Simonetta Soro

THE ROLE OF THE REACTION MEDIUM IN LIPASE-CATALYZED ESTERIFICATIONS AND TRANSESTERIFICATIONS

Abstract The nature of the reaction medium (different organic solvents and supercritical fluids) markedly influenced the microbial lipase activity in esterification and transesterification reactions employing as substrates natural and synthetic compounds in terms of reaction rates and lipase enantioselectivity. The experimental data obtained show that while there are no substantial correlations between enantioselectivity and some physicochemical characteristics of the solvent as hydrophobicity and dielectric constant, the solvent polarity and hydrophobicity is able to modulate greatly lipase activity. The possible effects of the solvent characteristics on the catalytic performance of the enzymes are discussed and a rationale is proposed to explain the results obtained.

The βI/βIII‐tubulin isoforms and their complexes with antimitotic agents

Both microtubule destabilizer and stabilizer agents are important molecules in anticancer therapy. In particular, paclitaxel has been demonstrated to be effective for the treatment of ovarian, breast, and nonsmall cell lung carcinomas. It has been shown that emergence of resistance against this agent correlates with an increase in the relative abundance of tubulin isoform βIII and that the more recently discovered IDN5390 can be effectively used once resistance has emerged. In this paper, we analyze the binding modes of these antimitotic agents to type I and III isoforms of β‐tubulin by computational methods. Our results are able to provide a molecular explanation of the experimental data. Using the same protocol, we could also show that no preference for any of the two isoforms can be detected for epothilone A, a potentially very interesting drug for which no data about the emergence of resistance is currently available. Our analysis provides structural insights about the recognition mode and the stabilization mechanism of these antimitotic agents and provides useful suggestions for the design of more potent and selective antimitotic agents.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

The prediction of protein function at CASP6

In the CASP6 experiment, the new “Function Prediction” category was tentatively introduced. Predictors were asked to provide functional information on the CASP targets, many of which were of unknown function. This article describes the setup of the experiment and its results, highlighting what was learned from it, and suggesting modifications to its format for the next rounds. The obvious limitation of such an experiment is that the results cannot be assessed in the standard CASP fashion, as all targets remain of unknown function. Furthermore, we had to face the expected difficulties due to the novelty of the experiment and to the problems connected with function definition. Nevertheless, and even with a limited number of participating groups, we believe that the results of the experiment can be useful both for its future and for experimentalists working on the functional assignment of the CASP6 targets. We found that, in a few cases, a consensus functional prediction could be derived for targets of unknown function. However, our analysis suggests that a general description of the method used should be made available together with the predictions so that a higher reliability can be assigned to cases where completely independent methods give the same or similar predictions. Proteins 2005;Suppl 7:201–213.

Lipolytic isoenzymes from Euphorbia latex

Abstract The activity and substrate specificity of latex lipases from Euphorbia species (E. characias, E. wulfenii, E. pinea, E. myrsinites and E. dendroides) were investigated. High lipolytic activity was found only in E. characias and for the first time in E. wulfenii latex. For both species the lipolytic activity on various triglycerides, and under different temperature and pH conditions, in both crude latex and in partially purified enzymes was quantified. Optimised extraction and purification methods permitted the recovery of the enzymatic fraction with high lipolytic activity. This fraction is probably constituted by a pool of different lipolytic enzymes. Finally, lipolytic activity was also measured for E. characias and E. wulfenii during vegetative and reproductive stages.

Probing the substrate specificity for lipases. II. Kinetic and modeling studies on the molecular recognition of 2-arylpropionic esters by Candida rugosa and Rhizomucor miehei lipases

Racemic arylpropionic esters 1-3, precursors of therapeutically important non-steroidal antiinflammatory drugs, were subjected to hydrolyses in the presence of either Candida rugosa or Rhizomucor miehei crude lipases. The hydrolyses of 1 and 2 proved to be highly enantioselective, whereas 3 was not transformed at all. Both the substrate specificity and the enantioselectivity of these lipases were explained through a molecular modeling study involving docking experiments between 1-3 and the amino acids forming the enzymes active-sites, whose three dimensional structures were obtained from X-ray crystallographic data, followed by extensive conformational analysis on their computer-generated complexes. The results of this study also account for the high enantioselective and good yielding hydrolysis of 3 (as the corresponding 2-chloroethyl ester) catalyzed by CRL pretreated with 2-propanol, recently reported in the literature, and lead to admit that such a treatment may operate very deep conformational changes on the amino acids of the enzyme active-site.

