Abstract
There is a pressing need for in silico tools that can aid in the identification of the complete repertoire of protein binding (SLiMs, MoRFs, miniMotifs) and modification (moiety ...attachment/removal, isomerization, cleavage) motifs. We have created PSSMSearch, an interactive web-based tool for rapid statistical modeling, visualization, discovery and annotation of protein motif specificity determinants to discover novel motifs in a proteome-wide manner. PSSMSearch analyses proteomes for regions with significant similarity to a motif specificity determinant model built from a set of aligned motif-containing peptides. