1,721,096 research outputs found
Structural and evolutionary insights into astacin metallopeptidases
No full textThe astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a “Met-turn” methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors
Table1_Structural and evolutionary insights into astacin metallopeptidases.xlsx
No full textThe astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a “Met-turn” methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.</p
DataSheet1_Structural and evolutionary insights into astacin metallopeptidases.DOCX
No full textThe astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a “Met-turn” methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.</p
Substrate specificity of a metalloprotease of the pappalysin family revealed by an inhibitor and a product complex
No full textDevelopment of efficient eukaryotic and bacterial expression systems for functional studies of recombinant proteins of biomedical interest
Full text linkDepartament responsable de la tesi: Departament de Bioquímica i Biologia Molecular.Al llarg aquesta tesi s'han fet molts esforços per desenvolupar un sistema d'expressió heteròloga de glicoproteïnes humanes d'alt rendiment, puresa i homogeneïtat, així amb la flexibilitat necessària per modificar la proteïna a voluntat. Hem desenvolupat dos sistemes d'expressió a eucariotes, uno a cèllules de Drosophila Schneider 2, per a l'expressió inductible mitjançant l'addició de Cu2SO4 i per a l'expressió transitòria amb transfecció de polietilenimina (PEI); i un per a mamífer, un sistema d'expressió transitòria basat en cèl·lules Expi293F i transfecció de PEI. La proteòlisi és una part fonamental de la rotació de proteïnes en organismes vius i el seu desequilibri pot comportar malalties, provocant inflamacions, malalties neurodegeneratives, càncer o mort cel·lular entre altres trastorns. Per tant, els mecanismes que regulen l'activitat de la peptidasa s'han de regular de manera estricta per garantir l'activitat proteolítica adequada en el moment i lloc adequats. La queilis-isozima 1 de la subtilisina humana (hSKI1) és la proteïna serina que catalitza el primer pas a l'activació proteolítica de les proteïnes d'unió de l'element regulador d'esterol (SREBPs), regulador clau del metabolisme dels lípids intracel·lulars. Aquesta proteasa és una proteïna transmembrana localitzada al reticle endoplasmàtic i l'aparell de Golgi i la seva maduració és un procés molt complex. Vam aconseguir una expressió soluble als sistemes d'expressió d'insectes i mamífers i establim les bases per a futurs estudis bioquímics i estructurals amb garanties d'èxit. Al segon projecte, el principal inhibidor de la proteasa del plasma (la α2-macroglobulina humana, hα2M), que neutralitza un ampli espectre d'endopeptidases, però també modula l'activitat de les citocines, hormones, factors de creixement i altres proteïnes i, per tant, per tant té un gran impacte en la fisiologia humana. Es van realitzar assajos bioquímics, biofísics, estructurals i d'unió amb altres dianes interessants (Streptococcus GRAB, TGFβ2 humà i LRP1 humà) en complexos amb hα2M autèntics i recombinants per a caracteritzar aquestes interaccions. Per tant, també vam expressar les variants hα2M en cèl·lules eucariotes i vam purificar l'autèntica proteïna de la sang. A més, expressem pro-TGFβ2 a cèllules d'insecte i mamífer i cristalizamos la forma madura de TGFβ2. I finalment, vam revisar la literatura disponible sobre la família de les metaloproteinases de matriu (MMP) fora de vertebrats i vam realitzar cerques a la base de dades de dominis catalítics de MMP potencials en invertebrats, plantes, fongs, virus, protistes, arqueus i bacteris. En general, la present tesi ha establert nous i prometedors sistemes d'expressió al laboratori, que han beneficiat i ha contribuït substancialment al camp als nivells d'expressió, purificació i cristal·lització de la proteïna serina hSKI1 i l'inhibidor de la proteasa hα2M, sols o interactuant amb GRAB i TGFβ2.En esta tesis, se han realizado esfuerzos para desarrollar un sistema para la expresión heteróloga de glicoproteínas humanas con alto rendimiento, pureza y homogeneidad, así como la flexibilidad necesaria para diseñar la proteína a voluntad. Desarrollamos dos sistemas de expresión eucariotas, uno en células de Drosophila