Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen

The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37 degrees C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the beta-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation inter-mediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour, which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism

Electrochemical studies on small electron transfer proteins using membrane electrodes

Journal of Electroanalytical Chemistry 541 (2003) 153-162Membrane electrodes (ME) were constructed using gold, glassy carbon and pyrolytic graphite supports and a dialysis membrane, and used to study the electrochemical behavior of small size electron transfer proteins: monohemic cytochrome c522 from Pseudomonas nautica and cytochrome c533 as well as rubredoxin from Desulfovibrio vulgaris . Different electrochemical techniques were used including cyclic voltammetry (CV), square wave voltammetry (SW) and differential pulse voltammetry (DP). A direct electrochemical response was obtained in all cases except with rubredoxin where a facilitator was added to the protein solution entrapped between the membrane and the electrode surface. Formal potentials and heterogeneous charge transfer rate constants were determined from the voltammetric data. The influence of the ionic strength and the pH of the medium on the electrochemical response at the ME were analyzed. The benefits from the use of the ME in protein electrochemistry and its role in modulating the redox behavior are analyzed. A critical comparison is presented with data obtained at non-MEs. Finally, the interactions that must be established between the proteins and the electrode surfaces are discussed, thereby modeling molecular interactions that occur in biological systems

Calpain 3 Is a Rapid-Action, Unidirectional Proteolytic Switch Central to Muscle Remodeling

Calpain 3 (CAPN3) is a cysteine protease that when mutated causes Limb Girdle Muscular Dystrophy 2A. It is thereby the only described Calpain family member that genetically causes a disease. Due to its inherent instability little is known of its substrates or its mechanism of activity and pathogenicity. In this investigation we define a primary sequence motif underlying CAPN3 substrate cleavage. This motif can transform non-related proteins into substrates, and identifies >300 new putative CAPN3 targets. Bioinformatic analyses of these targets demonstrate a critical role in muscle cytoskeletal remodeling and identify novel CAPN3 functions. Among the new CAPN3 substrates are three E3 SUMO ligases of the Protein Inhibitor of Activated Stats (PIAS) family. CAPN3 can cleave PIAS proteins and negatively regulates PIAS3 sumoylase activity. Consequently, SUMO2 is deregulated in patient muscle tissue. Our study thus uncovers unexpected crosstalk between CAPN3 proteolysis and protein sumoylation, with strong implications for muscle remodeling

A New Constraint Solver for 3D Lattices and Its Application to the Protein Folding Problem

The paper describes the formalization and implementation of an efficient constraint programming framework operating on 3D crystal lattices. The framework is motivated and applied to address the problem of solving the abinitio protein structure prediction problem - i.e., predicting the 3D structure of a protein from its amino acid sequence. Experimental results demonstrate that our novel approach offers up to a 3 orders of magnitude of speedup compared to other constraint-based solutions proposed for the problem at hand

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen

Modulation of the proteolytic activity of matrix metalloproteinase-2 (gelatinase A) on fibrinogen Susanna MONACO*, Magda GIOIA*†, Janet RODRIGUEZ‡, Giovanni Francesco FASCIGLIONE*, Donato DI PIERRO*, Giulio LUPIDI§, Ludwig KRIPPAHL_, Stefano MARINI* and Massimo COLETTA*†1 *Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy, †Interuniversity Consortium on the Metal Chemistry in Biological Systems (CIRCMSB), P.za Umberto I 1, I-70100 Bari, Italy, ‡Laboratorio de Farmacologia, Centro de Quimica Farmaceutica, 200 y 21, Atabey, 21, Atabey, Playa, P.O. Box 16042, La Habana, Cuba, §Department of Molecular, Cellular and Animal Biology, University of Camerino, Via F. Camerini 2, I-62032 Camerino, Italy, and _Departamento de Quimica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal The proteolytic processing of bovine fibrinogen by MMP-2 (gelatinase A), which brings about the formation of a product unable to form fibrin clots, has been studied at 37◦C. Catalytic parameters, although showing a somewhat lower catalytic efficiency with respect to thrombin and plasmin, indeed display values indicating a pathophysiological significance of this process. A parallel molecular modelling study predicts preferential binding of MMP-2 to the β-chain of fibrinogen through its haemopexin-like domain, which has been directly demonstrated by the inhibitory effect in the presence of the exogenous haemopexin-like domain. However, the removal of this domain does not impair the interaction between MMP-2 and fibrinogen, but it dramatically alters the proteolytic mechanism, producing different fragmentation intermediates. The investigation at various pH values between 6.0 and 9.3 indicates a proton-linked behaviour,which is relevant for interpreting the influence on the process by environmental conditions occurring at the site of an injury. Furthermore, the action of MMP-2 on peroxynitrite-treated fibrinogen has been investigated, a situation possibly occurring under oxidative stress. The chemical alteration of fibrinogen, which has been shown to abolish its clotting activity, brings about only limited modifications of the catalytic parameters without altering the main enzymatic mechanism. Key words: fibrinogen, fragmentation, gelatinase A, kinetics, molecular modelling, pH-dependence

Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome

Paracoccus denitrificans can accommodate horse cytochrome c and Paracoccus cytochrome c550 at different sites on its molecular surface. Here we use 1H NMR spectroscopy, analytical ultracentrifugation, molecular docking simulation, and microcalorimetry to investigate whether these small cytochromes can be accommodated simultaneously in the formation of a ternary complex. The pattern of perturbation of heme methyl and methionine methyl resonances in binary and ternary solutions shows that a ternary complex can be formed, and this is confirmed by the increase in the sedimentation coefficient upon addition of horse cytochrome c to a solution in which cytochrome c550 fully occupies its binding site on cytochrome c peroxidase. Docking experiments in which favored binary solutions of cytochrome c550 bound to cytochrome c peroxidase act as targets for horse cytochrome c and the reciprocal experiments in which favored binary solutions of horse cytochrome c bound to cytochrome c peroxidase act as targets for cytochrome c550 show that the enzyme can accommodate both cytochromes at the same time on adjacen

PROVA: Rule-based Java-scripting for a bioinformatics semantic web

Abstract. Transparent information integration across distributed and heterogeneous data sources and computational tools is a prime concern for bioinformatics. Recently, there have been proposals for a semantic web addressing these requirements. A promising approach for such a semantic web are the integration of rules to specify and implement workflows and object-orientation to cater for computational aspects. We present PROVA, a Java-based rule-engine, which realises this integration. It enables one to separate a declarative description of information workflows from any implementation details and thus easily create and maintain code. We show how PROVA is used to compose an information and computation workflow involving – rules for specifying the workflow, – rules for reasoning over the data, – rules for accessing flat files, databases, and other services, and – rules involving heavy-duty computations. The resulting code is very compact and re-usable. We give a detailed account of PROVA and document its use with a example of a system, PSIMAP, which derives domain-domain interactions from multidomain structures in the PDB using the SCOP domain and superfamily definitions. PSIMAP is a typical bioinformatics application in that it integrates disparate information resources in different formats (flat files (PDB) and database (SCOP)) requiring additional computations. PROVA is available at comas.soi.city.ac.uk/prova Keywords: Rules, Reasoning, Declarative and Object oriented Programming, Workflows.