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Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 A resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins. The electron cryo-microscopy structure of Sulfolobus islandicus pili enabled the identification of SiL_2606 as the main pilin in these filaments and revealed that the pili are glycosylated, which probably explains how these structures remain soluble and stable even when cells grow at pH 3 and 80 °C.

International audience; RNA sequencing (RNA-seq) has become one of the most powerful tools to unravel the genomic basis of biological adaptation and diversity. Although challenging, RNA-seq is particularly promising for research on non-model, secretive species that cannot be observed in nature easily and therefore remain comparatively understudied. Among such animals, the caecilians (order Gymnophiona) likely constitute the least known group of vertebrates, despite being an old and remarkably distinct lineage of amphibians. Here, we characterize multi-tissue transcriptomes for five species of caecilians that represent a broad level of diversity across the order. We identified vertebrate homologous elements of caecilian functional genes of varying tissue specificity that reveal a great number of unclassified gene families, especially for the skin. We annotated several protein domains for those unknown candidate gene families to investigate their function. We also conducted supertree analyses of a phylogenomic dataset of 1,955 candidate orthologous genes among five caecilian species and other major lineages of vertebrates, with the inferred tree being in agreement with current views of vertebrate evolution and systematics. Our study provides insights into the evolution of vertebrate protein-coding genes, and a basis for future research on the molecular elements underlying the particular biology and adaptations of caecilian amphibians.

International audience; In a previous study, we purified Sm-PLGV an heterodimeric phospholipase A2, from the venom glands of the Tunisian scorpion Scorpio maurus. This enzyme contains a Long chain, a penta-peptide insertion, which is cut out during the maturation process, followed by a short chain. A disulfide bridge links the two chains. Three recombinant forms of this enzyme were produced in Escherichia coli: rPLA2(+5) with a penta-peptide insert, rPLA2(-5) without the penta-peptide, and the Long chain alone without the short one. In the present study, we showed that Sm-PLGV, rPLA2(+5) and rPLA2(-5) displayed more potent anti-angiogenic activity in vitro than the recombinant Long chain and the short one obtained by chemical synthesis. These phospholipases A2 inhibited in a dose-dependent manner adhesion, migration and invasion of Human Umbilical Vein Endothelial Cells. Using Matrigel™, we demonstrated that Sm-PLGV, rPLA2(+5) and rPLA2(-5) significantly inhibited tubulogensesis. We also showed a clear dissociation between the anti-angiogenic effect of Sm-PLGV and its catalytic activity.

Metagenomic sequence analysis is rapidly becoming the primary source of virus discovery 1–3 . A substantial majority of the currently available virus genomes come from metagenomics, and some of these represent extremely abundant viruses, even if never grown in the laboratory. A particularly striking case of a virus discovered via metagenomics is crAssphage, which is by far the most abundant human-associated virus known, comprising up to 90% of sequences in the gut virome 4 . Over 80% of the predicted proteins encoded in the approximately 100 kilobase crAssphage genome showed no significant similarity to available protein sequences, precluding classification of this virus and hampering further study. Here we combine a comprehensive search of genomic and metagenomic databases with sensitive methods for protein sequence analysis to identify an expansive, diverse group of bacteriophages related to crAssphage and predict the functions of the majority of phage proteins, in particular those that comprise the structural, replication and expression modules. Most, if not all, of the crAss-like phages appear to be associated with diverse bacteria from the phylum Bacteroidetes, which includes some of the most abundant bacteria in the human gut microbiome and that are also common in various other habitats. These findings provide for experimental characterization of the most abundant but poorly understood members of the human-associated virome.

International audience; With the immense growth in the number of available protein structures, fast and accurate structure comparison has been essential. We propose an efficient method for structure comparison, based on a structural alphabet. Protein Blocks (PBs) is a widely used structural alphabet with 16 pentapeptide conformations that can fairly approximate a complete protein chain. Thus a 3D structure can be translated into a 1D sequence of PBs. With a simple Needleman-Wunsch approach and a raw PB substitution matrix, PB-based structural alignments were better than many popular methods. iPBA web server presents an improved alignment approach using (i) specialized PB Substitution Matrices (SM) and (ii) anchor-based alignment methodology. With these developments, the quality of ∼88% of alignments was improved. iPBA alignments were also better than DALI, MUSTANG and GANGSTA(+) in >80% of the cases. The webserver is designed to for both pairwise comparisons and database searches. Outputs are given as sequence alignment and superposed 3D structures displayed using PyMol and Jmol. A local alignment option for detecting subs-structural similarity is also embedded. As a fast and efficient 'sequence-based' structure comparison tool, we believe that it will be quite useful to the scientific community. iPBA can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/ipba/.

International audience; The hepatitis C virus (HCV) genome shows remarkable sequence variability, leading to the classification of at least six major genotypes, numerous subtypes and a myriad of quasispecies within a given host. A database allowing researchers to investigate the genetic and structural variability of all available HCV sequences is an essential tool for studies on the molecular virology and pathogenesis of hepatitis C as well as drug design and vaccine development. We describe here the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr), a collection of computer-annotated sequences based on reference genomes. The annotations include genome mapping of sequences, use of recommended nomenclature, subtyping as well as three-dimensional (3D) molecular models of proteins. A WWW interface has been developed to facilitate database searches and the export of data for sequence and structure analyses. As part of an international collaborative effort with the US and Japanese databases, the European HCV Database (euHCVdb) is mainly dedicated to HCV protein sequences, 3D structures and functional analyses.

