Structural characterization of the M* partly folded intermediate of wild-type and P138A EcAspAT

A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M*partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M*intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposabl

Inhibition of bone resorption by Tanshinone VI isolated from Salvia miltiorrhiza Bunge

During the last decade, a more detailed knowledge of molecular mechanisms involved in osteoclastogenesis has driven research efforts in the development and screening of compound libraries of several small molecules that specifically inhibit the pathway involved in the commitment of the osteoclast precursor cells. Natural compounds that suppress osteoclast differentiation may have therapeutic value in treating osteoporosis and other bone erosive diseases such as rheumatoid arthritis or metastasis associated with bone loss. In ongoing investigation into anti-osteoporotic compounds from natural products we have analyzed the effect of Tanshinone VI on osteoclasts differentiation, using a physiologic three-dimensional osteoblast/bone marrow model of cell co-culture. Tanshinone VI is an abietane diterpene extracted from the root of Salvia miltiorrhiza Bunge (Labiatae), a Chinese traditional crude drug, “Tan-Shen”. Tashinone has been widely used in clinical practice for the prevention of cardiac diseases, arthritis and other inflammation-related disorders based on its pharmacological actions in multiple tissues. Although Tanshinone VI A has been used as a medicinal agent in the treatment of many diseases, its role in osteoclast-related bone diseases remains unknown. We showed previously that Tanshinone VI greatly inhibits osteoclast differentiation and suppresses bone resorption through disruption of the actin ring; subsequently, we intended to examine the precise inhibitory mechanism of Tanshinone VI on osteoclast differentiating factor. This study shows, for the first time, that Tanshinone VI prevents osteoclast differentiation by inhibiting RANKL expression and NFkB induction

Zircon dissolution in a ductile shear zone, Monte Rosa granite gneiss, northern Italy

The sizes, distributions and shapes of zircon grains within variably deformed granite gneiss from the western Alps have been studied. Zircon shows numerous indicators of a metamorphic response in both the host gneiss and a 5 cm wide continuous ductile shear zone, within which the zircon grain sizes range from <1 µm to >50 µm. However, the very fine grain sizes are virtually absent from grain boundaries. Within this zone, zircons consistently have more rounded and embayed margins, which are interpreted as evidence of dissolution in response to fluid influx during shearing. Zircons are preferentially located near metamorphic muscovite in both the host gneiss and the shear zone and tend to show the poorest crystal shape, indicating that fluids linked to the formation and presence of muscovite may enhance both the crystallization of zircon and its subsequent dissolution. Larger zircon crystals typically show a brittle response to deformation when adjacent to phyllosilicates, with fractures consistently perpendicular to the (001) mica cleavage. The variety of metamorphic behaviour observed for zircon indicates that it may be highly reactive in sub-solidus mid-crustal metamorphic environments

The effect of prime-site occupancy on the hepatitis C virus NS3 protease structure.

We recently reported a new class of inhibitors of the chymotrypsin-like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S′ site of the protease, which is not generally used by the natural substrates. The effect of prime-site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis-mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S′ site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S′ site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N-terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P-P′ inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistanc

Antiproliferative Oleanane Saponins from Dizygotheca elegantissima

Four new oleanane saponins (1-4), together with three known saponins, were isolated from the aerial parts of Dizygotheca elegantissima R. Vig. & Guillaumin. Their structures were elucidated by 1D and 2D NMR experiments including 1D TOCSY, DQF-COSY, HSQC, and HMBC spectroscopy, as well as ESIMS analysis. The antiproliferative activity of all isolated compounds was evaluated

PROTEOMICS AS USEFUL TOOL FOR STUDING BIOMARKERS OF EARLY EXPOSURE TO POLYCHLORINATED BIPHENILS

Polychlorinated biphenyls (PCBs) are toxic persistent organic pollutants present in the hydrosphere. Mussels are used as sentinel organisms for the monitoring of marine pollution for their ability to bioaccumulate contaminants, their sessile nature, filter-feeding habits and widespread distribution. Alteration in protein expression is often indicative of toxic exposure or effect and changes in protein expression profiles derived from environmental stress may be used for the identification of novel biomarkers. In this study, proteomics has been used in mussels as a preliminary screening of quantitative changes in protein expression caused by a mixture of PCBs, potentially useful as new biomarkers, following the identification of the proteins more drastically altered. Our data demonstrated that proteomic approach represents a valuable tool for identifying biomarkers of exposure to environmental contaminants

Proteomic approach for the detection of biomarkers ofexposure in mussels exposed to PCB

In the current study, a preliminary proteomic approach has been used in Mytilus galloprovincialis as a screening of changes in protein expression caused by a mixture of polychlorinated biphenyls (PCBs), in order to characterize the effects of PCBs on the protein profile and to develop new molecular biomarkers, after identifying the proteins more drastically altered. Methods:Mussels were exposed for three weeks to three polychlorinated biphenyls under controlled conditions. The edible parts were homogenized and lyophilized. Extracted proteins were quantified and separated by two-dimensional electrophoresis (2-DE) in IPG strips (pH 3–10). The protein spots in gels were visualized by Coomassie Brilliant Blue staining. Gel images were obtained using a Image Scanner. Image analysis included spot detection, quantification and matching. The volume of each spot from each gel was normalized to the total gel spot volume in order to correct it for differences in gel staining. More than 500 spots were resolved and altered expression was qualitatively detected. Results and conclusions of the study: Our results showed a well conserved protein pattern regardless of the treatments, demonstrating that the exposition to the PCB mixture did not impair the normal physiological function of the mussels. However, the levels of a restricted number of proteins were clearly and reproducibly affected by the treatment; therefore, these polypeptides were considered promising biomarker candidates. In conclusion, even if further studies are needed to validate these findings, our data demonstrated that proteomic approach represents a valuable tool for identifying biomarkers of exposure to environmental contaminants

Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M.

Petrosaspongiolide M (PM) is an anti-inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A2 (PLA2) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA2 and γ-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA2 has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA2 adduct is consistent with the formation of a Schiff base by reaction of a PLA2 amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM γ-hydroxybutenolide ring. Proteolysis of the modified PLA2 by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA2 α-amino terminal group of the lle-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA2 was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the ε-amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA2 binding site were obtained for manoalide, a paradigm for irreversible PLA2 inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the γ-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA2 binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA2 inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA2 secretory enzymes