Nou mètode per identificar els dos extrems de les proteïnes

Investigadors de la Universitat de Gant (Bèlgica) i de la UAB han desenvolupat un nou procediment per identificar els dos extrems de les molècules de proteïnes, i el seu processament-maduració, en estudis de proteòmica massiva in-vivo o ex-vivo. El treball ha estat publicat on line a Nature Methods, amb el títol "Complementary Positional Proteomics for Screening of Endo- and Exoproteases". El nou mètode desenvolupat permet ampliar aquests estudis a l'extrem C-terminal de les proteïnes, aquell que té el grup carboxil, que es considera que constitueix el final de les cadenes lineals d'aminoàcids que les formen.Investigadores de la Universidad de Gante (Bélgica) y de la UAB han desarrollado un nuevo procedimiento para identificar los dos extremos de las moléculas de proteínas, y su procesamiento-maduración, en estudios de proteómica masiva in-vivo o ex-vivo. El trabajo ha sido publicado on line en Nature Methods, con el título "ComplementaryPositional Proteomics for Screening of Endo- and Exoproteases". El nuevo método desarrollado permite ampliar estos estudios al extremo C-terminal de las proteínas, aquel que tiene el grupo carboxilo, que se considera constituye el final de las cadenas lineales de aminoácidos que las forman.Researchers at University of Ghent, Belgium, and Universitat Autòno made Barcelona developed a new procedure to identify the two extremes of protein molecules and their processing and maturing in in-vivo and ex-vivo massive proteomic studies. The research has been published online at Nature Methods, under the title of "Complementary PositionalProteomics for Screening of Endo- and Exoproteases". The new method developed by scientists helps to expand studies to C-terminal proteins, proteins with a free carboxyl group, at the end of the linear chain of amino acid

Descrit un nou sistema de regulació de proteïnes

Una recerca internacional amb participació de l'IBB i la UAB ha caracteritzat un nou i potent inhibidor proteic d'un mol·lusc tropical marí anomenat Nerita versicolor. La recerca ha estat publicada al Journal of Biological Chemistry i s'ha centrat en les carboxipeptidasesUna investigación internacional con participación del IBB y la UAB ha caracterizado un nuevo y potente inhibidor proteico de un molusco tropical marino llamado Nerita versicolor. La investigación, que ha sido publicada en el Journal of Biological Chemistry, se ha centrado en las carboxipeptidasas, unas proteínas cuya actividad es clave para numerosos procesos biológicos.An international research with the participation of IBB and UAB characterised a new and strong protein inhibitor of the tropical marine mollusc Nerita versicolor. The research was published in the Journal of Biological Chemistry and is focused on carboxypeptidases, proteins playing a key role in numerous biological processes

Molècules fluorescents per a crear inhibidors de proteases

Un grup d'investigadors de l'Institut de Biotecnologia i de Biomedicina i del Departament de Bioquímica i de Biologia Molecular de la UAB, dirigit pel catedràtic Francesc Xavier Avilés, està coordinant un projecte europeu que estudia les proteases en situacions normals i de malaltia mitjançant marcadors fluorescents que permeten monitoritzar-les. Es tracta del projecte Chemical Genomics by Activity Monitoring of Proteases (CAMP), i té com a objectiu final produir fàrmacs contra les proteases en situacions d'inflamació, malalties cardiovasculars, càncer i neurodegeneració.Un grupo de investigadores del Instituto de Biotecnología y Biomedicina y del Departamento de Bioquímica y de Biología Molecular de la UAB, dirigido por el catedrátic Francesc Xavier Avilés, está coordinando un proyecto europeo que estudia las proteasas en situaciones normales y de enfermedad mediante marcadores fluorescentes que permiten monitoritzarlas. Se trata del proyecto Chemical Genomics by Activity Monitoring of Proteases (CAMP), y tiene como objetivo final producir fármacos contra las proteasas en situaciones de inflamación, enfermedades cardiovasculares, cáncer y neurodegeneración.A research group of the UAB Institute of Biotechnology and Biomedicine and the UAB Department of Biochemistry and Molecular Biology, directed by Francesc Xavier Avilés, is the coordinator of a European project aimed at studying proteases with fluorescent molecules that allow their monitoring in healthy and pathological situations. Entitled "Chemical Genomics by Activity Monitoring of Proteases" (CAMP), the project was designed with the final objective of developing drugs to control proteases in inflammations, cardiovascular diseases, cancer and neurodegeneration

