Study of the peptidasic site of cholinesterase: preliminary results

AbstractThe peptidasic site of highly purified human plasma cholinesterase was investigated using active-site-directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non-polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of ‘aging’ when inhibited by DFP or soman

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

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

The ESTHER Database (ESTerases and alpha/beta-Hydrolase Enzymes and Relatives) is dedicated to the analysis of proteins belonging to the superfamily of alpha/beta-hydrolases

The ESTHER Database (ESTerases and alpha/beta-Hydrolase Enzymes and Relatives) is dedicated to the analysis of proteins belonging to the superfamily of alpha/beta-hydrolases (bioweb.supagro.inra.fr/esther/) Alpha/beta hydrolases constitute one of the largest and most diverse protein superfamily, with so far more than 800 000 sequences (of which 42000 non-redundant) grouped in 175 subfamilies (Lenfant et al. 2013. They play fundamental roles in virtually all physiological processes and are targeted by drugs aimed at treating diseases such as diabetes, obesity, and neurodegenerative disorders (24 genes have been found mutated in 22 genetic diseases). Yet, for most of the 120+ predicted mammalian alpha/beta hydrolases we lack biological understanding. Also, despite what their generic name would suggest, many of these proteins are not enzymes (some have lost all residues necessary to constitute an active site) (Marchot and Chatonnet 2012). The functions of few representatives of this latest group are known: intracellular receptors of small molecules, endo cellular membrane trafficking agents, inter-actors with partners at cell-cell interface, hormone precursors. The database was launched in 1994 as a Gopher server and quickly moved to WWW. The underlying system of the database is the venerable ACeDB system. AceDB is a genome database system developed since 1989 primarily by Jean Thierry-Mieg (CNRS, Montpellier) and Richard Durbin (Sanger Institute) originally developed for the C.elegansgenome project and is still the kernel of the wormbase database and server. One of the advantages of the system is that the structure of the database (models of relationship between objects) can be retrieved as simple text. All the data can be retrieved as text or xml files. The initial interface to the web is based on CGI scripts AcePerl developed by Lincoln Stein. As a database engine ACeDB compares favorably with Mysql or even commercial database systems Complex queries can be easily built Many are used to generate on the fly the different chapters of the server but many examples are also given whih can be customized by users. Examples ofdedicated searches have been published for example to analyse the mutations of cholinesterases or carboxylesterases responsible for insecticide resistance

Hydrolase versus other functions of members of the alpha/beta-hydrolase fold superfamily of proteins.

International audienceGenes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones... The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold protein

Enzymatic activity and protein interactions in alpha/beta hydrolase fold proteins: moonlighting versus promiscuity.

Contact: [email protected], [email protected] audienceGenes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones... The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold proteins

Breathing without acetylcholinesterase.

ISSN : 0065-259

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, no