Fundacion ARAID

Molina de Aragón, Spain

Fundacion ARAID

Molina de Aragón, Spain

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Hurtado-Guerrero R.,Fundacion ARAID | Hurtado-Guerrero R.,University of Zaragoza
Biochemical Society Transactions | Year: 2016

Protein O-GalNAcylation is an abundant post-translational modification and predicted to occur in over 80 % of the proteins passing through the Golgi apparatus. This modification is driven by 20 polypeptide GaINAc (N-acetylgalactosamine)-transferases (GalNAc-Ts), which are unique in that they possess both catalytic and lectin domains. The peptide substrate specificities of GalNAc-Ts are still poorly defined and our understanding of the sequence and structural features that direct O-glycosylation of proteins is limited. Part of this may be attributed to the complex regulation by coordinated action of multiple GalNAc-T isoforms, and part of this may also be attributed to the two functional domains of GalNAc-Ts that both seems to be involved in directing the substrate specificities. Recent studies have resulted in 3D structures of GalNAc-Ts and determination of the reaction mechanism of this family of enzymes. Key advances include the trapping of binary/ternary complexes in combination with computational simulations and AFM/small-SAXS experiments, which have allowed for the dissection of the reaction coordinates and the mechanism by which the lectin domains modulate the glycosylation. These studies not only broaden our knowledge of the modes-of-action of this family of enzymes but also open up potential avenues for the rational design of effective and selective inhibitors of O-glycosylation. © 2016 Authors; published by Portland Press Limited.


Lucas I.,Fundacion ARAID | Lucas I.,University of Zaragoza | Vila-Fungueirino J.M.,University of Santiago de Compostela | Jimenez-Cavero P.,University of Zaragoza | And 5 more authors.
ACS Applied Materials and Interfaces | Year: 2014

We report magnetic and electronic transport measurements across epitaxial bilayers of ferromagnetic insulator LaCoO3 and half-metallic ferromagnet La2/3Sr1/3MnO3 (LCO/LSMO: 3.5 nm/20 nm) fabricated by a chemical solution method. The I-V curves at room temperature and 4K measured with conducting atomic force microscopy (CAFM) on well-defined patterned areas exhibit the typical features of a tunneling process. The curves have been fitted to the Simmons model to determine the height (φ) and width (s) of the insulating LCO barrier. The results yield φ = 0.40 ± 0.05 eV (0.50 ± 0.01 eV) at room temperature (4K) and s = 3 nm, in good agreement with the structural analysis. Our results demonstrate that this chemical method is able to produce epitaxial heterostructures with the quality required for this type of fundamental studies and applications. © 2014 American Chemical Society.


Hurtado-Guerrero R.,Fundacion ARAID | Hurtado-Guerrero R.,University of Zaragoza | Davies G.J.,University of York
Current Opinion in Chemical Biology | Year: 2012

Enzymatic glycosylation of proteins, a post-transitional modification of great significance, is carried out by diverse glycosyltransferases (GTs) that harness activated sugar donors, typically nucleotide or lipid-phosphate linked species. Recent work has seen a major increase in the study of the 3D structure and reaction mechanism of these enzymes. Key advances include the dissection of the classical O-glycosylating and N-glycosylating apparatus, revealing unusual folds and hitherto unconsidered chemical mechanisms for acceptor activation. There has been considerable success in the application of kinetic isotope effects and quantum simulations to address the controversial issue of the reaction mechanism of retaining GTs. New roles for old modifications, exemplified by potential epigenetic roles for glycosylation, have been discovered and there has also been a plethora of studies into important mammalian glycosylations that play key roles in cellular biology, opening up new targets for chemical intervention approaches. © 2012 Elsevier Ltd.


Bryson J.F.J.,University of Cambridge | Herrero-Albillos J.,Fundacion ARAID | Herrero-Albillos J.,Centro Universitario Of La Defensa | Herrero-Albillos J.,University of Zaragoza | And 6 more authors.
Earth and Planetary Science Letters | Year: 2014