Schneider 2, (expresión inducible mediante la adición de Cu2SO4 y expresión transitoria con transfección con polietilenimina (PEI)); y otro en células de mamífero, (un sistema de expresión transitoria basado en células Expi293F y transfección con PEI). La proteólisis es una parte fundamental del recambio proteico en los organismos vivos y su desequilibrio puede conducir enfermedades, causando inflamación, enfermedades neurodegenerativas, cáncer o muerte celular, entre otros trastornos. Por lo tanto, los mecanismos que regulan la actividad de peptidasas deben estar exquisitamente regulados para garantizar una actividad proteolítica adecuada en el momento y lugar correctos. La subtilisina kexina-isozima 1 humana (hSKI1) es la serina proteasa que cataliza el primer paso en la activación proteolítica de las proteínas de unión al elemento regulador de esteroles (SREBP), regulador clave del metabolismo de los lípidos intracelulares. Esta proteasa es una proteína transmembrana localizada en el retículo endoplásmico y el aparato de Golgi y su maduración es un proceso muy complejo. Logramos una expresión soluble en los sistemas de expresión de insecto y mamífero y sentamos las bases para futuros estudios bioquímicos y estructurales. En el segundo proyecto, se ha estudiado el principal inhibidor de proteasas del plasma (la α2-macroglobulina humana, hα2M), que tiene un gran impacto en la fisiología humana ya que neutraliza un amplio espectro de endopeptidasas, pero también modula la actividad de las citocinas, hormonas, factores de crecimiento y otras proteínas. Se realizaron ensayos bioquímicos, biofísicos, estructurales y de unión con otras proteínas interesantes (G-related α2-macroglobulin binding protein de Streptococcus pyogenes; human transforming growth factor-β2, TGFβ2; and human lipoprotein receptor-related protein 1, LRP1) en complejo con endógena y recombinante hα2Ms para caracterizar estas interacciones. Por lo tanto, también expresamos las variantes hα2M en células eucariotas y purificamos la proteína endógena de sangre. Además, expresamos pro-TGFβ2 en células de insecto y mamífero y cristalizamos la forma madura de TGFβ2. Y finalmente, revisamos la literatura disponible sobre la familia de metaloproteinasas de matriz (MMP) fuera de los vertebrados y realizamos búsquedas en la base de datos para potenciales dominios catalíticos de MMP en invertebrados, plantas, hongos, virus, protistas, arqueas y bacterias. En general, la presente tesis ha establecido nuevos y prometedores sistemas de expresión en el laboratorio y ha contribuido sustancialmente al campo en los niveles de expresión, purificación y cristalización en la serina proteasa hSKI1 y el inhibidor de proteasas de la sangre hα2M, solo o interactuando con GRAB y TGFβ2.In this thesis, efforts have been made to develop a system for heterologous expression of human glycoproteins with high yield, purity and homogeneity, as well as the necessary flexibility to engineer the protein at will. We established two eukaryotic expression systems, based in Drosophila Schneider 2 cells (inducible expression of stable cell lines by addition of Cu2SO4 and transient expression with polyethylenimine (PEI) transfection) and in mammalian cells (transient expression on Expi293F cells with PEI transfection). Proteolysis is a fundamental part of the protein turnover in living organisms and its imbalance may lead to diseases, causing inflammation, neurodegenerative diseases, cancer or cell death among other disorders. Thus, mechanisms that regulate peptidase activity must be exquisitely regulated to ensure proper proteolytic activity at the right time and place. The human subtilisin kexin-isozyme 1 (hSKI1) is the serine protease which catalyzes the first step in the proteolytic activation of the sterol regulatory element binding proteins (SREBPs), key regulator of the intracellular lipid metabolism. This protease is a transmembrane protein localized in endoplasmic reticulum and Golgi apparatus and its maturation is a very complex process. We achieved to express it soluble in the insect and mammalian expression systems and laid the foundations for future biochemical and structural studies. In the second project, the main protease inhibitor of the plasma blood has been studied (the human α2-macroglobulin, hα2M), which has great impact on human physiology by participating in inhibition of a broad spectrum of endopeptidases, but also by modulating the activity of cytokines, hormones, growth factors and other proteins. Biochemical, biophysical, structural and binding assays with other interesting targets (G-related α2-macroglobulin binding protein from Streptococcus pyogenes, GRAB; human transforming growth factor-β2, TGFβ2; and human lipoprotein receptor-related protein 1, LRP1) in complex with wild-type and recombinant hα2Ms were performed to characterize these interactions. Therefore, we expressed the hα2M variants in eukaryotic cells and purified the endogenous protein from blood. Furthermore, we achieved to express soluble pro-TGFβ2 in insect and mammalian cells and we managed to crystallize the mature part of this protein. And finally, we reviewed the available literature on the matrix metalloproteinase (MMP) family outside vertebrates and performed database searches for potential MMP catalytic domains in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. Overall, the present thesis has established new and promising expression systems at the laboratory and has contributed substantially to the field at the expression, purification and crystallization levels on the serine protease hSKI1 and protease inhibitor hα2M, alone or interacting with GRAB and TGFβ2
Biologia Molecular i del Desenvolupament
No full textComunicación presentada en el II Congrés de Biologia de l'sbc, celebrado en Barcelona (España) del 3 al 4 de mayo de 2018
The Human Microbiome. Present Status and Future Prospects
No full textConferencia presentada en B-Debate. International Center for Science Debate, celebrada en Barcelona, el 2 y 3 de julio de 2015Peer Reviewe
A different look for AB5 toxins
No full textMetzincins are a distinct clan of metallopeptidases encompassing several families. In this issue of Structure, Ng and colleagues describe the results of the structural analysis of the toxilysins, a novel family of metzincins employed by gastroinfective bacteria as intracellular virulence factors following host cell invasion. © 2013 Elsevier Ltd.Peer Reviewe
Tannerella forsythia-derived protease inhibitors ¿ Foes or allies in the pathobiology of periodontitis?
No full textTrabajo presentado en FEBS: Design and engineering of synthetic biosystems, celebrado en la Isla Spetses (Grecia) del 9 al 17 de septiembre de 2018
Structure and mechanism of metallocarboxypeptidases
No full textMetallocarboxpeptidases cleave C-terminal residues from peptide substrates and participate in a wide range of physiological processes, but they also contribute to human pathology. On the basis of structural information, we can distinguish between two groups of such metallopeptidases: cowrins and funnelins. Cowrins comprise protozoan, prokaryotic, and mammalian enzymes related to both neurolysin and angiotensin-converting enzyme and their catalytic domains contain 500-700 residues. They are ellipsoidal and traversed horizontally by a long, deep, narrow active-site cleft, in which the C-terminal residues are cut from oligopeptides and unstructured protein tails. The consensus cowrin structure contains a common core of 17 helices and a three-stranded β-sheet, which participates in substrate binding. This protease family is characterized by a set of spatially conserved amino acids involved in catalysis, HEXXH+EXXS/G+H+Y/R+Y. Funnelins comprise structural relatives of the archetypal bovine carboxypeptidase A1 and feature mammalian, insect and bacterial proteins with strict carboxypeptidase activity. Their ∼ 300-residue catalytic domains evince a consensus central eight-stranded β-sheet flanked on either side by a total of eight helices. They also contain a characteristic set of conserved residues, HXXE+R+NR+H+Y+E, and their active-site clefts are rather shallow and lie at the bottom of a funnel-like cavity. Therefore, these enzymes act on a large variety of well-folded proteins. In both cowrins and funnelins, substrate hydrolysis follows a common general base/acid mechanism. A metal-bound solvent molecule ultimately performs the attack on the scissile peptide bond with the assistance of a strictly conserved glutamate residue. Copyright © Informa UK Ltd.Peer Reviewe
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