) The conductances of the porins’ channel are obtained from the slope of their I-V curve. The raw channel conductances are shown. (C) The ion selectivity is determined as the ratio of K⁺ to Cl^- current. The value shown at each voltage is the averaged ratio from three monomers and the error bar corresponds to the standard deviation among the three.</p

Electrostatic properties of Omp-Pst1 and Omp-Pst2.

<p>(A) Charge distribution at constriction zone. The constriction zone is defined as the area within 5 Å of the narrowest point of the channel. The negatively charged residues are shown in red spheres and the positively charged in blue spheres. (B) Mode vectors of cations (blue) and anions (red) for every 1 ns within the first 100 ns in the voltage-applied simulations. (C) Electrostatic surface of anti-L3 side and L3 side. Contour color set as ± 100 kT/e. (D) Electrostatic potentials along the pores calculated by HOLE. The values are normalized to intracellular entrance.</p

Conductance, ion selectivity and critical voltage of Omp-Pst1 and Omp-Pst2 measured in single channel.

<p>Conductance, ion selectivity and critical voltage of Omp-Pst1 and Omp-Pst2 measured in single channel.</p

Comparison of hydrogen-bonding network on Omp-Pst2 L3 at two voltages.

<p>A) Hydrogen bonds on L3 as a function of time. The color scale indicates whether the hydrogen bond is seen in 0, 1, 2 or 3 monomers of the trimer. The hydrogen bond W111-D312, which anchors L3 tip to barrel wall, is highlighted. B) Snapshots of Omp-Pst2 L3 at 200 ns at V_TM = +/- 1 V. The three charged residues D114, D117, E119, and their hydrogen-bonding residues are shown in sticks.</p