Prediction of enzyme function by combining sequence similarity and protein interactions

Background: A number of studies have used protein interaction data alone for protein function prediction. Here, we introduce a computational approach for annotation of enzymes, based on the observation that similar protein sequences are more likely to perform the same function if they share similar interacting partners. Results: The method has been tested against the PSI-BLAST program using a set of 3,890 protein sequences from which interaction data was available. For protein sequences that align with at least 40% sequence identity to a known enzyme, the specificity of our method in predicting the first three EC digits increased from 80% to 90% at 80% coverage when compared to PSI-BLAST. Conclusion: Our method can also be used in proteins for which homologous sequences with known interacting partners can be detected. Thus, our method could increase 10% the specificity of genome-wide enzyme predictions based on sequence matching by PSI-BLAST alone.

Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

Background: The trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each other's phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.Results: The analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).Conclusion: The similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

The quest for a message: budding yeast, a model organism to study the control of pre-mRNA splicing

Removal of introns during pre-mRNA splicing is a critical process in gene expression, and understanding its control at both single-gene and genomic levels is one of the great challenges in Biology. Splicing takes place in a dynamic, large ribonucleoprotein complex known as the spliceosome. Combining Genetics and Biochemistry, Saccharomyces cerevisiae provides insights into its mechanisms, including its regulation by RNA-protein interactions. Recent genome-wide analyses indicate that regulated splicing is broad and biologically relevant even in organisms with a relatively simple intronic structure, such as yeast. Furthermore, the possibility of coordination in splicing regulation at genomic level is becoming clear in this model organism. This should provide a valuable system to approach the complex problem of the role of regulated splicing in genomic expression.

Biana: a software framework for compiling biological interactions and analyzing networks

Background: The analysis and usage of biological data is hindered by the spread of information across multiple repositories and the difficulties posed by different nomenclature systems and storage formats. In particular, there is an important need for data unification in the study and use of protein-protein interactions. Without good integration strategies, it is difficult to analyze the whole set of available data and its properties.Results: We introduce BIANA (Biologic Interactions and Network Analysis), a tool for biological information integration and network management. BIANA is a Python framework designed to achieve two major goals: i) the integration of multiple sources of biological information, including biological entities and their relationships, and ii) the management of biological information as a network where entities are nodes and relationships are edges. Moreover, BIANA uses properties of proteins and genes to infer latent biomolecular relationships by transferring edges to entities sharing similar properties. BIANA is also provided as a plugin for Cytoscape, which allows users to visualize and interactively manage the data. A web interface to BIANA providing basic functionalities is also available. The software can be downloaded under GNU GPL license from http://sbi.imim.es/web/BIANA.php.Conclusions: BIANA's approach to data unification solves many of the nomenclature issues common to systems dealing with biological data. BIANA can easily be extended to handle new specific data repositories and new specific data types. The unification protocol allows BIANA to be a flexible tool suitable for different user requirements: non-expert users can use a suggested unification protocol while expert users can define their own specific unification rules.

Gene Ontology Function prediction in Mollicutes using Protein-Protein Association Networks

Abstract Background: Many complex systems can be represented and analysed as networks. The recent availability of large-scale datasets, has made it possible to elucidate some of the organisational principles and rules that govern their function, robustness and evolution. However, one of the main limitations in using protein-protein interactions for function prediction is the availability of interaction data, especially for Mollicutes. If we could harness predicted interactions, such as those from a Protein-Protein Association Networks (PPAN), combining several protein-protein network function-inference methods with semantic similarity calculations, the use of protein-protein interactions for functional inference in this species would become more potentially useful. Results: In this work we show that using PPAN data combined with other approximations, such as functional module detection, orthology exploitation methods and Gene Ontology (GO)-based information measures helps to predict protein function in Mycoplasma genitalium. Conclusions: To our knowledge, the proposed method is the first that combines functional module detection among species, exploiting an orthology procedure and using information theory-based GO semantic similarity in PPAN of the Mycoplasma species. The results of an evaluation show a higher recall than previously reported methods that focused on only one organism network.

Protein interaction studies point to new functions for Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein-protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of E. coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3"-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.

Estudis estructurals dels canvis conformacionals de les E3 ubiquitin lligases per RMN

L'estudi a nivell molecular de la ruta de senyalització activada per TGF-B té un impacte notable en el panorama actual donada la seva implicació en processos autoimmunitaris i carcinogènics. D'altra banda, l'elucidació a nivell estructural dels mecanismes moleculars que permeten a les ubiquitin lligases de tipus E3 marcar específicament llurs dianes per a la degradació proteosòmica - entre d'altres - resulta fonamental donada la seva importància pel que fa al control sobre el turnover proteic a nivell intracel•lular. En aquest projecte es pretén elucidar els mecanismes d'activació i catlàlisi de les ubiquitin lligases E3 tipus HECT Smurf1 i Nedd4L al mateix temps que se n'estudia la implicació en la regulació dels agents missatgers de la ruta del TGF-B. Així, la tasca es divideix en tres sub-projectes els quals se centren en a) l'estudi de la interacció d'aquestes lligases amb llurs dianes; b) l'elucidació del mecanisme d'activació i c) del de catàlisi d'aquests enzims. Per tal d'assolir aquests objectius ens servim principalment dels avantatges que ens aporta la Ressonància Magnètica Nuclear i altres tècniques biofísiques, principalment la ITC. Durant el temps que he gaudit de la beca FI, m'he centrat principalment en la preparació de pèptids per SPPS, llur purificació per HPLC i caracterització per MS i RMN. Aquests pèptids representen diferents patrons de fosforilació de certes dianes de les lligases esmentades, de manera que han estat emprats per a l'estudi d'interaccions proteïna-proteïna per ITC i RMN. M'he iniciat doncs en l'ús d'aquestes tècniques. D'altra banda, també he preparat mostres protèiques mitjançant l'ús de sistemes d'expressió bacterians basats en E.coli, incloent l'amplificació i clonació del gen que codifica per la proteïna d'interés així com la seva expressió, purificació i caracterització per MS i RMN.

Predicting cancer involvement of genes from heterogeneous data

Background: Systematic approaches for identifying proteins involved in different types of cancer are needed. Experimental techniques such as microarrays are being used to characterize cancer, but validating their results can be a laborious task. Computational approaches are used to prioritize between genes putatively involved in cancer, usually based on further analyzing experimental data. Results: We implemented a systematic method using the PIANA software that predicts cancer involvement of genes by integrating heterogeneous datasets. Specifically, we produced lists of genes likely to be involved in cancer by relying on: (i) protein-protein interactions; (ii) differential expression data; and (iii) structural and functional properties of cancer genes. The integrative approach that combines multiple sources of data obtained positive predictive values ranging from 23% (on a list of 811 genes) to 73% (on a list of 22 genes), outperforming the use of any of the data sources alone. We analyze a list of 20 cancer gene predictions, finding that most of them have been recently linked to cancer in literature. Conclusion: Our approach to identifying and prioritizing candidate cancer genes can be used to produce lists of genes likely to be involved in cancer. Our results suggest that differential expression studies yielding high numbers of candidate cancer genes can be filtered using protein interaction networks.

Time-scale and other invariants of integrative mechanical behavior in living cells.

In dealing with systems as complex as the cytoskeleton, we need organizing principles or, short of that, an empirical framework into which these systems fit. We report here unexpected invariants of cytoskeletal behavior that comprise such an empirical framework. We measured elastic and frictional moduli of a variety of cell types over a wide range of time scales and using a variety of biological interventions. In all instances elastic stresses dominated at frequencies below 300 Hz, increased only weakly with frequency, and followed a power law; no characteristic time scale was evident. Frictional stresses paralleled the elastic behavior at frequencies below 10 Hz but approached a Newtonian viscous behavior at higher frequencies. Surprisingly, all data could be collapsed onto master curves, the existence of which implies that elastic and frictional stresses share a common underlying mechanism. Taken together, these findings define an unanticipated integrative framework for studying protein interactions within the complex microenvironment of the cell body, and appear to set limits on what can be predicted about integrated mechanical behavior of the matrix based solely on cytoskeletal constituents considered in isolation. Moreover, these observations are consistent with the hypothesis that the cytoskeleton of the living cell behaves as a soft glassy material, wherein cytoskeletal proteins modulate cell mechanical properties mainly by changing an effective temperature of the cytoskeletal matrix. If so, then the effective temperature becomes an easily quantified determinant of the ability of the cytoskeleton to deform, flow, and reorganize.

Cognate peptide-receptor ligand mapping by directed phage display

Background: A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described. The approach may be readily integrated into the interface of genomic and proteomic studies to identify biologically relevant ligands.Methods: A gene fragment library from influenza coat protein haemagglutinin (HA) gene was constructed by treating HA cDNA with DNAse I to create 50 ¿ 100 bp fragments. These fragments were cloned into plasmid pORFES IV and in-frame inserts were selected. These in-frame fragment inserts were subsequently cloned into a filamentous phage display vector JC-M13-88 for surface display as fusions to a synthetic copy of gene VIII. Two well characterized antibodies, mAb 12CA5 and pAb 07431, directed against distinct known regions of HA were used to pan the library. Results: Two linear epitopes, HA peptide 112 ¿ 126 and 162¿173, recognized by mAb 12CA5 and pAb 07431, respectively, were identified as the cognate epitopes.Conclusion: This approach is a useful alternative to conventional methods such as screening of overlapping synthetic peptide libraries or gene fragment expression libraries when searching for precise peptide protein interactions, and may be applied to functional proteomics.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BACKGROUND: The bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species. RESULTS: We sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an omega ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence. CONCLUSION: The models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection. REVIEWERS: This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.