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Boya P.,CSIC - Biological Research Center | Reggiori F.,University Utrecht | Codogno P.,University of Paris Descartes
Nature Cell Biology | Year: 2013

Autophagy maintains cell, tissue and organism homeostasis through degradation. Complex post-translational modulation of the Atg (autophagy-related) proteins adds additional entry points for crosstalk with other cellular processes and helps define cell-type-specific regulations of autophagy. Beyond the simplistic view of a process exclusively dedicated to the turnover of cellular components, recent data have uncovered unexpected functions for autophagy and the autophagy-related genes, such as regulation of metabolism, membrane transport and modulation of host defenses-indicating the novel frontiers lying ahead. © 2013 Macmillan Publishers Limited. All rights reserved.

Asensio J.L.,CSIC - Biological Research Center | Arda A.,Institute Quimica Organica General | Canada F.J.,Institute Quimica Organica General | Jimenez-Barbero J.,Institute Quimica Organica General
Accounts of Chemical Research | Year: 2013

The recognition of saccharides by proteins has far reaching implications in biology, technology, and drug design. Within the past two decades, researchers have directed considerable effort toward a detailed understanding of these processes. Early crystallographic studies revealed, not surprisingly, that hydrogen-bonding interactions are usually involved in carbohydrate recognition. But less expectedly, researchers observed that despite the highly hydrophilic character of most sugars, aromatic rings of the receptor often play an important role in carbohydrate recognition.With further research, scientists now accept that noncovalent interactions mediated by aromatic rings are pivotal to sugar binding. For example, aromatic residues often stack against the faces of sugar pyranose rings in complexes between proteins and carbohydrates. Such contacts typically involve two or three CH groups of the pyranoses and the π electron density of the aromatic ring (called CH/π bonds), and these interactions can exhibit a variety of geometries, with either parallel or nonparallel arrangements of the aromatic and sugar units.In this Account, we provide an overview of the structural and thermodynamic features of protein-carbohydrate interactions, theoretical and experimental efforts to understand stacking in these complexes, and the implications of this understanding for chemical biology. The interaction energy between different aromatic rings and simple monosaccharides based on quantum mechanical calculations in the gas phase ranges from 3 to 6 kcal/mol range. Experimental values measured in water are somewhat smaller, approximately 1.5 kcal/mol for each interaction between a monosaccharide and an aromatic ring. This difference illustrates the dependence of these intermolecular interactions on their context and shows that this stacking can be modulated by entropic and solvent effects. Despite their relatively modest influence on the stability of carbohydrate/protein complexes, the aromatic platforms play a major role in determining the specificity of the molecular recognition process.The recognition of carbohydrate/aromatic interactions has prompted further analysis of the properties that influence them. Using a variety of experimental and theoretical methods, researchers have worked to quantify carbohydrate/aromatic stacking and identify the features that stabilize these complexes. Researchers have used site-directed mutagenesis, organic synthesis, or both to incorporate modifications in the receptor or ligand and then quantitatively analyzed the structural and thermodynamic features of these interactions. Researchers have also synthesized and characterized artificial receptors and simple model systems, employing a reductionistic chemistry-based strategy. Finally, using quantum mechanics calculations, researchers have examined the magnitude of each property's contribution to the interaction energy. © 2012 American Chemical Society.

Penalva M.A.,CSIC - Biological Research Center
Molecular Microbiology | Year: 2015

The Spitzenkörper (SPK) is an accumulation of vesicles interleaved with actin microfilaments present at the cytosolic side of the apical plasma membrane (PM) of hyphal tips of many species of filamentous fungi. The physiological role of the SPK has captivated fungal biologists over the years, but only very recently this 'organelle' is starting to be understood in the molecular terminology used for cell biological models. One aspect that has received little attention is the role of cellular membrane asymmetry in the organization of membrane traffic, in particular in the genetic and cell biological model Aspergillus nidulans. The paper by Schultzhaus etal. (2015) in this issue breaks the ice, providing original insight that may foster research in phospholipid composition in the context of intracellular traffic and the organization of the SPK. Notably, it shows that like the stout Neurospora crassaSPK, the much slimmer one of A.nidulans, appears to be formed by different strata, altogether suggesting that the SPK might be a mosaic of exocytic carriers with different functional specializations, and a major sorting hub for intracellular membranes. © 2015 John Wiley & Sons Ltd.

Oeste C.L.,CSIC - Biological Research Center
Histochemistry and cell biology | Year: 2013

Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.