New class of poly(vinyl alcohol) polymers as column-chromatography stationary phases for Candida rugosa lipase isoforms separation

The preparation of new stationary phases of cross-linked polyvinyl alcohol esterified with various linear fatty acids is described. The physico-chemical properties of these polymers are reported, including electron microscopy and swelling measurements. Batch adsorption experiments were performed in order to characterize the basic separative properties of these phases. Cross-linked polyvinyl alcohol esterified with dodecanoic acid was used for hydrophobic interaction chromatography of a commercial crude preparation of Candida rugosa lipase. Characterization of the purified fractions was carried out via native electrophoresis and sodium dodecyl sulfate-polyacrylamide electrophoresis.

A proangiogenic peptide derived from vascular endothelial growth factor receptor-1 acts through α5β1 integrin

Vascular endothelial growth factor receptor-1 (VEGFR-1) is a tyrosine kinase receptor for growth factors of the VEGF family. Endothelial cells express a membrane-bound and a soluble variant of this protein, the latter being mainly considered as a negative regulator of VEGF-A signaling. We previously reported that the soluble form is deposited in the extracellular matrix produced by endothelial cells in culture and is able to promote cell adhesion and migration through binding to alpha5beta1 integrin. In this study, we demonstrate that the Ig-like domain II of VEGFR-1, which contains the binding determinants for the growth factors, is involved in the interaction with alpha5beta1 integrin. To identify domain regions involved in integrin binding, we designed 12 peptides putatively mimicking the domain II surface and tested their ability to inhibit alpha5beta1-mediated endothelial cell adhesion to soluble VEGFR-1 and directly support cell adhesion. One peptide endowed with both these properties was identified and shown to inhibit endothelial cell migration toward soluble VEGFR-1 as well. This peptide directly binds alpha5beta1 integrin, but not VEGF-A, inducing endothelial cell tubule formation in vitro and neoangiogenesis in vivo. Alanine scanning mutagenesis of the peptide defined which residues were responsible for its biologic activity and integrin binding.

Inhibition of endothelial cell migration and angiogenesis by a vascular endothelial growth factor receptor-1 derived peptide.

Vascular endothelial growth factor receptor-1 (VEGFR-1) exists in two isoforms: a membrane-bound isoform (mVEGFR-1) and a soluble one (sVEGFR-1). mVEGFR-1 is involved in endothelial cell migration and survival supported by VEGF-A and placenta growth factor (PlGF), whereas the biologic function of sVEGFR-1 has not been fully elucidated. We previously reported that sVEGFR-1 induces endothelial cell motility and promotes endothelial cell adhesion. In this study, we tested a set of VEGFR-1-derived peptides for their ability to interfere with endothelial cell migration. Peptide B3 was found to specifically inhibit cell migration induced by sVEGFR-1 and by mVEGFR-1-specific ligands. Moreover, peptide B3 markedly hampered angiogenesis in vitro and in vivo and was found to interfere with VEGFR-1 homodimerisation. Altogether, these data demonstrate that peptide B3 might be a useful tool for the specific inhibition of VEGFR-1 function and might represent a basis for the development of new anti-angiogenic compounds.

Revisiting the prediction of protein function at CASP6

The ability to predict the function of a protein, given its sequence and/or 3D structure, is an essential requirement for exploiting the wealth of data made available by genomics and structural genomics projects and is therefore raising increasing interest in the computational biology community. To foster developments in the area as well as to establish the state of the art of present methods, a function prediction category was tentatively introduced in the 6th edition of the Critical Assessment of Techniques for Protein Structure Prediction (CASP) worldwide experiment. The assessment of the performance of the methods was made difficult by at least two factors: (a) the experimentally determined function of the targets was not available at the time of assessment; (b) the experiment is run blindly, preventing verification of whether the convergence of different predictions towards the same functional annotation was due to the similarity of the methods or to a genuine signal detectable by different methodologies. In this work, we collected information about the methods used by the various predictors and revisited the results of the experiment by verifying how often and in which cases a convergent prediction was obtained by methods based on different rationale. We propose a method for classifying the type and redundancy of the methods. We also analyzed the cases in which a function for the target protein has become available. Our results show that predictions derived from a consensus of different methods can reach an accuracy as high as 80%. It follows that some of the predictions submitted to CASP6, once reanalyzed taking into account the type of converging methods, can provide very useful information to researchers interested in the function of the target proteins.