Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping

Four residues Pro19, Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins, studied by means of CD measurements, proved reversible and well represented by the two-state ND transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.     

Synthesis,activity and molecular modeling of a new series of chromones as low molecular weight protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTP) are crucial elements in eukaryotic signal transduction. Several reports suggested that the LMW-PTP family has oncogenic relevance. Moreover, LMW-PTP has been recognized as a negative regulator of insulin-mediated mitotic and metabolic signaling. Thus, inhibition of the LMW-PTP can be considered an attractive approach for the design of new therapeutic agents for the treatment of type II diabetes and for new antitumoral drugs. To date very few (and weak) inhibitors of LMW-PTP have been identified. On the basis of the reported weak activity of some flavonoids on phosphatases, we discovered a lead that originated a new class of highly active LMW-PTP inhibitors; these compounds inhibit also PTP-1B and are active in cellular assays. Docking experiments and SAR highlighted the possible binding mode of these compounds to the enzyme, putting the background for the future optimization of their inhibitory activity and selectivity towards the closely related enzyme PTP-1B.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >10⁵-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.     

Effect of reline material and denture base surface treatment on the impact strength of a denture base acrylic resin

doi:10.1111/j.1741‐2358.2009.00292.x
Effect of reline material and denture base surface treatment on the impact strength of a denture base acrylic resin Objective: In this study, the effect of relining and surface treatment on the impact strength (IS) of a heat‐polymerising denture base acrylic resin (Lucitone 550‐L) was evaluated. Materials and methods: Rectangular bars of L were made (60 × 6 × 2 mm) and relined (2 mm) with the relining resins Ufi Gel Hard (UH) and Tokuso Rebase Fast (TR). Specimens relined with L and intact L, TR and UH specimens were also made (60 × 6 × 4 mm), for comparison. Before relining, the L surface was left untreated or wetted with methyl methacrylate monomer and/or the bonding agents (BA) supplied by manufacturers of the reline resins. V‐notches were machined at the midpoint of the length of all specimens. The notches were made either across the width (Nw) or across the thickness of the specimens (Nth). The Charpy impact test was performed using a 0.5‐J pendulum, which had been specially designed and constructed. Data were analysed separately for each notch position using one‐way analysis of variance and Tukey honestly significant difference post‐hoc test (p = 0.05). Results: The IS of L was similar to that of L/L. For the Nw notch, treating the denture base L with TR BA and relining with TR reline material produced the highest IS. Conclusion: The IS of specimens made from heat polymerising acrylic resin Lucitone 550 was increased after relining using the hard chairside reline resin TR with its proprietary BA.     

Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells

Celiac disease is characterized by the secretion of IgA-class autoantibodies that target tissue transglutaminase (tTG). It is now recognized that anti-tTG antibodies are functional and not mere bystanders in the pathogenesis of celiac disease. Here we report that interaction between anti-tTG antibodies and extracellular membrane-bound tTG inhibits peptide 31–43 (but not peptide 57–68) uptake by cells, thereby impairing the ability of p31–43 to drive Caco-2 cells into S-phase. This effect did not involve tTG catalytic activity. Because anti-tTG antibodies interfered with epidermal growth factor endocytosis, we assume that they exert their effect by reducing peptide 31–43 endocytosis. Our results suggest that cell-surface tTG plays a hitherto unknown role in the regulation of gliadin peptide uptake and endocytosis.     

Fluorescence emission and enhanced photochemical stability of Zn-5-triethyl ammonium methyl salicylidene ortho-phenylendiiminate interacting with native DNA

The photophysical and photochemical properties of the cationic Zn^II complex of 5-triethyl ammonium methyl salicylidene ortho-phenylendiimine (ZnL²⁺) interacting with native DNA were investigated by steady state and time-resolved fluorescence spectroscopies. Experimental results indicate that, in the presence of DNA, ZnL²⁺ is efficiently protected from a photochemical process, which occurs when it is in the free state dispersed in aqueous solution. The analysis of the absorption and emission spectra of ZnL²⁺, both stored in the dark and after exposure to tungsten lamp light for 24 h, corroborated by quantum chemical calculations, allowed us to point out that ZnL²⁺ undergoes a photoinduced two-electron oxidation process. According to this picture, the protective action of DNA toward the intercalated ZnL²⁺ was attributed to an effective inhibition of the ZnL²⁺ photooxidation. In this context, it can be considered that DNA-intercalated ZnL²⁺ is located in a region more hydrophobic than that sensed in the bulk water solvent. Moreover, by a thorough analysis of steady state and time-resolved fluorescence spectra, the interaction process can be consistently explained in terms of a complete intercalation of the complex molecules and that the polarity of the environment sensed by intercalated ZnL²⁺ is comprised between that of methanol and ethanol.     

Kinetic mechanism of the Zn-dependent aryl-phosphatase activity of myo-inositol-1-phosphatase

Myo-inositol-1-phosphatase (EC 3.1.3.25) is able to hydrolyze myo-inositol-1-phosphate in the presence of Mg(2+) ions at neutral pH, and also p-nitrophenyl phosphate in the presence of Zn(2+)-ions at acidic pH. This enzyme plays a role in phosphatidylinositol cell signalling and is a putative target of lithium therapy in manic depression. We elucidate here the kinetic mechanism of the Zn-dependent activity of myo-inositol-1-phosphatase. As part of this analysis it was necessary to determine the basicity constants of p-nitrophenyl phosphate and the stability constant of its metal-complex in the presence of zinc chloride. We find that the Zn-dependent reaction may be described either by a rapid-equilibrium random mechanism or an ordered steady-state mechanism in which the substrate binds to the free enzyme prior to the metal ion. In both models the Zn-substrate complex acts as a high affinity inhibitor, yielding a dead-end species through its binding to the enzyme-Zn-substrate in rapid-equilibrium or to the enzyme-phosphate complexes in a steady-state model. Phosphate is a competitive inhibitor of the enzyme with respect to the substrate and an uncompetitive inhibitor with respect to zinc ions.