Canas A.I.,CSIC - Biological Research Center | Camarero S.,CSIC - Biological Research Center
Biotechnology Advances | Year: 2010

Laccases are oxidoreductases which oxidize a variety of aromatic compounds using oxygen as the electron acceptor and producing water as by-product. The interest for these old enzymes (first described in 19th century) has progressively increased due to their outstanding biotechnological applicability. The presence of redox mediators is required for a number of biotechnological applications, providing the oxidation of complex substrates not oxidized by the enzyme alone. The efficiency of laccase-mediator systems to degrade recalcitrant compounds has been demonstrated, but still the high cost and possible toxicity of artificial mediators hamper their application at the industrial scale. Here, we present a general outlook of how alternative mediators can change this tendency. We focus on phenolic compounds related to lignin polymer that promotes the in vitro transformation of recalcitrant non-phenolic structures by laccase and are seemingly the natural mediators of laccases. The use of eco-friendly mediators easily available from lignocellulose, could contribute to the industrial implementation of laccases and the development of the 21th century biorefineries. © 2010 Elsevier Inc.

Giraldo R.,CSIC - Biological Research Center
ChemBioChem | Year: 2010

One of the major objectives that bottom-up synthetic biology shares with chemical biology is to engineer extant biological molecules to implement novel functionalities in living systems. Proteins, due to their astonishing structural and functional versatility and to their central roles in the biology of cells, should be cornerstones of synthetic biology. In particular, protein amyloid cross-β assemblies constitute one of the most stable, conceptually simple and universal macromolecular architectures ever found in Nature and thus have enormous potential to be explored. This article focuses on the concepts behind the use of the amyloid cross-β-structural framework as a synthetic biology part, underlining recent basic findings and ideas. The pros and the cons associated with the polymorphism and the cellular toxicity of protein amyloids are also discussed, keeping in mind the possible suitability of these protein assemblies for scaffolding novel orthogonal macromolecular devices in vivo.The many sides of amyloids: Engineering amyloid cross-β-sheet protein assemblies (in blue, on an electron micrograph of RepA-WH1 fibres) poses a number of challenges, such as a broad structural polymorphism. Progress has recently been made towards modulating amyloidogenesis at quasi-physiological conditions. Amyloids are potentially suitable as scaffold parts in the assembly of fused proteins (e.g., mCherry fluorescent protein, in red) into synthetic macromolecular devices in bacteria. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Penalva M.T.,CSIC - Biological Research Center
Current Opinion in Microbiology | Year: 2010

Endocytosis has been the Cinderella of membrane trafficking studies in filamentous fungi until recent work involving genetically tractable models has boosted interest in the field. Endocytic internalization predominates in the hyphal tips, spatially coupled to secretion. Early endosomes (EEs) show characteristic long-distance motility, riding on microtubule motors. The fungal tip contains a region baptised the 'dynein loading zone' where acropetally moving endosomes reaching the tip shift from a kinesin to dynein, reversing the direction of their movement. Multivesicular body biogenesis starts from these motile EEs. Maturation of EEs into late endosomes and vacuoles appears to be essential. The similarities between fungal and mammalian endocytic trafficking suggest that conditional mutant genetic screens would yield valuable information. © 2010 Elsevier Ltd.

Ruiz-Duenas F.J.,CSIC - Biological Research Center
Mycologia | Year: 2013

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in species of the order Polyporales.

Llave C.,CSIC - Biological Research Center
Trends in Plant Science | Year: 2010

Once a virus enters a cell, viral double-stranded RNA (dsRNA) is targeted by the RNA silencing machinery to initiate a cascade of regulatory events directed by viral small interfering RNAs (vsiRNAs). Recent genetic and functional studies along with the high-throughput sequencing of vsiRNAs have shed light on the genetic and structural requirements for virus targeting, the origins and compositions of vsiRNAs and their potential for controlling gene expression. The precise nature of the triggering molecules of virus-induced RNA silencing or the targeting constraints for viral genome recognition and processing represent outstanding questions that will be discussed in this review. The contribution of vsiRNAs to antiviral defense and host genome modifications has profound implications for our understanding of viral pathogenicity and host specificity in plants. © 2010 Elsevier Ltd.

Boya P.,CSIC - Biological Research Center
Antioxidants and Redox Signaling | Year: 2012

Significance: Lysosomes are organelles in which cellular degradation occurs in a controlled manner, separated from other cellular components. As several pathways terminate in the lysosome, lysosomal dysfunction has a profound impact on cell homeostasis, resulting in manifold pathological situations, including infectious diseases, neurodegeneration, and aging. Recent Advances: Lysosomal biology demonstrates that in addition to regulating the final steps of catabolic processes, lysosomes are essential up-stream modulators of autophagy and other essential lysosomal pathways. Future Directions and Critical Issues: Lysosomal membrane permeabilization offers therapeutic potential in the treatment of cancer, though the molecular regulators of this process remain obscure. This review focuses on recent discoveries in lysosomal function and dysfunction, primarily in in vivo situations. Antioxid. Redox Signal. © 2012 Mary Ann Liebert, Inc.

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