Tracking the origin and divergence of cholinesterases and neuroligins: the evolution of synaptic proteins

14. International Symposium on Cholinergic Mechanisms (ISCM), Hangzhou, 2013/05/05-9A cholinesterase activity can be found in all kingdoms of living organism, yet cholinesterases involved in cholinergic transmission appeared only recently in the animal phylum. Among various proteins homologous to cholinesterases, one finds neuroligins. These proteins, with an altered catalytic triad and no known hydrolytic activity, display well-identified cell adhesion properties. The availability of complete genomes of a few metazoans provides opportunities to evaluate when these two protein families emerged during evolution. In bilaterian animals, acetylcholinesterase co-localizes with proteins of cholinergic synapses while neuroligins co-localize and may interact with proteins of excitatory glutamatergic or inhibitory GABAergic/glycinergic synapses. To compare evolution of the cholinesterases and neuroligins with other proteins involved in the architecture and functioning of synapses, we devised a method to search for orthologs of these partners in genomes of model organisms representing distinct stages of metazoan evolution. Our data point to a progressive recruitment of synaptic components during evolution. This finding may shed light on the common or divergent developmental regulation events involved into the setting and maintenance of the cholinergic versus glutamatergic and GABAergic/glycinergic synapses

ESTHER, the database of the α/β-hydrolase fold superfamily of proteins: tools to explore diversity of functions

The ESTHER database, which is freely available via a web server (http://bioweb.ensam.inra.fr/esther) and is widely used, is dedicated to proteins with an a/b-hydrolase fold, and it currently contains >30 000 manually curated proteins. Herein, we report those substantial changes towards improvement that we have made to improve ESTHER during the past 8 years since our 2004 update. In particular, we generated 87 new families and increased the coverage of the UniProt Knowledgebase (UniProtKB). We also renewed the ESTHER website and added new visualization tools, such as the Overall Table and the Family Tree. We also address two topics of particular interest to the ESTHER users. First, we explain how the different enzyme classifications (bacterial lipases, peptidases,carboxylesterases) used by different communities of users are combined in ESTHER. Second, we discuss how variations of core architecture or in predicted active site residues result in a more precise clustering of families, and whether this strategy provides trustable hints to identify enzymelike proteins with no catalytic activity

A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding

<p>Abstract</p> <p>Background</p> <p>Proteins may evolve through the recruitment and modification of discrete domains, and in many cases, protein action can be dissected at the domain level. PDZ domains are found in many important structural and signaling complexes, and are generally thought to interact with their protein partners through a C-terminal consensus sequence. We undertook a comprehensive search for protein partners of all individual PDZ domains in <it>C. elegans </it>to characterize their function and mode of interaction.</p> <p>Results</p> <p>Coupling high-throughput yeast two-hybrid screens with extensive validation by co-affinity purification, we defined a domain-orientated interactome map. This integrates PDZ domain proteins in numerous cell-signaling pathways and shows that PDZ domain proteins are implicated in an unexpectedly wide range of cellular processes. Importantly, we uncovered a high frequency of non-canonical interactions, not involving the C-terminus of the protein partner, which were directly confirmed in most cases. We completed our study with the generation of a yeast array representing the entire set of PDZ domains from <it>C. elegans </it>and provide a proof-of-principle for its application to the discovery of PDZ domain targets for any protein or peptide of interest.</p> <p>Conclusions</p> <p>We provide an extensive domain-centered dataset, together with a clone resource, that will help future functional study of PDZ domains. Through this unbiased approach, we revealed frequent non-canonical interactions between PDZ domains and their protein partners that will require a re-evaluation of this domain's molecular function.</p> <p>[The protein interactions from this publication have been submitted to the IMEx (<url>http://www.imexconsortium.org</url>) consortium through IntAct (PMID: 19850723) and assigned the identifier IM-14654]</p

CAZyme content of Pochonia chlamydosporia reflects that chitin and chitosan modification are involved in nematode parasitism

