Interactome-wide prediction of short, disordered protein interaction motifs in humans

Edwards RJ, Davey NE, O’Brien K & Shields DC (2012): Interactome-wide prediction of short, disordered protein interaction motifs in humans. Molecular Biosystems 8: 282-95.

Abstract

Many of the specific functions of intrinsically disordered protein segments are mediated by Short Linear Motifs (SLiMs) interacting with other proteins. Well known examples include SLiMs that interact with 14-3-3, PDZ, SH2, SH3, and WW domains but the true extent and diversity of SLiM-mediated interactions is largely unknown. Here, we attempt to expand our knowledge of human SLiMs by applying in silico SLiM prediction to the human interactome. Combining data from seven different interaction databases, we analysed approximately 6000 protein-centred and 1600 domain-centred human interaction datasets of 3+ unrelated proteins that interact with a common partner. Results were placed in context through comparison to randomised datasets of similar size and composition. The search returned thousands of evolutionarily conserved, intrinsically disordered occurrences of hundreds of significantly enriched recurring motifs, including many that have never been previously identified (). In addition to True Positive results for at least 25 different known SLiMs, a striking number of “off-target” proteins/domains also returned significantly enriched known motifs. Often, this was due to the non-independence of the datasets, with many proteins sharing interaction partners or contributing interactions to multiple domain datasets. The majority of these motif classes, however, were also found to be significantly enriched in one or more randomised datasets. This highlights the need for care when interpreting motif predictions of this nature but also raises the possibility that SLiM occurrences may be successfully identified independently of interaction data. Although not as compositionally biased as previous studies, patterns matching known SLiMs tended to cluster into a few large groups of similar sequence, while novel predictions tended to be more distinctive and less abundant. Whether this is due to ascertainment bias or a true functional composition bias of SLiMs is not clear and warrants further investigation.

PMID: 21879107

Shotgun proteomic analysis of Emiliania huxleyi, a marine phytoplankton species of major biogeochemical importance

Jones BM*, Edwards RJ*, Skipp PJ, O’Connor CD & Iglesias-Rodriguez MD (2011): Shotgun Proteomic Analysis of Emiliania huxleyi, a Marine Phytoplankton Species of Major Biogeochemical Importance. Marine Biotechnology 13(3): 496-504. *Joint first authors

Abstract

Emiliania huxleyi is a unicellular marine phytoplankton species known to play a significant role in global biogeochemistry. Through the dual roles of photosynthesis and production of calcium carbonate (calcification), carbon is transferred from the atmosphere to ocean sediments. Almost nothing is known about the molecular mechanisms that control calcification, a process that is tightly regulated within the cell. To initiate proteomic studies on this important and phylogenetically remote organism, we have devised efficient protein extraction protocols and developed a bioinformatics pipeline that allows the statistically robust assignment of proteins from MS/MS data using preexisting EST sequences. The bioinformatics tool, termed BUDAPEST (Bioinformatics Utility for Data Analysis of Proteomics using ESTs), is fully automated and was used to search against data generated from three strains. BUDAPEST increased the number of identifications over standard protein database searches from 37 to 99 proteins when data were amalgamated. Proteins involved in diverse cellular processes were uncovered. For example, experimental evidence was obtained for a novel type I polyketide synthase and for various photosystem components. The proteomic and bioinformatic approaches developed in this study are of wider applicability, particularly to the oceanographic community where genomic sequence data for species of interest are currently scarce.

PMID: 20924652

SLiMSearch 2.0: biological context for short linear motifs in proteins

Davey NE, Haslam NJ, Shields DC & Edwards RJ (2011): SLiMSearch 2.0: biological context for short linear motifs in proteins. Nucleic Acids Research 39: W56-W60.

Abstract

Short, linear motifs (SLiMs) play a critical role in many biological processes. The SLiMSearch 2.0 (Short, Linear Motif Search) web server allows researchers to identify occurrences of a user-defined SLiM in a proteome, using conservation and protein disorder context statistics to rank occurrences. User-friendly output and visualizations of motif context allow the user to quickly gain insight into the validity of a putatively functional motif occurrence. For each motif occurrence, overlapping UniProt features and annotated SLiMs are displayed. Visualization also includes annotated multiple sequence alignments surrounding each occurrence, showing conservation and protein disorder statistics in addition to known and predicted SLiMs, protein domains and known post-translational modifications. In addition, enrichment of Gene Ontology terms and protein interaction partners are provided as indicators of possible motif function. All web server results are available for download. Users can search motifs against the human proteome or a subset thereof defined by Uniprot accession numbers or GO term. The SLiMSearch server is available at: http://bioware.ucd.ie/slimsearch2.html.

PMID: 21622654

Selective toxicity of the anthelmintic emodepside revealed by heterologous expression of human KCNMA1 in Caenorhabditis elegans

Crisford A, Murray C, O’Connor V, Edwards RJ, Kruger N, Welz C, von Samson-Himmelstjerna G, Harder A, Walker RJ & Holden-Dye L. (2011): Selective Toxicity of the Anthelmintic Emodepside Revealed by Heterologous Expression of Human KCNMA1 in Caenorhabditis elegans. Molecular Pharmacology 79 (6): 1031-43.

Abstract

Emodepside is a resistance-breaking anthelmintic of a new chemical class, the cyclooctadepsipeptides. A major determinant of its anthelmintic effect is the calcium-activated potassium channel SLO-1. SLO-1 belongs to a family of channels that are highly conserved across the animal phyla and regulate neurosecretion, hormone release, muscle contraction, and neuronal network excitability. To investigate the selective toxicity of emodepside, we performed transgenic experiments in which the nematode SLO-1 channel was swapped for a mammalian ortholog, human KCNMA1. Expression of either the human channel or Caenorhabditis elegans slo-1 from the native slo-1 promoter in a C. elegans slo-1 functional null mutant rescued behavioral deficits that otherwise resulted from loss of slo-1 signaling. However, worms expressing the human channel were 10- to 100-fold less sensitive to emodepside than those expressing the nematode channel. Strains expressing the human KCNMA1 channel were preferentially sensitive to the mammalian channel agonists NS1619 and rottlerin. In the C. elegans pharyngeal nervous system, slo-1 is expressed in neurons, not muscle, and cell-specific rescue experiments have previously shown that emodepside inhibits serotonin-stimulated feeding by interfering with SLO-1 signaling in the nervous system. Here we show that ectopic overexpression of slo-1 in pharyngeal muscle confers sensitivity of the muscle to emodepside, consistent with a direct interaction of emodepside with the channel. Taken together, these data predict an emodepside-selective pharmacophore harbored by SLO-1. This has implications for the development of this drug/target interface for the treatment of helminth infections.

PMID: 21415309

SLiMSearch: a webserver for finding novel occurrences of short linear motifs in proteins, incorporating sequence context

Davey NE, Haslam NJ, Shields DC & Edwards RJ (2010): SLiMSearch: a webserver for finding novel occurrences of short linear motifs in proteins, incorporating sequence context. In: Pattern Recognition in Bioinformatics Edited by Dijkstra TMH, Tsivtsivadze E, Marchiori E & Heskes T. Springer-Verlag, Berlin. Lecture Notes in Bioinformatics 6282: 50-61.

Abstract

Short, linear motifs (SLiMs) play a critical role in many biological processes. The SLiMSearch (Short, Linear Motif Search) webserver is a flexible tool that enables researchers to identify novel occurrences of predefined SLiMs in sets of proteins. Numerous masking options give the user great control over the contextual information to be included in the analyses, including evolutionary filtering and protein structural disorder. User-friendly output and visualizations of motif context allow the user to quickly gain insight into the validity of a putatively functional motif occurrence. Users can search motifs against the human proteome, or submit their own datasets of UniProt proteins, in which case motif support within the dataset is statistically assessed for over- and under-representation, accounting for evolutionary relationships between input proteins. SLiMSearch is freely available as open source Python modules and all webserver results are available for download. The SLiMSearch server is available at: http://bioware.ucd.ie/slimsearch.html.

Protein interactions with the platelet integrin alpha(IIb) regulatory motif

Raab M, Daxecker H, Edwards RJ, Treumann A, Murphy D & Moran N (2010): Protein interactions with the platelet integrin alpha(IIb) regulatory motif. Proteomics 10: 2790-2800.

Abstract

Integrins are transmembrane proteins regulating cellular shape, mobility and the cell cycle. A highly conserved signature motif in the cytoplasmic tail of the integrin alpha-subunit, KXGFFKR, plays a critical role in regulating integrin function. To date, six proteins have been identified that target this motif of the platelet-specific integrin alpha(IIb)beta(3). We employ peptide-affinity chromatography followed-up with LC-MS/MS analysis as well as protein chips to identify new potential regulators of integrin function in platelets and put them into their biological context using information from protein:protein interaction (PPI) databases. Totally, 44 platelet proteins bind with high affinity to an immobilized LAMWKVGFFKR-peptide. Of these, seven have been reported in the PPI literature as interactors with integrin alpha-subunits. 68 recombinant human proteins expressed on the protein chip specifically bind with high affinity to biotin-tagged alpha-integrin cytoplasmic peptides. Two of these proteins are also identified in the peptide-affinity experiments, one is also found in the PPI databases and a further one is present in the data to all three approaches. Finally, novel short linear interaction motifs are common to a number of proteins identified.

PMID: 20486118

Computational identification and analysis of protein short linear motifs

Davey NE, Edwards RJ & Shields DC (2010): Computational identification and analysis of protein short linear motifs. Frontiers in Bioscience 15: 801-25.

Abstract

Short linear motifs (SLiMs) in proteins can act as targets for proteolytic cleavage, sites of post-translational modification, determinants of sub-cellular localization, and mediators of protein-protein interactions. Computational discovery of SLiMs involves assembling a group of proteins postulated to share a potential motif, masking out residues less likely to contain such a motif, down-weighting shared motifs arising through common evolutionary descent, and calculation of statistical probabilities allowing for the multiple testing of all possible motifs. Much of the challenge for motif discovery lies in the assembly and masking of datasets of proteins likely to share motifs, since the motifs are typically short (between 3 and 10 amino acids in length), so that potential signals can be easily swamped by the noise of stochastically recurring motifs. Focusing on disordered regions of proteins, where SLiMs are predominantly found, and masking out non-conserved residues can reduce the level of noise but more work is required to improve the quality of high-throughput experimental datasets (e.g. of physical protein interactions) as input for computational discovery.

PMID: 20515727