How to Annotate and Submit a Short Linear Motif to the Eukaryotic Linear Motif Resource.

Over the past few years, it has become apparent that approximately 35% of the human proteome consists of intrinsically disordered regions. Many of these disordered regions are rich in short linear motifs (SLiMs) which mediate protein-protein interactions. Although these motifs are short and often partially conserved, they are involved in many important aspects of protein function, including cleavage, targeting, degradation, docking, phosphorylation, and other posttranslational modifications. The Eukaryotic Linear Motif resource (ELM) was established over 15 years ago as a repository to store and catalogue the scientific discoveries of motifs. Each motif in the database is annotated and curated manually, based on the experimental evidence gathered from publications. The entries themselves are submitted to ELM by filling in two annotation templates designed for motif class and motif instance annotation. In this protocol, we describe the steps involved in annotating new motifs and how to submit them to ELM.

The eukaryotic linear motif resource - 2018 update.

Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome.

Exploring Short Linear Motifs Using the ELM Database and Tools.

The Eukaryotic Linear Motif (ELM) resource is dedicated to the characterization and prediction of short linear motifs (SLiMs). SLiMs are compact, degenerate peptide segments found in many proteins and essential to almost all cellular processes. However, despite their abundance, SLiMs remain largely uncharacterized. The ELM database is a collection of manually annotated SLiM instances curated from experimental literature. In this article we illustrate how to browse and search the database for curated SLiM data, and cover the different types of data integrated in the resource. We also cover how to use this resource in order to predict SLiMs in known as well as novel proteins, and how to interpret the results generated by the ELM prediction pipeline. The ELM database is a very rich resource, and in the following protocols we give helpful examples to demonstrate how this knowledge can be used to improve your own research. © 2017 by John Wiley & Sons, Inc.

ELM 2016--data update and new functionality of the eukaryotic linear motif resource.

The Eukaryotic Linear Motif (ELM) resource (http://elm.eu.org) is a manually curated database of short linear motifs (SLiMs). In this update, we present the latest additions to this resource, along with more improvements to the web interface. ELM 2016 contains more than 240 different motif classes with over 2700 experimentally validated instances, manually curated from more than 2400 scientific publications. In addition, more data have been made available as individually searchable pages and are downloadable in various formats.

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.

Proteome-wide analysis of human disease mutations in short linear motifs: neglected players in cancer?

Disease mutations are traditionally thought to impair protein functionality by disrupting the folded globular structure of proteins. However, 22% of human disease mutations occur in natively unstructured segments of proteins known as intrinsically disordered regions (IDRs). This therefore implicates defective IDR functionality in various human diseases including cancer. The functionality of IDRs is partly attributable to short linear motifs (SLiMs), but it remains an open question how much defects in SLiMs contribute to human diseases. A proteome-wide comparison of the distribution of missense mutations from disease and non-disease mutation datasets revealed that, in IDRs, disease mutations are more likely to occur within SLiMs than neutral missense mutations. Moreover, compared to neutral missense mutations, disease mutations more frequently impact functionally important residues of SLiMs, cause changes in the physicochemical properties of SLiMs, and disrupt more SLiM-mediated interactions. Analysis of these mutations resulted in a comprehensive list of experimentally validated or predicted SLiMs disrupted in disease. Furthermore, this in-depth analysis suggests that 'prostate cancer pathway' is particularly enriched for proteins with disease-related SLiMs. The contribution of mutations in SLiMs to disease may currently appear small when compared to mutations in globular domains. However, our analysis of mutations in predicted SLiMs suggests that this contribution might be more substantial. Therefore, when analysing the functional impact of mutations on proteins, SLiMs in proteins should not be neglected. Our results suggest that an increased focus on SLiMs in the coming decades will improve our understanding of human diseases and aid in the development of targeted treatments.

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.

An open science peer review oath.

One of the foundations of the scientific method is to be able to reproduce experiments and corroborate the results of research that has been done before. However, with the increasing complexities of new technologies and techniques, coupled with the specialisation of experiments, reproducing research findings has become a growing challenge. Clearly, scientific methods must be conveyed succinctly, and with clarity and rigour, in order for research to be reproducible. Here, we propose steps to help increase the transparency of the scientific method and the reproducibility of research results: specifically, we introduce a peer-review oath and accompanying manifesto. These have been designed to offer guidelines to enable reviewers (with the minimum friction or bias) to follow and apply open science principles, and support the ideas of transparency, reproducibility and ultimately greater societal impact. Introducing the oath and manifesto at the stage of peer review will help to check that the research being published includes everything that other researchers would need to successfully repeat the work. Peer review is the lynchpin of the publishing system: encouraging the community to consciously (and conscientiously) uphold these principles should help to improve published papers, increase confidence in the reproducibility of the work and, ultimately, provide strategic benefits to authors and their institutions.

The switches.ELM resource: a compendium of conditional regulatory interaction interfaces.

Short linear motifs (SLiMs) are protein interaction sites that play an important role in cell regulation by controlling protein activity, localization, and local abundance. The functionality of a SLiM can be modulated in a context-dependent manner to induce a gain, loss, or exchange of binding partners, which will affect the function of the SLiM-containing protein. As such, these conditional interactions underlie molecular decision-making in cell signaling. We identified multiple types of pre- and posttranslational switch mechanisms that can regulate the function of a SLiM and thereby control its interactions. The collected examples of experimentally characterized SLiM-based switch mechanisms were curated in the freely accessible switches.ELM resource (http://switches.elm.eu.org). On the basis of these examples, we defined and integrated rules to analyze SLiMs for putative regulatory switch mechanisms. We applied these rules to known validated SLiMs, providing evidence that more than half of these are likely to be pre- or posttranslationally regulated. In addition, we showed that posttranslationally modified sites are enriched around SLiMs, which enables cooperative and integrative regulation of protein interaction interfaces. We foresee switches.ELM complementing available resources to extend our knowledge of the molecular mechanisms underlying cell signaling.

Biggest challenges in bioinformatics.

The third Heidelberg Unseminars in Bioinformatics (HUB) was held on 18th October 2012, at Heidelberg University, Germany. HUB brought together around 40 bioinformaticians from academia and industry to discuss the 'Biggest Challenges in Bioinformatics' in a 'World Café' style event.

iELM--a web server to explore short linear motif-mediated interactions.

The recent expansion in our knowledge of protein-protein interactions (PPIs) has allowed the annotation and prediction of hundreds of thousands of interactions. However, the function of many of these interactions remains elusive. The interactions of Eukaryotic Linear Motif (iELM) web server provides a resource for predicting the function and positional interface for a subset of interactions mediated by short linear motifs (SLiMs). The iELM prediction algorithm is based on the annotated SLiM classes from the Eukaryotic Linear Motif (ELM) resource and allows users to explore both annotated and user-generated PPI networks for SLiM-mediated interactions. By incorporating the annotated information from the ELM resource, iELM provides functional details of PPIs. This can be used in proteomic analysis, for example, to infer whether an interaction promotes complex formation or degradation. Furthermore, details of the molecular interface of the SLiM-mediated interactions are also predicted. This information is displayed in a fully searchable table, as well as graphically with the modular architecture of the participating proteins extracted from the UniProt and Phospho.ELM resources. A network figure is also presented to aid the interpretation of results. The iELM server supports single protein queries as well as large-scale proteomic submissions and is freely available at http://i.elm.eu.org.

Attributes of short linear motifs.

Traditionally, protein-protein interactions were thought to be mediated by large, structured domains. However, it has become clear that the interactome comprises a wide range of binding interfaces with varying degrees of flexibility, ranging from rigid globular domains to disordered regions that natively lack structure. Enrichment for disorder in highly connected hub proteins and its correlation with organism complexity hint at the functional importance of disordered regions. Nevertheless, they have not yet been extensively characterised. Shifting the attention from globular domains to disordered regions of the proteome might bring us closer to elucidating the dense and complex connectivity of the interactome. An important class of disordered interfaces are the compact mono-partite, short linear motifs (SLiMs, or eukaryotic linear motifs (ELMs)). They are evolutionarily plastic and interact with relatively low affinity due to the limited number of residues that make direct contact with the binding partner. These features confer to SLiMs the ability to evolve convergently and mediate transient interactions, which is imperative to network evolution and to maintain robust cell signalling, respectively. The ability to discriminate biologically relevant SLiMs by means of different attributes will improve our understanding of the complexity of the interactome and aid development of bioinformatics tools for motif discovery. In this paper, the curated instances currently available in the Eukaryotic Linear Motif (ELM) database are analysed to provide a clear overview of the defining attributes of SLiMs. These analyses suggest that functional SLiMs have higher levels of conservation than their surrounding residues, frequently evolve convergently, preferentially occur in disordered regions and often form a secondary structure when bound to their interaction partner. These results advocate searching for small groupings of residues in disordered regions with higher relative conservation and a propensity to form the secondary structure. Finally, the most interesting conclusions are examined in regard to their functional consequences.

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 instances.

Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors.

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, (1)H NMR and (1)H(15)N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.

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

The Phospho.ELM resource (http://phospho.elm.eu.org) is a relational database designed to store in vivo and in vitro phosphorylation data extracted from the scientific literature and phosphoproteomic analyses. The resource has been actively developed for more than 7 years and currently comprises 42,574 serine, threonine and tyrosine non-redundant phosphorylation sites. Several new features have been implemented, such as structural disorder/order and accessibility information and a conservation score. Additionally, the conservation of the phosphosites can now be visualized directly on the multiple sequence alignment used for the score calculation. Finally, special emphasis has been put on linking to external resources such as interaction networks and other databases.

Intracellular localization map of human herpesvirus 8 proteins.

Human herpesvirus 8 (HHV-8) is the etiological agent of Kaposi's sarcoma. We present a localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading frames were cloned with a Myc tag in expression plasmids, confirmed by full-length sequencing, and expressed in HeLa cells. Protein localizations were analyzed by immunofluorescence microscopy. Fifty-one percent of all proteins were localized in the cytoplasm, 22% were in the nucleus, and 27% were found in both compartments. Surprisingly, we detected viral FLIP (v-FLIP) in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally localized exclusively in the cytoplasm. This suggested that v-FLIP may exert additional or alternative functions compared to cellular FLIPs. In addition, it has been shown recently that the K10 protein can bind to at least 15 different HHV-8 proteins. We noticed that K10 and only five of its 15 putative binding factors were localized in the nucleus when the proteins were expressed in HeLa cells individually. Interestingly, in coexpression experiments K10 colocalized with 87% (13 of 15) of its putative binding partners. Colocalization was induced by translocation of either K10 alone or both proteins. These results indicate active intracellular translocation processes in virus-infected cells. Specifically in this framework, the localization map may provide a useful reference to further elucidate the function of HHV-8-encoded genes in human diseases.

A computational strategy for the prediction of functional linear peptide motifs in proteins.

MOTIVATION: Short linear peptide motifs mediate protein-protein interaction, cell compartment targeting and represent the sites of post-translational modification. The identification of functional motifs by conventional sequence searches, however, is hampered by the short length of the motifs resulting in a large number of hits of which only a small portion is functional. RESULTS: We have developed a procedure for the identification of functional motifs, which scores pattern conservation in homologous sequences by taking explicitly into account the sequence similarity to the query sequence. For a further improvement of this method, sequence filters have been optimized to mask those sequence regions containing little or no linear motifs. The performance of this approach was verified by measuring its ability to identify 576 experimentally validated motifs among a total of 15 563 instances in a set of 415 protein sequences. Compared to a random selection procedure, the joint application of sequence filters and the novel scoring scheme resulted in a 9-fold enrichment of validated functional motifs on the first rank. In addition, only half as many hits need to be investigated to recover 75% of the functional instances in our dataset. Therefore, this motif-scoring approach should be helpful to guide experiments because it allows focusing on those short linear peptide motifs that have a high probability to be functional.