Pochonia chlamydosporia is a soil fungus with a multitrophic lifestyle combining endophytic and saprophytic behaviors, in addition to a nematophagous activity directed against eggs of root-knot and other plant parasitic nematodes. The carbohydrate-active enzymes encoded by the genome of P. chlamydosporia suggest that the endophytic and saprophytic lifestyles make use of a plant cell wall polysaccharide degradation machinery that can target cellulose, xylan and, to a lesser extent, pectin. This enzymatic machinery is completed by a chitin breakdown system that involves not only chitinases, but also chitin deacetylases and a large number of chitosanases. P. chlamydosporia can degrade and grow on chitin and is particularly efficient on chitosan. The relevance of chitosan breakdown during nematode egg infection is supported by the immunolocalization of chitosan in Meloidogyne javanica eggs infected by P. chlamydosporia and by the fact that the fungus expresses chitosanase and chitin deacetylase genes during egg infection. This suggests that these enzymes are important for the nematophagous activity of the fungus and they are targets for improving the capabilities of P. chlamydosporia as a biocontrol agent in agriculture.This research was funded by the Spanish Ministry of Economy and Competitiveness Grant AGL2015-66833-R, with a grant from the Generalitat Valenciana to A. Aranda-Martinez (ACIF/2013/120) as well as a sabbatical grant to L.V. Lopez-Llorca (PR2015-0008)

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes.This work was supported by a grant from the European Research Agency—Industrial Biotechnology Initiative as financed by the national research councils: Biotechnology and Biological Sciences Research Council (grant number BB/L000423) and Agence Française de l'Environnement et de la Maîtrise de l'Energie (grant number 1201C102). The Danish Council for Strategic Research (grant numbers 12-134923 and 12-134922). The Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570) funded travel to synchrotrons. P.H.W. acknowledges the experimental assistance of Rebecca Gregory and Dr Victor Chechik. L.L.L. acknowledges the experimental assistance of Dorthe Boelskifte and the ESRF and MAXLAB staff for assistance with data collection.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms696

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.We thank K. Rasmussen and R.M. Borup for experimental assistance, and MAXLAB, Sweden and the European Synchrotron Radiation Facility (ESRF), France, for synchrotron beam time and assistance. This work was supported by the UK Biotechnology and Biological Sciences Research Council (grant numbers BB/L000423 to P.D., G.J.D. and P.H.W., and BB/L021633/1 to G.J.D. and P.H.W.), Agence Française de l'Environnement et de la Maîtrise de l'Energie (grant number 1201C102 to B.H.), the Danish Council for Strategic Research (grant numbers 12-134923 to L.L.L. and 12-134922 to K.S.J.). Travel to synchrotrons was supported by the Danish Ministry of Higher Education and Science through the Instrument Center DANSCATT and the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement 283570). L.M., S.F., S.C. and H.D. were supported by Institut de Chimie Moléculaire de Grenoble FR 2607, LabEx ARCANE (ANR-11-LABX-0003-01), the PolyNat Carnot Institute and the French Agence Nationale de la Recherche (PNRB2005-11).This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nchembio.202

Creating a specialist protein resource network:a meeting report for the protein bioinformatics and community resources retreat

During 11–12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxida-tive cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications

Network of Caenorhabditis elegan's PDZ domains

Le domaine PDZ participe aux réseaux moléculaires à l’origine de fonctions cellulaires touchées lors de pathologies diverses. L’exploration de ce réseau par double hybride a permis d’attribuer de nouvelles fonctions putatives aux ligands protéiques des domaines PDZ du ver Caenorhabditis elegans. Les interactions ont laissé apparaitre une proportion inattendue de ligands atypiques interagissant par une séquence interne. Nous avons ensuite validé fonctionnellement in silico des groupes d’interactions de notre interactome qui forment des micro-réseaux co-exprimés par l’intégration de données de profils d’expression. Finalement, ce travail a permis la construction d’un outil exploratoire, le PIPE (PDZ Interacting Protein Explorer) qui permet de cribler l’ensemble des domaines PDZ du ver à la recherche d’interactions avec une protéine d’intérêt révélant déjà de nombreuses interactions supplémentaires entre domaines PDZ et ligandsPDZ domains allow the organization of molecular networks responsible for cellular functions essential for multicellularity as polarization or transduction of extracellular signals. Exploration of this network by two-hybrid revealed a functional diversity for ligands of Caenorhabditis elegans’s PDZ domains. New putative functions were being observed through GO-terms and an unexpected proportion of internal ligands appeared, confirmed by Co-IP. We then functionally validated in silico groups of interactions that form our interactome microarrays co-expressed by the integration of data from expression profiles. Finally, this work has enabled the construction of an exploratory tool, the PIPE (PDZ Interacting Protein Explorer) that allows screening of all PDZ domains looking for interactions with a protein of interest and had already showed many additional interactions between PDZ domains and ligand