X-ray photoemission electron microscopy (XPEEM) enables natural remanent magnetisation to be imaged with ~30nm resolution across a field of view of 5-20 μm. The method is applied to structural features typical of the Widmanstätten microstructure (kamacite - tetrataenite rim - cloudy zone - plessite) in the Tazewell IIICD iron meteorite. Kamacite lamellae and the tetrataenite rim are multidomain, whereas plessite consists of laths of different phases displaying a range of stable magnetisation directions. The cloudy zone (CZ) displays a complex interlocking domain pattern resulting from nanoscale islands of tetrataenite with easy axes distributed along three possible crystallographic directions. Quantitative analysis of the coarse and intermediate CZ was achieved using a combination of image simulations and histogram profile matching. Remanence information was extracted from individual regions of interest ~400nm wide, demonstrating for the first time the capability of XPEEM to perform quantitative paleomagnetic analysis at sub-micron length scales. The three tetrataenite easy axis orientations occur with equal probability in the coarse and intermediate CZ, suggesting that spinodal decomposition in these regions was not strongly influenced by internal interaction fields, and that they are suitable candidates for future paleomagnetic studies. The fine CZ shows a strong dominance of one easy axis. This effect is attributed to island-island exchange interactions that render the fine CZ unsuitable for paleomagnetic study. Variations in the relative strength (proportion of dominant easy axis) and direction (direction of dominant easy axis) of a paleomagnetic field can be resolved from different regions of the CZ using XPEEM, raising the prospect of obtaining a time-resolved measurement of the active dynamo period in meteorites originating from the upper unmelted regions of differentiated asteroids (e.g. chondrites, pallasites, mesosiderites). © 2014.


Raich L.,University of Barcelona | Borodkin V.,University of Dundee | Fang W.,University of Dundee | Castro-Lopez J.,University of Zaragoza | And 5 more authors.
Journal of the American Chemical Society | Year: 2016

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (G = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from 4C1 → [4E]‡ → 1,4B/4E to 4C1 → [4H3]‡ → 4C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs. © 2016 American Chemical Society.


Driver S.L.,University of Cambridge | Herrero-Albillos J.,Fundacion ARAID | Herrero-Albillos J.,Centro Universitario Of La Defensa | Herrero-Albillos J.,University of Zaragoza | And 6 more authors.
Journal of Physics Condensed Matter | Year: 2014

Magnetic phase transitions in RCo2 Laves phases with R as a rare earth element are accompanied by changes in crystallographic space group. For purely structural transitions they would be described as improper ferroelastic and therefore fulfil the condition for multiferroic phase transitions in combining two out of three properties, ferro/antiferromagnetism, ferroelectricity and ferroelasticity. Here lattice parameter data from the literature and new measurements of elastic and anelastic properties, by resonant ultrasound spectroscopy, for NdCo2 and ErCo2 have been analysed from this perspective. The temperature dependence of symmetry-breaking shear strains is consistent with the cubic ↔ tetragonal transition in NdCo2 being close to tricritical in character and the cubic ↔ rhombohedral transition in ErCo2 being first order. Elastic softening and acoustic loss within the stability ranges of the ferroelastic phases can be understood in terms of a combination of intrinsic softening due to strain/order parameter coupling and ferroelastic twin-wall motion. Softening ahead of the transitions does not fit with standard macroscopic descriptions of dynamic effects from other systems but, rather, in the case of NdCo2, might be attributed to the involvement of a second zone centre order parameter related to a separate instability driven by cooperative Jahn-Teller distortions. In ErCo2, acoustic loss in the temperature interval above the transition point is discussed in terms of a possible tweed microstructure associated with strain coupling to local magnetic ordering. The overall multiferroic behaviour can be understood in terms of a single magnetic order parameter (irrep of magnetic space group ) which couples with a structural order parameter (irrep or ). The coupling is linear/quadratic which, in the case of two separate instabilities, causes them to combine in a single multiferroic phase transition. © 2014 IOP Publishing Ltd.


PubMed | University of Bordeaux Segalen, Fundacion ARAID, University of Cordoba, Spain, University of Hamburg and Baylor College of Medicine
Type: Journal Article | Journal: Journal of proteome research | Year: 2016

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.


PubMed | Fundacion ARAID
Type: Journal Article | Journal: ACS applied materials & interfaces | Year: 2014

We report magnetic and electronic transport measurements across epitaxial bilayers of ferromagnetic insulator LaCoO3 and half-metallic ferromagnet La2/3Sr1/3MnO3 (LCO/LSMO: 3.5 nm/20 nm) fabricated by a chemical solution method. The I-V curves at room temperature and 4K measured with conducting atomic force microscopy (CAFM) on well-defined patterned areas exhibit the typical features of a tunneling process. The curves have been fitted to the Simmons model to determine the height () and width (s) of the insulating LCO barrier. The results yield = 0.40 0.05 eV (0.50 0.01 eV) at room temperature (4K) and s = 3 nm, in good agreement with the structural analysis. Our results demonstrate that this chemical method is able to produce epitaxial heterostructures with the quality required for this type of fundamental studies and applications.


PubMed | Fundacion ARAID and French Institute of Health and Medical Research
Type: Journal Article | Journal: Biochemistry | Year: 2015

The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.


PubMed | University of Zaragoza, University of Barcelona and Fundacion ARAID
Type: Journal Article | Journal: Journal of the American Chemical Society | Year: 2016

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (G() = 12 kcal/mol). The 2-OHnucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 [(4)E]() (1,4)B/(4)E to (4)C1 [(4)H3]() (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

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