Plant viruses of the recently recognized family Amalgaviridae have monopartite double-stranded (ds) RNA genomes and encode two proteins: an RNA-dependent RNA polymerase (RdRp) and a putative capsid protein (CP). Whereas the RdRp of amalgaviruses has been found to be most closely related to the RdRps of dsRNA viruses of the family Partitiviridae, the provenance of their CP remained obscure. Here we show that the CP of amalgaviruses is homologous to the nucleocapsid proteins of negative-strand RNA viruses of the genera Phlebovirus (Bunyaviridae) and Tenuivirus. The chimeric genomes of amalgaviruses are a testament to the effectively limitless gene exchange between viruses that shaped the evolution of the virosphere. Reviewers: This article was reviewed by Lakshminarayan M. Iyer and Nick V. Grishin. For complete reviews, see the Reviewers’ Reports section. Electronic supplementary material The online version of this article (doi:10.1186/s13062-015-0047-8) contains supplementary material, which is available to authorized users.

LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast.

Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels. PBE server is freely available at http://bioinformatics.univ-reunion.fr/PBE/.

In the cuttlefish, Sepia officinalis, the ovary appears to be one of the main sources of regulatory peptides involved in the successive steps of egg-laying. Following the identification of the SepCRP-1, which is a peptide extracted from ovary and involved in egg capsule secretion, investigations were focused on the identification of related peptides. Seven related-Sepia Capsule Releasing Peptides (R-SepCRPs) were identified by means of mass spectrometry and characterized using MS/MS spectra and/or Edman degradation. Finally, primary structures were verified by the comparison of MS/MS spectra from endogenic and synthetic peptides. This new ovarian peptide family exhibits a conserved SLXKD tag involved in the biological activity. LC-MS/MS screening clearly demonstrates that R-SepCRPs are restricted to the female genital tract. Expressed during vitellogenesis, they are released by vitellogenic follicles and full-grown oocytes (FGO) in the genital coelom. Biological activities suggest that R-SepCRPs would be responsible for the storage of FGO before mating and would take part in the mechanical secretion of egg capsule products, as previously described for SepCRP-1.

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.

Motivation: Remote homology detection between protein sequences is a central problem in computational biology. Discriminative methods involving support vector machines (SVMs) are currently the most effective methods for the problem of superfamily recognition in the Structural Classification Of Proteins (SCOP) database. The performance of SVMs depends critically on the kernel function used to quantify the similarity between sequences. Results: We propose new kernels for strings adapted to biological sequences, which we call local alignment kernels. These kernels measure the similarity between two sequences by summing up scores obtained from local alignments with gaps of the sequences. When tested in combination with SVM on their ability to recognize SCOP superfamilies on a benchmark dataset, the new kernels outperform state-of-the-art methods for remote homology detection. Availability: Software and data available upon request.

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.

International audience; This article is an introduction to the special issue of the journal PROTEINS, dedicated to the tenth Critical Assessment of Structure Prediction (CASP) experiment to assess the state of the art in protein structure modeling. The article describes the conduct of the experiment, the categories of prediction included, and outlines the evaluation and assessment procedures. The 10 CASP experiments span almost 20 years of progress in the field of protein structure modeling, and there have been enormous advances in methods and model accuracy in that period. Notable in this round is the first sustained improvement of models with refinement methods, using molecular dynamics. For the first time, we tested the ability of modeling methods to make use of sparse experimental three-dimensional contact information, such as may be obtained from new experimental techniques, with encouraging results. On the other hand, new contact prediction methods, though holding considerable promise, have yet to make an impact in CASP testing. The nature of CASP targets has been changing in recent CASPs, reflecting shifts in experimental structural biology, with more irregular structures, more multi-domain and multi-subunit structures, and less standard versions of known folds. When allowance is made for these factors, we continue to see steady progress in the overall accuracy of models, particularly resulting from improvement of non-template regions.

International audience; We present the results of the assessment of the intramolecular residue-residue contact predictions from 26 prediction groups participating in the 10th round of the CASP experiment. The most recently developed direct coupling analysis methods did not take part in the experiment likely because they require a very deep sequence alignment not available for any of the 114 CASP10 targets. The performance of contact prediction methods was evaluated with the measures used in previous CASPs (i.e., prediction accuracy and the difference between the distribution of the predicted contacts and that of all pairs of residues in the target protein), as well as new measures, such as the Matthews correlation coefficient, the area under the precision-recall curve and the ranks of the first correctly and incorrectly predicted contact. We also evaluated the ability to detect interdomain contacts and tested whether the difficulty of predicting contacts depends upon the protein length and the depth of the family sequence alignment. The analyses were carried out on the target domains for which structural homologs did not exist or were difficult to identify. The evaluation was performed for all types of contacts (short, medium, and long-range), with emphasis placed on long-range contacts, i.e. those involving residues separated by at least 24 residues along the sequence. The assessment suggests that the best CASP10 contact prediction methods perform at approximately the same level, and comparably to those participating in CASP9.

