The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase.

Alpha-synuclein ferrireductase activity is detectible in vivo, is altered in Parkinson's disease and increases the neurotoxicity of DOPAL.

The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease.

Steady-State Kinetics of α-Synuclein Ferrireductase Activity Identifies the Catalytically Competent Species.

α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson's disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.

Bacterial formate hydrogenlyase complex.

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Bioinformatics Training Network (BTN): a community resource for bioinformatics trainers.

Funding bodies are increasingly recognizing the need to provide graduates and researchers with access to short intensive courses in a variety of disciplines, in order both to improve the general skills base and to provide solid foundations on which researchers may build their careers. In response to the development of 'high-throughput biology', the need for training in the field of bioinformatics, in particular, is seeing a resurgence: it has been defined as a key priority by many Institutions and research programmes and is now an important component of many grant proposals. Nevertheless, when it comes to planning and preparing to meet such training needs, tension arises between the reward structures that predominate in the scientific community which compel individuals to publish or perish, and the time that must be devoted to the design, delivery and maintenance of high-quality training materials. Conversely, there is much relevant teaching material and training expertise available worldwide that, were it properly organized, could be exploited by anyone who needs to provide training or needs to set up a new course. To do this, however, the materials would have to be centralized in a database and clearly tagged in relation to target audiences, learning objectives, etc. Ideally, they would also be peer reviewed, and easily and efficiently accessible for downloading. Here, we present the Bioinformatics Training Network (BTN), a new enterprise that has been initiated to address these needs and review it, respectively, to similar initiatives and collections.

iAnn: an event sharing platform for the life sciences.

SUMMARY: We present iAnn, an open source community-driven platform for dissemination of life science events, such as courses, conferences and workshops. iAnn allows automatic visualisation and integration of customised event reports. A central repository lies at the core of the platform: curators add submitted events, and these are subsequently accessed via web services. Thus, once an iAnn widget is incorporated into a website, it permanently shows timely relevant information as if it were native to the remote site. At the same time, announcements submitted to the repository are automatically disseminated to all portals that query the system. To facilitate the visualization of announcements, iAnn provides powerful filtering options and views, integrated in Google Maps and Google Calendar. All iAnn widgets are freely available. AVAILABILITY: http://iann.pro/iannviewer CONTACT: manuel.corpas@tgac.ac.uk.

InterPro in 2011: new developments in the family and domain prediction database.

InterPro (http://www.ebi.ac.uk/interpro/) is a database that integrates diverse information about protein families, domains and functional sites, and makes it freely available to the public via Web-based interfaces and services. Central to the database are diagnostic models, known as signatures, against which protein sequences can be searched to determine their potential function. InterPro has utility in the large-scale analysis of whole genomes and meta-genomes, as well as in characterizing individual protein sequences. Herein we give an overview of new developments in the database and its associated software since 2009, including updates to database content, curation processes and Web and programmatic interfaces.

Analysis of hydrogenase 1 levels reveals an intimate link between carbon and hydrogen metabolism in Escherichia coli K-12.

Two of the three [NiFe]-hydrogenases (Hyd) of Escherichia coli have a hydrogen-uptake function in anaerobic metabolism. While Hyd-2 is maximally synthesized when the bacterium grows by fumarate respiration, Hyd-1 synthesis shows a correlation with fermentation of sugar substrates. In an attempt to advance our knowledge on the physiological function of Hyd-1 during fermentative growth, we examined Hyd-1 activity and levels in various derivatives of E. coli K-12 MC4100 with specific defects in sugar utilization. MC4100 lacks a functional fructose phosphotransferase system (PTS) and therefore grows more slowly under anaerobic conditions in rich medium in the presence of d-fructose compared with d-glucose. Growth in the presence of fructose resulted in an approximately 10-fold increase in Hyd-1 levels in comparison with growth under the same conditions with glucose. This increase in the amount of Hyd-1 was not due to regulation at the transcriptional level. Reintroduction of a functional fruBKA-encoded fructose PTS into MC4100 restored growth on d-fructose and reduced Hyd-1 levels to those observed after growth on d-glucose. Reducing the rate of glucose uptake by introducing a mutation in the gene encoding the cAMP receptor protein, or consumption through glycolysis, by introducing a mutation in phosphoglucose isomerase, increased Hyd-1 levels during growth on glucose. These results suggest that the ability to oxidize hydrogen by Hyd-1 shows a strong correlation with the rate of carbon flow through glycolysis and provides a direct link between hydrogen, carbon and energy metabolism.

Bioinformatics training: a review of challenges, actions and support requirements.

As bioinformatics becomes increasingly central to research in the molecular life sciences, the need to train non-bioinformaticians to make the most of bioinformatics resources is growing. Here, we review the key challenges and pitfalls to providing effective training for users of bioinformatics services, and discuss successful training strategies shared by a diverse set of bioinformatics trainers. We also identify steps that trainers in bioinformatics could take together to advance the state of the art in current training practices. The ideas presented in this article derive from the first Trainer Networking Session held under the auspices of the EU-funded SLING Integrating Activity, which took place in November 2009.

InterPro: the integrative protein signature database.

