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Phospho.ELM: a database of phosphorylation sites--update 2011.

Phospho.ELM is a manually curated database of eukaryotic phosphorylation sites. The resource includes data collected from published literature as well as high-throughput data sets. The current release of Phospho.ELM (version 7.0, July 2007) contains 4078 phospho-protein sequences covering 12 025 phospho-serine, 2362 phospho-threonine and 2083 phospho-tyrosine sites. The entries provide information about the phosphorylated proteins and the exact position of known phosphorylated instances, the kinases responsible for the modification (where known) and links to bibliographic references. The database entries have hyperlinks to easily access further information from UniProt, PubMed, SMART, ELM, MSD as well as links to the protein interaction databases MINT and STRING. A new BLAST search tool, complementary to retrieval by keyword and UniProt accession number, allows users to submit a protein query (by sequence or UniProt accession) to search against the curated data set of phosphorylated peptides. Phospho.ELM is available on line at: http://phospho.elm.eu.org.The Phospho.ELM resource (http://phospho.elm.eu.org) is a relational database designed to store in vivo and in vitro phosphorylation data extracted from the scientific literature and phosphoproteomic analyses. The resource has been actively developed for more than 7 years and currently comprises 42u2009574 serine, threonine and tyrosine non-redundant phosphorylation sites. Several new features have been implemented, such as structural disorder/order and accessibility information and a conservation score. Additionally, the conservation of the phosphosites can now be visualized directly on the multiple sequence alignment used for the score calculation. Finally, special emphasis has been put on linking to external resources such as interaction networks and other databases.

A structure filter for the Eukaryotic Linear Motif Resource

BackgroundMany proteins are highly modular, being assembled from globular domains and segments of natively disordered polypeptides. Linear motifs, short sequence modules functioning independently of protein tertiary structure, are most abundant in natively disordered polypeptides but are also found in accessible parts of globular domains, such as exposed loops. The prediction of novel occurrences of known linear motifs attempts the difficult task of distinguishing functional matches from stochastically occurring non-functional matches. Although functionality can only be confirmed experimentally, confidence in a putative motif is increased if a motif exhibits attributes associated with functional instances such as occurrence in the correct taxonomic range, cellular compartment, conservation in homologues and accessibility to interacting partners. Several tools now use these attributes to classify putative motifs based on confidence of functionality.ResultsCurrent methods assessing motif accessibility do not consider much of the information available, either predicting accessibility from primary sequence or regarding any motif occurring in a globular region as low confidence. We present a method considering accessibility and secondary structural context derived from experimentally solved protein structures to rectify this situation. Putatively functional motif occurrences are mapped onto a representative domain, given that a high quality reference SCOP domain structure is available for the protein itself or a close relative. Candidate motifs can then be scored for solvent-accessibility and secondary structure context. The scores are calibrated on a benchmark set of experimentally verified motif instances compared with a set of random matches. A combined score yields 3-fold enrichment for functional motifs assigned to high confidence classifications and 2.5-fold enrichment for random motifs assigned to low confidence classifications. The structure filter is implemented as a pipeline with both a graphical interface via the ELM resource http://elm.eu.org/ and through a Web Service protocol.ConclusionNew occurrences of known linear motifs require experimental validation as the bioinformatics tools currently have limited reliability. The ELM structure filter will aid users assessing candidate motifs presenting in globular structural regions. Most importantly, it will help users to decide whether to expend their valuable time and resources on experimental testing of interesting motif candidates.

On the Mechanism of Chloroquine Resistance in Plasmodium falciparum

Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections

BackgroundMonitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins.MethodsPlasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1.ResultsResistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 – 10.7) for A623/S769 versus 623E/769u2009N 13.5 (9.8 – 17.3) nM with a mean increase of 65%; pu2009=u20090.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 – 11.6) versus 16.3 (10.7 – 21.8) nM with a mean increase of 87%; pu2009=u20090.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins.ConclusionsThese findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed.

Ten simple rules for developing a short bioinformatics training course

This paper considers what makes a short course in bioinformatics successful. In todays research environment, exposure to bioinformatics training is something that anyone embarking on life sciences research is likely to need at some point. Furthermore, as research technologies evolve, this need will continue to grow. In fact, as a consequence of the introduction of high-throughput technologies, there has already been an increase in demand for training relating to the use of computational resources and tools designed for high-throughput data storage, retrieval, and analysis. Biologists and computational scientists alike are seeking postgraduate learning opportunities in various bioinformatics topics that meet the needs and time restrictions of their schedules. Short, intensive bioinformatics courses (typically from a couple of days to a week in length, and covering a variety of topics) are available throughout the world, and more continue to be developed to meet the growing training needs. The challenges, however, when planning, organising, and delivering such courses, are not trivial [1], especially considering the heterogeneous backgrounds of participants. Here, we address such challenges and present a consensus of rules derived from the shared expertise of several bioinformatics trainers. While the rules apply broadly to bioinformatics training, aspects addressing specific audiences are also discussed in order to make these rules pragmatic and applicable to a wide range of readers. Delivering bioinformatics training is both crucial to facilitate the use of, and to exploit the investment in, bioinformatics tools and resources, and an excellent opportunity to solicit user evaluation and feedback to improve them. One point of crucial interest to the training course community concerns material preparation and distribution. Preparing effective materials (slides, notes, references, etc.) entails a huge effort that would be enormously facilitated if course developers could start from a body of available materials, for example if they could gain access to repositories of materials deposited by trainers of other courses. This was one of the reasons motivating the Bioinformatics Training Network (BTN) to set up the BTN website (http://www.biotnet.org/), which has been planned as a vessel for the training community to share and disseminate course information and materials. Course developers are warmly welcome to subscribe to the site and make available their materials to the community [2].

Data integration in biological research: an overview

Data sharing, integration and annotation are essential to ensure the reproducibility of the analysis and interpretation of the experimental findings. Often these activities are perceived as a role that bioinformaticians and computer scientists have to take with no or little input from the experimental biologist. On the contrary, biological researchers, being the producers and often the end users of such data, have a big role in enabling biological data integration. The quality and usefulness of data integration depend on the existence and adoption of standards, shared formats, and mechanisms that are suitable for biological researchers to submit and annotate the data, so it can be easily searchable, conveniently linked and consequently used for further biological analysis and discovery. Here, we provide background on what is data integration from a computational science point of view, how it has been applied to biological research, which key aspects contributed to its success and future directions.

Investigation of a potential mechanism for the inhibition of SmTGR by Auranofin and its implications for Plasmodium falciparum inhibition

Schistosoma mansoni and Plasmodium falciparum are pathogen parasites that spend part of their lives in the blood stream of the human host and are therefore heavily exposed to fluxes of toxic reactive oxygen species (ROS). SmTGR, an essential enzyme of the S. mansoni ROS detoxification machinery, is known to be inhibited by Auranofin although the inhibition mechanism has not been completely clarified. Auranofin also kills P. falciparum, even if its molecular targets are unknown. Here, we used computational and docking techniques to investigate the molecular mechanism of interaction between SmTGR and Auranofin. Furthermore, we took advantage of the homology relationship and of docking studies to assess if PfTR, the SmTGR malaria parasite homologue, can be a putative target for Auranofin. Our findings support a recently hypothesized molecular mechanism of inhibition for SmTGR and suggest that PfTR is indeed a possible and attractive drug target in P. falciparum.

Identification of the Schistosoma mansoni molecular target for the antimalarial drug artemether.

Plasmodium falciparum and Schistosoma mansonii are the parasites responsible for most of the malaria and schistosomiasis cases in the world. Notwithstanding their many differences, the two agents have striking similarities in that they both are blood feeders and are targets of an overlapping set of drugs, including the well-known artemether molecule. Here we explore the possibility of using the known information about the mode of action of artemether in Plasmodium to identify the molecular target of the drug in Schistosoma and provide evidence that artemether binds to SmSERCA, a putative Ca²⁺-ATPase of Schistosoma . We also predict the putative binding mode of the molecule for both its Plasmodium and Schistosoma targets. Our analysis of the mode of binding of artemether to Ca²⁺-ATPases also provides an explanation for the apparent paradox that, although the molecule has no side effect in humans, it has been shown to possess antitumoral activity.

Four simple recommendations to encourage best practices in research software

Scientific research relies on computer software, yet software is not always developed following practices that ensure its quality and sustainability. This manuscript does not aim to propose new software development best practices, but rather to provide simple recommendations that encourage the adoption of existing best practices. Software development best practices promote better quality software, and better quality software improves the reproducibility and reusability of research. These recommendations are designed around Open Source values, and provide practical suggestions that contribute to making research software and its source code more discoverable, reusable and transparent. This manuscript is aimed at developers, but also at organisations, projects, journals and funders that can increase the quality and sustainability of research software by encouraging the adoption of these recommendations.

The crystal structure of Giardia duodenalis 14-3-3 in the apo form: When protein post-translational modifications make the difference

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.