International audience; A putative genome duplication event within the Silurana lineage has given rise to the tetraploid Cameroon clawed frog Silurana epitropicalis (Fischberg, Colombelli, and Picard, 1982). Peptidomic analysis of norepinephrine-stimulated skin secretions of S. epitropicalis led to identification of 10 peptides with varying degrees of growth-inhibitory activity against Escherichia coli and Staphylococcus aureus. Structural characterization identified the peptides as belonging to the magainin family (magainin-SE1), the caerulein-precursor fragment family (CPF-SE1, -SE2 and -SE3), the xenopsin-precursor fragment family (XPF-SE1, SE-2, SE-3 and -SE4), and the peptide glycine-leucine-amide family (PGLa-SE1 and -SE2). In addition, peptide phenylalanine-glutamine-amide (FLGALLGPLMNLLQ·NH(2)) was isolated from the secretions that lacked antimicrobial activity. Comparison of the multiplicity of orthologous peptides in S. epitropicalis and the diploid Silurana tropicalis indicates that extensive nonfunctionalization (deletion or silencing) of antimicrobial peptide genes has occurred after polyploidization in the Silurana lineage, as in the Xenopus lineage. CPF-SE2 (GFLGPLLKLGLKGAAKLLPQLLPSRQQ; MIC=2.5μM) and CPF-SE3 (GFLGSLLKTGLKVGSNLL·NH(2); MIC=5μM) showed potent growth-inhibitory activity against a range of clinical isolates of methicillin-resistant S. aureus (MRSA). Their utility as systemic anti-infective drugs is limited by significant hemolytic activity against human erythrocytes (LC(50)=50μM for CPF-SE2 and 220μM for CPF-SE3) but the peptides may find application as topical agents in treatment of MRSA skin infections and decolonization of MRSA carriers.

International audience; Bacterial tyrosine-kinases share no resemblance with their eukaryotic counterparts and they have been unified in a new protein family named BY-kinases. These enzymes have been shown to control several biological functions in the bacterial cells. In recent years biochemical studies, sequence analyses and structure resolutions allowed the deciphering of a common signature. However, BY-kinase sequence annotations in primary databases remain incomplete. This prompted us to develop a specialized database of computer-annotated BY-kinase sequences: the Bacterial protein tyrosine-kinase database (BYKdb). BY-kinase sequences are first identified, thanks to a workflow developed in a previous work. A second workflow annotates the UniProtKB entries in order to provide the BYKdb entries. The database can be accessed through a web interface that allows static and dynamic queries and offers integrated sequence analysis tools. BYKdb can be found at http://bykdb.ibcp.fr.

International audience; Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, I:: and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, respectively), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana. We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.

International audience; Protein structures are necessary for understanding protein function at a molecular level. Dynamics and flexibility of protein structures are also key elements of protein function. So, we have proposed to look at protein flexibility using novel methods: (i) using a structural alphabet and (ii) combining classical X-ray B-factor data and molecular dynamics simulations. First, we established a library composed of structural prototypes (LSPs) to describe protein structure by a limited set of recurring local structures. We developed a prediction method that proposes structural candidates in terms of LSPs and predict protein flexibility along a given sequence. Second, we examine flexibility according to two different descriptors: X-ray B-factors considered as good indicators of flexibility and the root mean square fluctuations, based on molecular dynamics simulations. We then define three flexibility classes and propose a method based on the LSP prediction method for predicting flexibility along the sequence. This method does not resort to sophisticate learning of flexibility but predicts flexibility from average flexibility of predicted local structures. The method is implemented in PredyFlexy web server. Results are similar to those obtained with the most recent, cutting-edge methods based on direct learning of flexibility data conducted with sophisticated algorithms. PredyFlexy can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/predyflexy/.

International audience; With the immense growth in the number of available protein structures, fast and accurate structure comparison has been essential. We propose an efficient method for structure comparison, based on a structural alphabet. Protein Blocks (PBs) is a widely used structural alphabet with 16 pentapeptide conformations that can fairly approximate a complete protein chain. Thus a 3D structure can be translated into a 1D sequence of PBs. With a simple Needleman-Wunsch approach and a raw PB substitution matrix, PB-based structural alignments were better than many popular methods. iPBA web server presents an improved alignment approach using (i) specialized PB Substitution Matrices (SM) and (ii) anchor-based alignment methodology. With these developments, the quality of ∼88% of alignments was improved. iPBA alignments were also better than DALI, MUSTANG and GANGSTA(+) in >80% of the cases. The webserver is designed to for both pairwise comparisons and database searches. Outputs are given as sequence alignment and superposed 3D structures displayed using PyMol and Jmol. A local alignment option for detecting subs-structural similarity is also embedded. As a fast and efficient 'sequence-based' structure comparison tool, we believe that it will be quite useful to the scientific community. iPBA can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/ipba/.

International audience; With the immense growth in the number of available protein structures, fast and accurate structure comparison has been essential. We propose an efficient method for structure comparison, based on a structural alphabet. Protein Blocks (PBs) is a widely used structural alphabet with 16 pentapeptide conformations that can fairly approximate a complete protein chain. Thus a 3D structure can be translated into a 1D sequence of PBs. With a simple Needleman-Wunsch approach and a raw PB substitution matrix, PB-based structural alignments were better than many popular methods. iPBA web server presents an improved alignment approach using (i) specialized PB Substitution Matrices (SM) and (ii) anchor-based alignment methodology. With these developments, the quality of ∼88% of alignments was improved. iPBA alignments were also better than DALI, MUSTANG and GANGSTA(+) in >80% of the cases. The webserver is designed to for both pairwise comparisons and database searches. Outputs are given as sequence alignment and superposed 3D structures displayed using PyMol and Jmol. A local alignment option for detecting subs-structural similarity is also embedded. As a fast and efficient 'sequence-based' structure comparison tool, we believe that it will be quite useful to the scientific community. iPBA can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/ipba/.

