The eukaryotic linear motif resource ELM: 10 years and counting

The eukaryotic linear motif (ELM http://elm.eu.org) resource is a hub for collecting, classifying and curating information about short linear motifs (SLiMs). For >10 years, this resource has provided the scientific community with a freely accessible guide to the biology and function of linear motifs. The current version of ELM contains ∼200 different motif classes with over 2400 experimentally validated instances manually curated from >2000 scientific publications. Furthermore, detailed information about motif-mediated interactions has been annotated and made available in standard exchange formats. Where appropriate, links are provided to resources such as switches.elm.eu.org and KEGG pathways.Fil: Dinkel, Holder. European Molecular Biology Laboratory; AlemaniaFil: Van Roey, Kim. European Molecular Biology Laboratory; AlemaniaFil: Michael, Sushama. European Molecular Biology Laboratory; AlemaniaFil: Davey, Norman E.. University Of California ; Estados UnidosFil: Weatheritt, Robert J.. MRC. Laboratory of Molecular Biology; Estados UnidosFil: Born, Diana. Ruprecht-Karls-Universität; AlemaniaFil: Speck, Tobias. Ruprecht-Karls-Universität; AlemaniaFil: Kruger, Daniel. Ruprecht-Karls-Universität; AlemaniaFil: Grebnev, Gleb. University College Dublin; IrlandaFil: Kuban, Marta. Maria Sklodowska-Curie Memorial Cancer Center. Laboratory of Bioinformatics and Biostatistics; PoloniaFil: Strumillo, Marta. Maria Sklodowska-Curie Memorial Cancer Center. Laboratory of Bioinformatics and Biostatistics; PoloniaFil: Uyar, Bora. European Molecular Biology Laboratory; AlemaniaFil: Budd, Aidan. European Molecular Biology Laboratory; AlemaniaFil: Altenberg, Brigitte. European Molecular Biology Laboratory; AlemaniaFil: Seiler, Markus. European Molecular Biology Laboratory; AlemaniaFil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Glavina, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Diella, Francesca. European Molecular Biology Laboratory; AlemaniaFil: Gibson, Toby J. European Molecular Biology Laboratory; Alemani

ELM—the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instance

The MIntAct project—IntAct as a common curation platform for 11 molecular interaction databases

IntAct (freely available at http://www.ebi.ac.uk/intact) is an open-source, open data molecular interaction database populated by data either curated from the literature or from direct data depositions. IntAct has developed a sophisticated web-based curation tool, capable of supporting both IMEx- and MIMIx-level curation. This tool is now utilized by multiple additional curation teams, all of whom annotate data directly into the IntAct database. Members of the IntAct team supply appropriate levels of training, perform quality control on entries and take responsibility for long-term data maintenance. Recently, the MINT and IntAct databases decided to merge their separate efforts to make optimal use of limited developer resources and maximize the curation output. All data manually curated by the MINT curators have been moved into the IntAct database at EMBL-EBI and are merged with the existing IntAct dataset. Both IntAct and MINT are active contributors to the IMEx consortium (http://www.imexconsortium.org

Discovery of a novel ligand that modulates the protein-protein interactions of the AAA+ superfamily oncoprotein reptin

Financial support for this project was provided by Cancer Research UK (CRUK grant C21383/A6950). CRUK C483/A10706 and C483/A8033; EPSRC EP/F500421/1 doctoral training centre in cell and proteomic technologies; GACR P206/12/G151 and the state budget of the Czech Republic (LO1413).Developing approaches to discover protein-protein interactions (PPIs) remains a fundamental challenge. A chemical biology platform is applied here to identify novel PPIs for the AAA+ superfamily oncoprotein reptin. An in silico screen coupled with chemical optimization provided Liddean, a nucleotide-mimetic which modulates reptin’s oligomerization status, protein-binding activity and global conformation. Combinatorial peptide phage library screening of Liddean-bound reptin with next generation sequencing identified interaction motifs including a novel reptin docking site on the p53 tumor suppressor protein. Proximity ligation assays demonstrated that endogenous reptin forms a predominantly cytoplasmic complex with its paralog pontin in cancer cells and Liddean promotes a shift of this complex to the nucleus. An emerging view of PPIs in higher eukaryotes is that they occur through a striking diversity of linear peptide motifs. The discovery of a compound that alters reptin’s protein interaction landscape potentially leads to novel avenues for therapeutic development.Publisher PDFPeer reviewe

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs

Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG. Previously, we showed that cleavage by I-CreII leaves ends (2-nt 3′ overhangs) that are characteristic of GIY-YIG endonucleases, yet it has a relaxed metal requirement like H-N-H enzymes. Here we show that I-CreII can bind DNA without an added metal ion, and that it binds as a monomer, akin to GIY-YIG enzymes. Moreover, cleavage of supercoiled DNA, and estimates of strand-specific cleavage rates, suggest that I-CreII uses a sequential cleavage mechanism. Alanine substitution of a number of residues in the GIY-YIG motif, however, did not block cleavage activity, although DNA binding was substantially reduced in several variants. Substitution of conserved histidines in the H-N-H motif resulted in variants that did not promote DNA cleavage, but retained high-affinity DNA binding—thus identifying it as the catalytic motif. Unlike the non-specific H-N-H colicins, however; substitution of the conserved asparagine substantially reduced DNA binding (though not the ability to promote cleavage). These results indicate that, in I-CreII, two catalytic motifs have evolved to play important roles in specific DNA binding. The data also indicate that only the H-N-H motif has retained catalytic ability

TSLP-activated dendritic cells induce human T follicular helper cell differentiation through OX40-ligand.

T follicular helper cells (Tfh) are important regulators of humoral responses. Human Tfh polarization pathways have been thus far associated with Th1 and Th17 polarization pathways. How human Tfh cells differentiate in Th2-skewed environments is unknown. We show that thymic stromal lymphopoietin (TSLP)-activated dendritic cells (DCs) promote human Tfh differentiation from naive CD4 T cells. We identified a novel population, distinct from Th2 cells, expressing IL-21 and TNF, suggestive of inflammatory cells. TSLP-induced T cells expressed CXCR5, CXCL13, ICOS, PD1, BCL6, BTLA, and SAP, among other Tfh markers. Functionally, TSLP-DC-polarized T cells induced IgE secretion by memory B cells, and this depended on IL-4Rα. TSLP-activated DCs stimulated circulating memory Tfh cells to produce IL-21 and CXCL13. Mechanistically, TSLP-induced Tfh differentiation depended on OX40-ligand, but not on ICOS-ligand. Our results delineate a pathway of human Tfh differentiation in Th2 environments

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages

Mobile DNA elements in T4 and related phages

Mobile genetic elements are common inhabitants of virtually every genome where they can exert profound influences on genome structure and function in addition to promoting their own spread within and between genomes. Phage T4 and related phage have long served as a model system for understanding the molecular mechanisms by which a certain class of mobile DNA, homing endonucleases, promote their spread. Homing endonucleases are site-specific DNA endonucleases that initiate mobility by introducing double-strand breaks at defined positions in genomes lacking the endonuclease gene, stimulating repair and recombination pathways that mobilize the endonuclease coding region. In phage T4, homing endonucleases were first discovered as encoded within the self-splicing td, nrdB and nrdD introns of T4. Genomic data has revealed that homing endonucleases are extremely widespread in T-even-like phage, as evidenced by the astounding fact that ~11% of the T4 genome encodes homing endonuclease genes, with most of them located outside of self-splicing introns. Detailed studies of the mobile td intron and its encoded endonuclease, I-TevI, have laid the foundation for genetic, biochemical and structural aspects that regulate the mobility process, and more recently have provided insights into regulation of homing endonuclease function. Here, we summarize the current state of knowledge regarding T4-encoded homing endonucleases, with particular emphasis on the td/I-TevI model system. We also discuss recent progress in the biology of free-standing endonucleases, and present areas of future research for this fascinating class of mobile genetic elements

Experimental detection of short regulatory motifs in eukaryotic proteins: tips for good practice as well as for bad

It has become clear in outline though not yet in detail how cellular regulatory and signalling systems are constructed. The essential machines are protein complexes that effect regulatory decisions by undergoing internal changes of state. Subcomponents of these cellular complexes are assembled into molecular switches. Many of these switches employ one or more short peptide motifs as toggles that can move between one or more sites within the switch system, the simplest being on-off switches. Paradoxically, these motif modules (termed short linear motifs or SLiMs) are both hugely abundant but difficult to research. So despite the many successes in identifying short regulatory protein motifs, it is thought that only the “tip of the iceberg” has been exposed. Experimental and bioinformatic motif discovery remain challenging and error prone. The advice presented in this article is aimed at helping researchers to uncover genuine protein motifs, whilst avoiding the pitfalls that lead to reports of false discovery. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12964-015-0121-y) contains supplementary material, which is available to authorized users