The InterPro database (http://www.ebi.ac.uk/interpro/) integrates together predictive models or 'signatures' representing protein domains, families and functional sites from multiple, diverse source databases: Gene3D, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY and TIGRFAMs. Integration is performed manually and approximately half of the total approximately 58,000 signatures available in the source databases belong to an InterPro entry. Recently, we have started to also display the remaining un-integrated signatures via our web interface. Other developments include the provision of non-signature data, such as structural data, in new XML files on our FTP site, as well as the inclusion of matchless UniProtKB proteins in the existing match XML files. The web interface has been extended and now links out to the ADAN predicted protein-protein interaction database and the SPICE and Dasty viewers. The latest public release (v18.0) covers 79.8% of UniProtKB (v14.1) and consists of 16 549 entries. InterPro data may be accessed either via the web address above, via web services, by downloading files by anonymous FTP or by using the InterProScan search software (http://www.ebi.ac.uk/Tools/InterProScan/).

New developments in the InterPro database.

InterPro is an integrated resource for protein families, domains and functional sites, which integrates the following protein signature databases: PROSITE, PRINTS, ProDom, Pfam, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER. The latter two new member databases have been integrated since the last publication in this journal. There have been several new developments in InterPro, including an additional reading field, new database links, extensions to the web interface and additional match XML files. InterPro has always provided matches to UniProtKB proteins on the website and in the match XML file on the FTP site. Additional matches to proteins in UniParc (UniProt archive) are now available for download in the new match XML files only. The latest InterPro release (13.0) contains more than 13 000 entries, covering over 78% of all proteins in UniProtKB. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro). The InterProScan search tool is now also available via a web service at http://www.ebi.ac.uk/Tools/webservices/WSInterProScan.html.

InterPro, progress and status in 2005.

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created to integrate the major protein signature databases. Currently, it includes PROSITE, Pfam, PRINTS, ProDom, SMART, TIGRFAMs, PIRSF and SUPERFAMILY. Signatures are manually integrated into InterPro entries that are curated to provide biological and functional information. Annotation is provided in an abstract, Gene Ontology mapping and links to specialized databases. New features of InterPro include extended protein match views, taxonomic range information and protein 3D structure data. One of the new match views is the InterPro Domain Architecture view, which shows the domain composition of protein matches. Two new entry types were introduced to better describe InterPro entries: these are active site and binding site. PIRSF and the structure-based SUPERFAMILY are the latest member databases to join InterPro, and CATH and PANTHER are soon to be integrated. InterPro release 8.0 contains 11 007 entries, representing 2573 domains, 8166 families, 201 repeats, 26 active sites, 21 binding sites and 20 post-translational modification sites. InterPro covers over 78% of all proteins in the Swiss-Prot and TrEMBL components of UniProt. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).

Dissection and engineering of the Escherichia coli formate hydrogenlyase complex.

The Escherichia coli formate hydrogenlyase (FHL) complex is produced under fermentative conditions and couples formate oxidation to hydrogen production. In this work, the architecture of FHL has been probed by analysing affinity-tagged complexes from various genetic backgrounds. In a successful attempt to stabilize the complex, a strain encoding a fusion between FdhF and HycB has been engineered and characterised. Finally, site-directed mutagenesis of the hycG gene was performed, which is predicted to encode a hydrogenase subunit important for regulating sensitivity to oxygen. This work helps to define the core components of FHL and provides solutions to improving the stability of the enzyme.

InterPro protein classification.

Improvements in nucleotide sequencing technology have resulted in an ever increasing number of nucleotide and protein sequences being deposited in databases. Unfortunately, the ability to manually classify and annotate these sequences cannot keep pace with their rapid generation, resulting in an increased bias toward unannotated sequence. Automatic annotation tools can help redress the balance. There are a number of different groups working to produce protein signatures that describe protein families, functional domains or conserved sites within related groups of proteins. Protein signature databases include CATH-Gene3D, HAMAP, PANTHER, Pfam, PIRSF, PRINTS, ProDom, PROSITE, SMART, SUPERFAMILY, and TIGRFAMs. Their approaches range from characterising small conserved motifs that can identify members of a family or subfamily, to the use of hidden Markov models that describe the conservation of residues over entire domains or whole proteins. To increase their value as protein classification tools, protein signatures from these 11 databases have been combined into one, powerful annotation tool: the InterPro database (http://www.ebi.ac.uk/interpro/) (Hunter et al., Nucleic Acids Res 37:D211-D215, 2009). InterPro is an open-source protein resource used for the automatic annotation of proteins, and is scalable to the analysis of entire new genomes through the use of a downloadable version of InterProScan, which can be incorporated into an existing local pipeline. InterPro provides structural information from PDB (Kouranov et al., Nucleic Acids Res 34:D302-D305, 2006), its classification in CATH (Cuff et al., Nucleic Acids Res 37:D310-D314, 2009) and SCOP (Andreeva et al., Nucleic Acids Res 36:D419-D425, 2008), as well as homology models from ModBase (Pieper et al., Nucleic Acids Res 37:D347-D354, 2009) and SwissModel (Kiefer et al., Nucleic Acids Res 37:D387-D392, 2009), allowing a direct comparison of the protein signatures with the available structural information. This chapter reviews the signature methods found in the InterPro database, and provides an overview of the InterPro resource itself.

Diverse tetracycline resistance genotypes of Megasphaera elsdenii strains selectively cultured from swine feces.

A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 microg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.

An integrated, functionally annotated gene map of the DXS8026-ELK1 interval on human Xp11.3-Xp11.23: potential hotspot for neurogenetic disorders.

Human chromosome Xp11.3-Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an approximately 2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of approximately 5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of approximately 1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3-Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp.