The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP.P(i) being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P(i) release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se

Star forming regions of the southern galaxy

A catalog of southern dust cloud properties is being compiled to aid in the planning and analysis of radio spectral line surveys in the southern hemisphere. Ultimately, images of dust temperature and column density will be produced. For the interim, a list of the 60 and 100 micron fluxes was prepared for the cores and adjacent backgrounds of 65 prominent dust clouds. Dust temperatures and column densities were derived

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

Molecular architecture of softwood revealed by solid-state NMR

Economically important softwood from conifers is mainly composed of the polysaccharides cellulose, galactoglucomannan and xylan, and the phenolic polymer, lignin. The interactions between these polymers lead to wood mechanical strength and must be overcome in biorefining. Here, we use 13C multidimensional solid-state NMR to analyse the polymer interactions in never-dried cell walls of the softwood, spruce. In contrast to some earlier softwood cell wall models, most of the xylan binds to cellulose in the two-fold screw conformation. Moreover, galactoglucomannan alters its conformation by intimately binding to the surface of cellulose microfibrils in a semi-crystalline fashion. Some galactoglucomannan and xylan bind to the same cellulose microfibrils, and lignin is associated with both of these cellulose-bound polysaccharides. We propose a model of softwood molecular architecture which explains the origin of the different cellulose environments observed in the NMR experiments. Our model will assist strategies for improving wood usage in a sustainable bioeconomy

Molecular Mechanisms Used by Salmonella to Evade the Immune System

Human and animal pathogens are able to circumvent, at least temporarily, the sophisticated immune defenses of their hosts. Several serovars of the Gram-negative bacterium Salmonella enterica have been used as models for the study of pathogen-host interactions. In this review we discuss the strategies used by Salmonella to evade or manipulate three levels of host immune defenses: physical barriers, innate immunity and adaptive immunity. During its passage through the digestive system, Salmonella has to face the acidic pH of the stomach, bile and antimicrobial peptides in the intestine, as well as the competition with resident microbiota. After host cell invasion, Salmonella manipulates inflammatory pathways and the autophagy process. Finally, Salmonella evades the adaptive immune system by interacting with dendritic cells, and T and B lymphocytes. Mechanisms allowing the establishment of persistent infections are also discussed.European Regional Development Fund SAF2013-46229-R, SAF2016-75365-