Browsing by Author "Colombres, Marcela"

Now showing 1 - 3 of 3
  • No Thumbnail Available
    Item
    An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum
    (2008) Colombres, Marcela; Garate, Jose A.; Lagos, Carlos F.; Araya-Secchi, Raul; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Perez-Acle, Tomas; Eyzaguirre, Jaime
    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 angstrom resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.
  • Thumbnail Image
    Item
    Genome-wide identification of new Wnt/β-catenin target genes in the human genome using CART method
    (2010) Hodar, Christian; Assar, Rodrigo; Colombres, Marcela; Aravena, Andres; Pavez, Leonardo; Gonzalez, Mauricio; Martinez, Servet; Inestrosa, Nibaldo C.; Maass, Alejandro
    Background: The importance of in silico predictions for understanding cellular processes is now widely accepted, and a variety of algorithms useful for studying different biological features have been designed. In particular, the prediction of cis regulatory modules in non-coding human genome regions represents a major challenge for understanding gene regulation in several diseases. Recently, studies of the Wnt signaling pathway revealed a connection with neurodegenerative diseases such as Alzheimer's. In this article, we construct a classification tool that uses the transcription factor binding site motifs composition of some gene promoters to identify new Wnt/beta-catenin pathway target genes potentially involved in brain diseases.
  • Thumbnail Image
    Item
    Heparin activates Wnt signaling for neuronal morphogenesis
    (WILEY, 2008) Colombres, Marcela; Henriquez, Juan Pablo; Reig, German F.; Scheu, Jessica; Calderon, Rosario; Alvarez, Alejandra; Brandan, Enrique; Inestrosa, Nibaldo C.
    Writ factors are secreted ligands that affect different aspects of the nervous system behavior like neurodevelopment, synaptogenesis and neurodegeneration. In different model systems, Wnt signaling has been demonstrated to be regulated by heparan sulfate proteoglycans (HSPGs). Whether HSPGs modulate Writ signaling in the context of neuronal behavior is currently unknown. Here we demonstrate that activation of Wnt signaling with the endogenous ligand Wnt-7a results in an increased of neurite outgrowth in the neuroblastoma N2a cell line. Interestingly, heparin induces glycogen synthase kinase-3 beta (GSK-3 beta) inhibition, beta-catenin stabilization and morphological differentiation in both N2a cells and in rat primary hippocampal neuronal cultures. We also show that heparin modulates Wnt-3a-induced stabilization of beta-catenin. Several extracellular matrix and membrane-attached HSPGs were found to be expressed in both in vitro neuronal models. Changes in the expression of specific HSPGs were observed upon differentiation of N2a cells. Taken together, our findings suggest that HSPGs may modulate canonical Writ signaling for neuronal morphogenesis.