In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii., Author Summary Apicomplexa including Toxoplasma gondii are responsible for a variety of deadly infections. These intracellular parasites have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression in response to different environments. However, to date, little is known about nuclear factors that regulate their gene expression. Here, we have characterized parasite nuclear factors that bind to a stage-specific promoter. We identified several nuclear factors including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. We show that TgNF3 is predominantly a nucleolar, chromatin-associated protein that specifically binds to T. gondii nucleosome-associated histones and promoters. Genome-wide analysis identified promoter occupancies by TgNF3 and we demonstrated a direct role for this factor in transcriptional control of genes involved in parasite metabolism, transcription and translation. Ectopic expression of TgNF3 induces dynamic changes in the size of the nucleolus, and a severe attenuation of parasite virulence in vivo. In avirulent bradyzoites, TgNF3 is found exclusively in the cytoplasm, suggesting a potential role in regulating nucleolar and nuclear functions in the virulent tachyzoites of T. gondii.

International audience; Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)$_{206-214}$ epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP$_{206-214}$) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP$_{206-214}$. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.

Background The accurate determination of orthology and inparalogy relationships is essential for comparative sequence analysis, functional gene annotation and evolutionary studies. Various methods have been developed based on either simple blast all-versus-all pairwise comparisons and/or time-consuming phylogenetic tree analyses. Results We have developed OrthoInspector, a new software system incorporating an original algorithm for the rapid detection of orthology and inparalogy relations between different species. In comparisons with existing methods, OrthoInspector improves detection sensitivity, with a minimal loss of specificity. In addition, several visualization tools have been developed to facilitate in-depth studies based on these predictions. The software has been used to study the orthology/in-paralogy relationships for a large set of 940,855 protein sequences from 59 different eukaryotic species. Conclusion OrthoInspector is a new software system for orthology/paralogy analysis. It is made available as an independent software suite that can be downloaded and installed for local use. Command line querying facilitates the integration of the software in high throughput processing pipelines and a graphical interface provides easy, intuitive access to results for the non-expert.

Summary: We previously developed OrthoInspector, a package incorporating an original algorithm for the detection of orthology and inparalogy relations between different species. We have added new functionalities to the package. While its original algorithm was not modified, performing similar orthology predictions, we facilitated the prediction of very large databases (thousands of proteomes), refurbished its graphical interface, added new visualization tools for comparative genomics/protein family analysis and facilitated its deployment in a network environment. Finally, we have released three online databases of precomputed orthology relationships. Availability: Package and databases are freely available at http://lbgi.fr/orthoinspector with all major browsers supported. Contact: odile.lecompte@unistra.fr Supplementary information: Supplementary data are available at Bioinformatics online.

International audience; BACKGROUND: Protein fold recognition usually relies on a statistical model of each fold; each model is constructed from an ensemble of natural sequences belonging to that fold. A complementary strategy may be to employ sequence ensembles produced by computational protein design. Designed sequences can be more diverse than natural sequences, possibly avoiding some limitations of experimental databases. METHODOLOGY/PRINCIPAL FINDINGS: WE EXPLORE THIS STRATEGY FOR FOUR SCOP FAMILIES: Small Kunitz-type inhibitors (SKIs), Interleukin-8 chemokines, PDZ domains, and large Caspase catalytic subunits, represented by 43 structures. An automated procedure is used to redesign the 43 proteins. We use the experimental backbones as fixed templates in the folded state and a molecular mechanics model to compute the interaction energies between sidechain and backbone groups. Calculations are done with the Proteins@Home volunteer computing platform. A heuristic algorithm is used to scan the sequence and conformational space, yielding 200,000-300,000 sequences per backbone template. The results confirm and generalize our earlier study of SH2 and SH3 domains. The designed sequences ressemble moderately-distant, natural homologues of the initial templates; e.g., the SUPERFAMILY, profile Hidden-Markov Model library recognizes 85% of the low-energy sequences as native-like. Conversely, Position Specific Scoring Matrices derived from the sequences can be used to detect natural homologues within the SwissProt database: 60% of known PDZ domains are detected and around 90% of known SKIs and chemokines. Energy components and inter-residue correlations are analyzed and ways to improve the method are discussed. CONCLUSIONS/SIGNIFICANCE: For some families, designed sequences can be a useful complement to experimental ones for homologue searching. However, improved tools are needed to extract more information from the designed profiles before the method can be of general use.

Background The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. Results We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate β-globin locus, possibly arguing against a late transpositional origin of the β-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes. Conclusions The identification of putative orthologs of vertebrate globin variants in the B. floridae genome underlines the importance of cephalochordates for elucidating vertebrate genome evolution. The present study facilitates detailed functional studies of the amphioxus globins in order to trace conserved properties and specific adaptations of respiratory proteins at the base of chordate evolution.

The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the “surface-properties” of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity., PLoS Computational Biology, 6 (7), ISSN:1553-734X, ISSN:1553-7358

