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A lvaro-Benito M.,Autonomous University of Madrid | De Abreu M.,Autonomous University of Madrid | Portillo F.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | Sanz-Aparicio J.,CSIC - Institute of Physical Chemistry "Rocasolano" | Fernandez-Lobato M.,Autonomous University of Madrid
Applied and Environmental Microbiology | Year: 2010

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k cat/K m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymeś specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Rada P.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | Rojo A.I.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | Rojo A.I.,Complutense University of Madrid | Evrard-Todeschi N.,University of Paris Descartes | And 11 more authors.
Molecular and Cellular Biology | Year: 2012

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3βexhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3. © 2012, American Society for Microbiology.

Fraga R.,University of Santiago de Compostela | Zacconi F.,University of Santiago de Compostela | Sussman F.,University of Santiago de Compostela | Ordonez-Moran P.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | And 7 more authors.
Chemistry - A European Journal | Year: 2012

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D 3 (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3 a, 3 b, 4 a, 4 b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3 a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Maldonado A.A.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | Ribeiro J.M.,University of Extremadura | Sillero A.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC
Biochemistry and Molecular Biology Education | Year: 2010

The main object of this work is to present the pedagogical usefulness of the theoretical methods, developed in this laboratory, for the determination of the isoelectric point (pI) and the net electric charge of proteins together with some comments on the naming of the acid-base residues of proteins. © 2010 by The International Union of Biochemistry and Molecular Biology.

Herranz R.,Institute Investigaciones Biomedicas Alberto Sols UAM CSIC | Herranz R.,CSIC - Biological Research Center | BengurIa A.,CSIC - National Center for Biotechnology | BengurIa A.,Genomics Unit | And 6 more authors.
Molecular Ecology | Year: 2010

Genome-wide transcriptional profiling shows that reducing gravity levels during Drosophila metamorphosis in the International Space Station (ISS) causes important alterations in gene expression: a large set of differentially expressed genes (DEGs) are observed compared to 1g controls. However, the preparation procedures for spaceflight and the nonideal environmental conditions on board the ISS subject the organisms to additional environmental stresses that demonstrably affect gene expression. Simulated microgravity experiments performed on the ground, under ideal conditions for the flies, using the random position machine (RPM), show much more subtle effects on gene expression. However, when the ground experiments are repeated under conditions designed to reproduce the additional environmental stresses imposed by spaceflight procedures, 79% of the DEGs detected in the ISS are reproduced by the RPM experiment. Gene ontology analysis of them shows they are genes that affect respiratory activity, developmental processes and stress-related changes. Here, we analyse the effects of microgravity on gene expression in relation to the environmental stresses imposed by spaceflight. Analysis using 'gene expression dynamics inspector' (GEDI) self-organizing maps reveals a subtle response of the transcriptome to microgravity. Remarkably, hypergravity simulation induces similar response of the transcriptome, but in the opposite direction, i.e. the genes promoted under microgravity are usually suppressed under hypergravity. These results suggest that the transcriptome is finely tuned to normal gravity and that microgravity, together with environmental constraints associated with space experiments, can have profound effects on gene expression. © 2010 Blackwell Publishing Ltd.

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