Discovery and characterizatopn of small molecular weight metallocarboxypeptidase inhibitors

Descripció del recurs: el 02 de novembre de 2010Las hidrolasas son enzimas que catalizan la ruptura del enlace amida o peptódico, y por lo tanto son denominadas también proteasas o peptidasas. Las proteasas constituyen cerca del 2 % del genoma humano, lo que representa unos 600 productos génicos. De acuerdo con el residuo catalóticamente activo, existen seis grandes grupos de peptidasas. En este trabajo nos centraremos en la familia M14 de peptidasas, también denominadas metalocarboxipeptidasas (CPs) debido a que su actividad catalótica reside en el ion zinc presente en el sitio activo de la enzima. En el genoma humano, se han identificado al menos 26 genes que codifican carboxipeptidasas. Las peptidasas de la familia M14 que actúan en el tracto gastrointestinal son las principales metaloproteasas responsables de la obtención de aminoácidos libres de la proteína de la dieta. En otros compartimientos corporales, las CPs pueden llevar a cabo tareas especializadas y altamente reguladas como ser la maduración de neuropéptidos, citokinas y hormonas peptódicas. En algunos casos, una actividad catalótica fuera de control puede conducir a enfermedades. Cada vez existe una mayor evidencia experimental que demuestra la actividad carboxipeptidasa en procesos como la pancreatitis aguda, la diabetes, la inflamación, la fibrinólisis y el cáncer. A pesar de ciertos avances en algunos aspectos, la actividad específica de las CPs es pobremente conocida. Además, las carboxipeptidasas son blancos terapéuticos interesantes para el desarrollo de fármacos, y por lo tanto se ha decidido emplear una aproximación multi-disciplinaria para la identificación y caracterización de nuevas moléculas de bajo peso molecular capaces de interferir la actividad carboxipeptidasa. Así, en este trabajo se han combinado modernas herramientas computacionales, screening in vitro, modelado molecular y cristalografía de rayos X con el fin de obtener nuevas entidades quφmicas como base para el desarrollo de fármacos. Con base en herramientas computacionales, aplicando el método de Optimal Docking Areaö, se han caracterizado sitios de unión proteína-proteína y proteína-ligando en la superficie de las peptidasas de la familia M14. A partir de aquí, se identificó una nueva clase de compuestos químicos capaces de explotar las diferencias existentes entre enzimas de la familia por unión a regiones hidrofóbicas. Otros inhibidores fueron identificados mediante un screening in silico de grandes colecciones de compuestos. Ensayos in vitro demostraron que los compuestos líderes inhibieron de manera potente a las carboxipeptidasas blanco con otras características interesantes como la posibilidad de coordinación del ion zinc catalítico por intermedio de un anillo oxadiazol. A través de una colaboración con el Departamento de Química Orgánica se obtuvieron y caracterizaron nuevos compuestos químicos con conectividades atómicas novedosas que, inesperadamente, demostraron ser potentes inhibidores de carboxipeptidasas. Una clase adicional de molécula de bajo peso molecular caracterizada corresponde a inhibidores que se unen covalentemente al enzima blanco. En este caso, se logró obtener la estructura tridimensional del complejo a resolución atómica mediante cristalografφa de rayos X, lo que ha permitido el dise±o basado en la estructura de una nueva generación de compuestos. Basados en otros datos de cristalografía de rayos X y análisis computacional, se ha revisado y ampliado el mecanismo de acción catalítica de las peptidasas de la familia M14 a partir de una nueva forma cristalina de CPB a alta resolución. En conjunto, nuestro trabajo ha permitido la obtención de nuevas moléculas líderes de bajo peso molecular que podrían servir como base para futuros desarrollos en el diseño de fármacos y agentes de diagnóstico o imaginería dirigidos a metalocarboxipeptidasas fisiológicamente activas.Hydrolases are enzymes catalyzing the breakdown of the amide or peptide bond, and are therefore called proteases or peptidases as well. In the human genome, proteases made up about 2% of the genome, or about 600 gene products. There are six major groups of peptidases according to the catalytic residue. In our work we focused on the M14 family of peptidases, also called metallocarboxypeptidases (CPs) because of their catalytic activity hinges on the zinc ion present in the active site of the enzyme. In the human genome there are identified at least 26 genes encoding for CPs. M14 peptidases in the gastrointestinal tract are the main metalloproteases responsible of the liberation of free aminoacids from the protein content of the diet. In other compartments of the body, CPs may perform specialized and tightly controlled tasks such as neuropeptide, cytokine and hormone maturation. In some instances an imbalance in their activity leads to disease states in man. Increasing evidence shows carboxypeptidase involvement in acute pancreatitis, diabetes, inflammation, fibrinolysis and cancer. Although some aspects have become clearer, much of their activity remain poorly understood. Besides, carboxypeptidases are interesting targets for drug development, and therefore we pursued a multidisciplinary approach to identify and characterize novel small molecular weight compounds able to interfere carboxypeptidase activity. In this work we combined modern computational tools, in vitro screening, molecular modelling and X-ray crystallography to obtain new chemical entities useful as scaffolds for drug design. Based on a bioinformatics tools, the Optimal Docking Area method, we identified protein-protein and protein-ligand binding sites over the surface of M14 peptidases. This knowledge was employed to find out a new class of small molecular weight inhibitors which exploit the differential binding provided by hydrophobic patches. A further class of inhibitors was identified from in silico screening of collections of compounds. In vitro analysis revealed that the leads were potent inhibitors against the target proteases with interesting features like an oxadiazole zinc-chelating moiety. Compounds obtained from the Organic Chemistry Department were also screened, and unexpectedly, afforded some good inhibitors with unprecedented atomic bonding. One further class involved inhibitors that attach covalently to the target enzyme. In this case the structure of the complex obtained at high resolution by X-ray crystallography allowed the structure-guided design of new generation of compounds. The catalytic mechanism of M14 peptidases was also revisited based on our crystallographic and computational analysis of a new CPB crystal form at high resolution. Overall, our study provided new lead small molecular weight inhibitors which can be the foundation for further developments in the design of drugs and bioimaging or diagnostic agents targeted to physiologically-relevant metallocarboxypeptidases