Background Ribonucleotide reduction is the only de novo pathway for synthesis of deoxyribonucleotides, the building blocks of DNA. The reaction is catalysed by ribonucleotide reductases (RNRs), an ancient enzyme family comprised of three classes. Each class has distinct operational constraints, and are broadly distributed across organisms from all three domains, though few class I RNRs have been identified in archaeal genomes, and classes II and III likewise appear rare across eukaryotes. In this study, we examine whether this distribution is best explained by presence of all three classes in the Last Universal Common Ancestor (LUCA), or by horizontal gene transfer (HGT) of RNR genes. We also examine to what extent environmental factors may have impacted the distribution of RNR classes. Results Our phylogenies show that the Last Eukaryotic Common Ancestor (LECA) possessed a class I RNR, but that the eukaryotic class I enzymes are not directly descended from class I RNRs in Archaea. Instead, our results indicate that archaeal class I RNR genes have been independently transferred from bacteria on two occasions. While LECA possessed a class I RNR, our trees indicate that this is ultimately bacterial in origin. We also find convincing evidence that eukaryotic class I RNR has been transferred to the Bacteroidetes, providing a stunning example of HGT from eukaryotes back to Bacteria. Based on our phylogenies and available genetic and genomic evidence, class II and III RNRs in eukaryotes also appear to have been transferred from Bacteria, with subsequent within-domain transfer between distantly-related eukaryotes. Under the three-domains hypothesis the RNR present in the last common ancestor of Archaea and eukaryotes appears, through a process of elimination, to have been a dimeric class II RNR, though limited sampling of eukaryotes precludes a firm conclusion as the data may be equally well accounted for by HGT. Conclusions Horizontal gene transfer has clearly played an important role in the evolution of the RNR repertoire of organisms from all three domains of life. Our results clearly show that class I RNRs have spread to Archaea and eukaryotes via transfers from the bacterial domain, indicating that class I likely evolved in the Bacteria. However, against the backdrop of ongoing transfers, it is harder to establish whether class II or III RNRs were present in the LUCA, despite the fact that ribonucleotide reduction is an essential cellular reaction and was pivotal to the transition from RNA to DNA genomes. Instead, a general pattern of ongoing horizontal transmission emerges wherein environmental and enzyme operational constraints, especially the presence or absence of oxygen, are likely to be major determinants of the RNR repertoire of genomes.

International audience; We present a thorough analysis of the relation between amino acid sequence and local three-dimensional structure in proteins. A library of overlapping local structural prototypes was built using an unsupervised clustering approach called "hybrid protein model" (HPM). The HPM carries out a multiple structural alignment of local folds from a non-redundant protein structure databank encoded into a structural alphabet composed of 16 protein blocks (PBs). Following previous research focusing on the HPM protocol, we have considered gaps in the local structure prototype. This methodology allows to have variable length fragments. Hence, 120 local structure prototypes were obtained. Twenty-five percent of the protein fragments learnt by HPM had gaps. An investigation of tight turns suggested that they are mainly derived from three PB series with precise locations in the HPM. The amino acid information content of the whole conformational classes was tackled by multivariate methods, e.g., canonical correlation analysis. It points out the presence of seven amino acid equivalence classes showing high propensities for preferential local structures. In the same way, definition of "contrast factors" based on sequence-structure properties underline the specificity of certain structural prototypes, e.g., the dependence of Gly or Asn-rich turns to a limited number of PBs, or, the opposition between Pro-rich coils to those enriched in Ser, Thr, Asn and Glu. These results are so useful to analyze the sequence-structure relationships, but could also be used to improve fragment-based method for protein structure prediction from sequence.

International audience; We present a thorough analysis of the relation between amino acid sequence and local three-dimensional structure in proteins. A library of overlapping local structural prototypes was built using an unsupervised clustering approach called "hybrid protein model" (HPM). The HPM carries out a multiple structural alignment of local folds from a non-redundant protein structure databank encoded into a structural alphabet composed of 16 protein blocks (PBs). Following previous research focusing on the HPM protocol, we have considered gaps in the local structure prototype. This methodology allows to have variable length fragments. Hence, 120 local structure prototypes were obtained. Twenty-five percent of the protein fragments learnt by HPM had gaps. An investigation of tight turns suggested that they are mainly derived from three PB series with precise locations in the HPM. The amino acid information content of the whole conformational classes was tackled by multivariate methods, e.g., canonical correlation analysis. It points out the presence of seven amino acid equivalence classes showing high propensities for preferential local structures. In the same way, definition of "contrast factors" based on sequence-structure properties underline the specificity of certain structural prototypes, e.g., the dependence of Gly or Asn-rich turns to a limited number of PBs, or, the opposition between Pro-rich coils to those enriched in Ser, Thr, Asn and Glu. These results are so useful to analyze the sequence-structure relationships, but could also be used to improve fragment-based method for protein structure prediction from sequence.

Background The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. Results An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. Conclusion The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score.

Background Macromolecular docking is a challenging field of bioinformatics. Developing new algorithms is a slow process generally involving routine tasks that should be found in a robust library and not programmed from scratch for every new software application. Results We present an object-oriented Python/C++ library to help the development of new docking methods. This library contains low-level routines like PDB-format manipulation functions as well as high-level tools for docking and analyzing results. We also illustrate the ease of use of this library with the detailed implementation of a 3-body docking procedure. Conclusion The PTools library can handle molecules at coarse-grained or atomic resolution and allows users to rapidly develop new software. The library is already in use for protein-protein and protein-DNA docking with the ATTRACT program and for simulation analysis. This library is freely available under the GNU GPL license, together with detailed documentation.

The Joint Evolutionary Trees (JET) method detects protein interfaces, the core residues involved in the folding process, and residues susceptible to site-directed mutagenesis and relevant to molecular recognition. The approach, based on the Evolutionary Trace (ET) method, introduces a novel way to treat evolutionary information. Families of homologous sequences are analyzed through a Gibbs-like sampling of distance trees to reduce effects of erroneous multiple alignment and impacts of weakly homologous sequences on distance tree construction. The sampling method makes sequence analysis more sensitive to functional and structural importance of individual residues by avoiding effects of the overrepresentation of highly homologous sequences and improves computational efficiency. A carefully designed clustering method is parametrized on the target structure to detect and extend patches on protein surfaces into predicted interaction sites. Clustering takes into account residues' physical-chemical properties as well as conservation. Large-scale application of JET requires the system to be adjustable for different datasets and to guarantee predictions even if the signal is low. Flexibility was achieved by a careful treatment of the number of retrieved sequences, the amino acid distance between sequences, and the selective thresholds for cluster identification. An iterative version of JET (iJET) that guarantees finding the most likely interface residues is proposed as the appropriate tool for large-scale predictions. Tests are carried out on the Huang database of 62 heterodimer, homodimer, and transient complexes and on 265 interfaces belonging to signal transduction proteins, enzymes, inhibitors, antibodies, antigens, and others. A specific set of proteins chosen for their special functional and structural properties illustrate JET behavior on a large variety of interactions covering proteins, ligands, DNA, and RNA. JET is compared at a large scale to ET and to Consurf, Rate4Site, siteFiNDER|3D, and SCORECONS on specific structures. A significant improvement in performance and computational efficiency is shown., Author Summary Information obtained on the structure of macromolecular complexes is important for identifying functionally important partners but also for determining how such interactions will be perturbed by natural or engineered site mutations. Hence, to fully understand or control biological processes we need to predict in the most accurate manner protein interfaces for a protein structure, possibly without knowing its partners. Joint Evolutionary Trees (JET) is a method designed to detect very different types of interactions of a protein with another protein, ligands, DNA, and RNA. It uses a carefully designed sampling method, making sequence analysis more sensitive to the functional and structural importance of individual residues, and a clustering method parametrized on the target structure for the detection of patches on protein surfaces and their extension into predicted interaction sites. JET is a large-scale method, highly accurate and potentially applicable to search for protein partners.

Motivation: The biological solution for synthesis or remediation of organic compounds using living organisms, particularly bacteria and yeast, has been promoted because of the cost reduction with respect to the non-living chemical approach. In that way, computational frameworks can profit from the previous knowledge stored in large databases of compounds, enzymes and reactions. In addition, the cell behavior can be studied by modeling the cellular context. Results: We have implemented a Monte Carlo algorithm (DESHARKY) that finds a metabolic pathway from a target compound by exploring a database of enzymatic reactions. DESHARKY outputs a biochemical route to the host metabolism together with its impact in the cellular context by using mathematical models of the cell resources and metabolism. Furthermore, we provide the sequence of amino acids for the enzymes involved in the route closest phylogenetically to the considered organism. We provide examples of designed metabolic pathways with their genetic load characterizations. Here, we have used Escherichia coli as host organism. In addition, our bioinformatic tool can be applied for biodegradation or biosynthesis and its performance scales with the database size. Availability: Software, a tutorial and examples are freely available and open source at http://soft.synth-bio.org/desharky.html Contact: alfonso.jaramillo@polytechnique.fr Supplementary information: Supplementary data are available at Bioinformatics online.

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria -, Betaproteobacteria -, and Gammaproteobacteria -related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis., Author Summary The causative agent of the infectious disease, amoebiasis, is the parasite Entamoeba histolytica, which targets human intestine and liver. Once in the host, this parasite attaches to human cells and matrix components via factors at its surface such as the Gal/GalNAc lectin and proteophosphoglycans (PPGs). These factors are themselves anchored to the parasite surface by a glycosylphosphatidylinositol (GPI) moiety. To synthesise the GPI, a cascade of enzymes are necessary including the mannosyltransferase 1 (PIG-M1). A homolog of the PIG-M1 enzyme was shown to be present in E. histolytica (EhPIG-M1). To study the role of EhPIG-M1 in E. histolytica, parasites were constructed that had a reduced amount of mannosyltransferase. These parasites displayed a diminished production of GPI molecules and a lower amount of PPGs at the cell surface. Interestingly, the parasites were highly sensitive to the host blood complement and the formation of liver abscesses in hamsters was dramatically impaired. These results suggest that molecules anchored to the cell surface with the GPI moiety have a pivotal role in the survival of E. histolytica during pathogenesis.

Motivation: This work aims to develop computational methods to annotate protein structures in an automated fashion. We employ a support vector machine (SVM) classifier to map from a given class of structures to their corresponding structural (SCOP) or functional (Gene Ontology) annotation. In particular, we build upon recent work describing various kernels for protein structures, where a kernel is a similarity function that the classifier uses to compare pairs of structures.Results: We describe a kernel that is derived in a straightforward fashion from an existing structural alignment program, MAMMOTH. We find in our benchmark experiments that this kernel significantly out-performs a variety of other kernels, including several previously described kernels. Furthermore, in both benchmarks, classifying structures using MAMMOTH alone does not work as well as using an SVM with the MAMMOTH kernel.Availability: http://noble.gs.washington.edu/proj/3dkernelContact: noble@gs.washington.edu

The RBMY (RNA-binding motif gene on Y chromosome) protein encoded by the human Y chromosome is important for normal sperm development. Although its precise molecular RNA targets are unknown at present, it is suggested that human RBMY (hRBMY) participates in splicing in the testis. Using systematic evolution of ligands by exponential enrichment, we found that RNA stem-loops capped by a C(A)/(U)CAA pentaloop are high-affinity binding targets for hRBMY. Subsequent nuclear magnetic resonance structural determination of the hRBMY RNA recognition motif (RRM) in complex with a high-affinity target showed two distinct modes of RNA recognition. First, the RRM beta-sheet surface binds to the RNA loop in a sequence-specific fashion. Second, the beta2-beta3 loop of the hRBMY inserts into the major groove of the RNA stem. The first binding mode might be conserved in the paralogous protein heterogeneous nuclear RNP G, whereas the second mode of binding is found only in hRBMY. This structural difference could be at the origin of the function of RBMY in spermatogenesis.

Summary: Biskit is a modular, object-oriented python library that provides intuitive classes for many typical tasks of structural bioinformatics research. It facilitates the manipulation and analysis of macromolecular structures, protein complexes and molecular dynamics trajectories. At the same time, Biskit offers a software platform for the rapid integration of external programs and new algorithms into complex structural bioinformatics workflows. Calculations are thus often delegated to established programs like Xplor, Amber, Hex, Prosa, Hmmer and Modeller; interfaces to further software can be easily added. Moreover, Biskit simplifies the parallelization of time consuming calculations via PVM (Parallel Virtual Machine).Availability: The latest snapshot of Biskit, documentation and examples are freely available under the GNU General Public License at http://biskit.sf.net (alternate url http://biskit.pasteur.fr).Contact: johan.leckner@nmr.se, raik.gruenberg@crg.es

Protein point mutations are an essential component of the evolutionary and experimental analysis of protein structure and function. While many manually curated databases attempt to index point mutations, most experimentally generated point mutations and the biological impacts of the changes are described in the peer-reviewed published literature. We describe an application, Mutation GraB (Graph Bigram), that identifies, extracts, and verifies point mutations from biomedical literature. The principal problem of point mutation extraction is to link the point mutation with its associated protein and organism of origin. Our algorithm uses a graph-based bigram traversal to identify these relevant associations and exploits the Swiss-Prot protein database to verify this information. The graph bigram method is different from other models for point mutation extraction in that it incorporates frequency and positional data of all terms in an article to drive the point mutation–protein association. Our method was tested on 589 articles describing point mutations from the G protein–coupled receptor (GPCR), tyrosine kinase, and ion channel protein families. We evaluated our graph bigram metric against a word-proximity metric for term association on datasets of full-text literature in these three different protein families. Our testing shows that the graph bigram metric achieves a higher F-measure for the GPCRs (0.79 versus 0.76), protein tyrosine kinases (0.72 versus 0.69), and ion channel transporters (0.76 versus 0.74). Importantly, in situations where more than one protein can be assigned to a point mutation and disambiguation is required, the graph bigram metric achieves a precision of 0.84 compared with the word distance metric precision of 0.73. We believe the graph bigram search metric to be a significant improvement over previous search metrics for point mutation extraction and to be applicable to text-mining application requiring the association of words., Author Summary In biological research, new information is often presented in the form of peer-reviewed published journal articles. Despite the best efforts of electronic database curators, a majority of this information is still found only in textual form, and thus excluded from direct computational analysis. One such type of information that is abundant in scientific literature is protein point mutations. We seek to extract protein point mutation examples from the literature and to associate them with a unique protein name and species of origin in a standardized protein database. To do this, we have created an application that searches for and retrieves full-text articles from publishers, identifies point mutation terms, protein name terms, and organism name terms within the articles. We describe Mutation GraB, an application that utilizes a graph shortest-distance search in concert with word bigram analysis that is used to find significant associations between these terms in the text. This graph bigram search metric was found to be reasonably effective at identifying correct protein point mutation pairs and represents a good compromise between accuracy and broad applicability. The application can be applied to a large set of journal literature from a protein family to generate a database of point mutations.

International audience; The members of the Aquarana (or Rana catesbeiana species group) form a monophyletic group comprising seven species: R. catesbeiana, Rana clamitans, Rana grylio, Rana virgatipes, Rana septentrionalis, Rana heckscheri and Rana okaloosae. Previous work has led to structural characterization of the antimicrobial peptides present in electrically-stimulated skin secretions from the first five species listed and this study presents the primary structures of orthologs from the river frog R. heckscheri and the Florida bog frog R. okaloosae. Peptidomic analysis of R. heckscheri and R. okaloosae skin secretions led to the identification of peptides with antimicrobial activity belonging to the ranalexin, ranatuerin-2, and temporin families. In addition, a peptide (GFLDIIKDTGKDFAVKILNNLKCKLAGGCPR) was isolated from R. okaloosae whose primary structure identified it as a member of the palustrin-2 family. Consistent with previous data based upon morphological analysis and comparisons of the nucleotide sequences of mitochondrial and ribosomal genes, cladistic analysis based upon a comparison of the amino acid sequences of antimicrobial peptides indicates a sister-group relationship between R. heckscheri and R. grylio and a close, but less well defined, phylogenetic relationship between R. okaloosae and R. clamitans.

We investigated venoms from members of the genus Atheris (Serpentes, Viperidae), namely the rough scale bush viper (Atheris squamigera), the green bush viper (A. chlorechis) and the great lakes bush viper (A. nitschei), using mass spectrometry-based strategies, relying on matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) with de novo peptide sequencing. We discovered a set of novel peptides with masses in the 2-3 kDa range and containing poly-His and poly-Gly segments (pHpG). Complete primary structural elucidation and confirmation of two sequences by Edman degradation indicated the consensus sequence EDDH(9)GVG(10). Bioinformatic investigations in protein sequence databanks did not show relevant homology with known peptides or proteins. However, a more extensive investigation of data in nucleic acid databases revealed some similarities to the precursor sequences of bradykinin potentiating peptides (BPP) and C-type natriuretic peptides (CNP), agents that are known to affect the cardiovascular system by acting on specific metalloproteases and receptors. The novel pHpG peptides found in Atheris venoms might also act on the cardiovascular system by inhibiting particular metalloproteases, which however remain to be identified.

In structural genomics, an important goal is the detection and classification of protein-protein interactions, given the structures of the interacting partners. We have developed empirical energy functions to identify native structures of protein-protein complexes among sets of decoy structures. To understand the role of amino acid diversity, we parameterized a series of functions, using a hierarchy of amino acid alphabets of increasing complexity, with 2, 3, 4, 6, and 20 amino acid groups. Compared to previous work, we used the simplest possible functional form, with residue-residue interactions and a stepwise distance-dependence. We used increased computational resources, however, constructing 290,000 decoys for 219 protein-protein complexes, with a realistic docking protocol where the protein partners are flexible and interact through a molecular mechanics energy function. The energy parameters were optimized to correctly assign as many native complexes as possible. To resolve the multiple minimum problem in parameter space, over 64000 starting parameter guesses were tried for each energy function. The optimized functions were tested by cross validation on subsets of our native and decoy structures, by blind tests on series of native and decoy structures available on the Web, and on models for 13 complexes submitted to the CAPRI structure prediction experiment., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Background The post-genomic era is characterised by a torrent of biological information flooding the public databases. As a direct consequence, similarity searches starting with a single query sequence frequently lead to the identification of hundreds, or even thousands of potential homologues. The huge volume of data renders the subsequent structural, functional and evolutionary analyses very difficult. It is therefore essential to develop new strategies for efficient sampling of this large sequence space, in order to reduce the number of sequences to be processed. At the same time, it is important to retain the most pertinent sequences for structural and functional studies. Results An exhaustive analysis on a large scale test set (284 protein families) was performed to compare the efficiency of four different sampling methods aimed at selecting the most pertinent sequences. These four methods sample the proteins detected by BlastP searches and can be divided into two categories: two customisable methods where the user defines either the maximal number or the percentage of sequences to be selected; two automatic methods in which the number of sequences selected is determined by the program. We focused our analysis on the potential information content of the sampled sets of sequences using multiple alignment of complete sequences as the main validation tool. The study considered two criteria: the total number of sequences in BlastP and their associated E-values. The subsequent analyses investigated the influence of the sampling methods on the E-value distributions, the sequence coverage, the final multiple alignment quality and the active site characterisation at various residue conservation thresholds as a function of these criteria. Conclusion The comparative analysis of the four sampling methods allows us to propose a suitable sampling strategy that significantly reduces the number of homologous sequences required for alignment, while at the same time maintaining the relevant information concerning the active site residues.

Analysis of protein structures based on backbone structural patterns known as structural alphabets have been shown to be very useful. Among them, a set of 16 pentapeptide structural motifs known as protein blocks (PBs) has been identified and upon which backbone model of most protein structures can be built. PBs allows simplification of 3D space onto 1D space in the form of sequence of PBs. Here, for the first time, substitution probabilities of PBs in a large number of aligned homologous protein structures have been studied and are expressed as a simplified 16 × 16 substitution matrix. The matrix was validated by benchmarking how well it can align sequences of PBs rather like amino acid alignment to identify structurally equivalent regions in closely or distantly related proteins using dynamic programming approach. The alignment results obtained are very comparable to well established structure comparison methods like DALI and STAMP. Other interesting applications of the matrix have been investigated. We first show that, in variable regions between two superimposed homologous proteins, one can distinguish between local conformational differences and rigid-body displacement of a conserved motif by comparing the PBs and their substitution scores. Second, we demonstrate, with the example of aspartic proteinases, that PBs can be efficiently used to detect the lobe/domain flexibility in the multidomain proteins. Lastly, using protein kinase as an example, we identify regions of conformational variations and rigid body movements in the enzyme as it is changed to the active state from an inactive state. Proteins 2006. © 2006 Wiley-Liss, Inc.

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.

International audience; The microsporidian Encephalitozoon cuniculi is a unicellular obligate intracellular parasite considered as an emerging opportunistic human pathogen. The differentiation phase of its life cycle leads to the formation of stress-resistant spores. The E. cuniculi genome (2.9 Mbp) having been sequenced, we undertook a descriptive proteomic study of a spore-rich cell population isolated from culture supernatants. A combination of 2-DE and 2-DE-free techniques was applied to whole-cell protein extracts. Protein identification was performed using an automated MALDI-TOF-MS platform and a nanoLC-MS/MS instrument. A reference 2-DE map of about 350 major spots with multiple isoforms was obtained, and for the first time in microsporidia, a large set of unique proteins (177) including proteins with unknown function in a proportion of 25.6% was identified. The data are mainly discussed with reference to secretion and spore structural features, energy and carbohydrate metabolism, cell cycle control and parasite survival in the environment.

International audience; The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

Background In the post-genomic era, systems-level studies are being performed that seek to explain complex biological systems by integrating diverse resources from fields such as genomics, proteomics or transcriptomics. New information management systems are now needed for the collection, validation and analysis of the vast amount of heterogeneous data available. Multiple alignments of complete sequences provide an ideal environment for the integration of this information in the context of the protein family. Results MACSIMS is a multiple alignment-based information management program that combines the advantages of both knowledge-based and ab initio sequence analysis methods. Structural and functional information is retrieved automatically from the public databases. In the multiple alignment, homologous regions are identified and the retrieved data is evaluated and propagated from known to unknown sequences with these reliable regions. In a large-scale evaluation, the specificity of the propagated sequence features is estimated to be >99%, i.e. very few false positive predictions are made. MACSIMS is then used to characterise mutations in a test set of 100 proteins that are known to be involved in human genetic diseases. The number of sequence features associated with these proteins was increased by 60%, compared to the features available in the public databases. An XML format output file allows automatic parsing of the MACSIM results, while a graphical display using the JalView program allows manual analysis. Conclusion MACSIMS is a new information management system that incorporates detailed analyses of protein families at the structural, functional and evolutionary levels. MACSIMS thus provides a unique environment that facilitates knowledge extraction and the presentation of the most pertinent information to the biologist. A web server and the source code are available at http://bips.u-strasbg.fr/MACSIMS/.