Self-assembly of human latexin into amyloid-like oligomers

Background: In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations. Results: Here, we show that human latexin, a protein that shares the same fold with cystatin C, assembles into stable spherical amyloid-like oligomers that bind thioflavin-T and congo red similarly to common amyloid structures but do not evolve into fibrils. Latexin self-assembly correlates with the formation of a mostly denaturated state rather than with the population of partially structured intermediates during the unfolding process. The results suggest that unfolding of α-helix 3 might be involved in the transition of latexin toward amyloidotic species, supporting the notion of the protective role of the native protein structure against polymerization. Conclusion: Overall the data herein indicate that latexin could be a good model for the study of the structural and sequential determinants of oligomeric assemblies in protein aggregation processes

The X-ray structure of carboxypeptidase a inhibited by a thiirane mechanism-based ihibitor

The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 Å resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1' specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.Fil: Fernández, Daniel. Universitat Autònoma de Barcelona; EspañaFil: Testero, Sebastian Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. University of Notre Dame; Estados UnidosFil: Vendrell, Josep. Universitat Autònoma de Barcelona; EspañaFil: Avilés, Francesc X.. Universitat Autònoma de Barcelona; EspañaFil: Mobashery, Shahriar. University of Notre Dame; Estados Unido

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale

Prediction of "hot spots" of aggregation in disease-linked polypeptides

BACKGROUND: The polypeptides involved in amyloidogenesis may be globular proteins with a defined 3D-structure or natively unfolded proteins. The first class includes polypeptides such as β2-microglobulin, lysozyme, transthyretin or the prion protein, whereas β-amyloid peptide, amylin or α-synuclein all belong to the second class. Recent studies suggest that specific regions in the proteins act as "hot spots" driving aggregation. This should be especially relevant for natively unfolded proteins or unfolded states of globular proteins as they lack significant secondary and tertiary structure and specific intra-chain interactions that can mask these aggregation-prone regions. Prediction of such sequence stretches is important since they are potential therapeutic targets. RESULTS: In this study we exploited the experimental data obtained in an in vivo system using β-amyloid peptide as a model to derive the individual aggregation propensities of natural amino acids. These data are used to generate aggregation profiles for different disease-related polypeptides. The approach detects the presence of "hot spots" which have been already validated experimentally in the literature and provides insights into the effect of disease-linked mutations in these polypeptides. CONCLUSION: The proposed method might become a useful tool for the future development of sequence-targeted anti-aggregation pharmaceuticals

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale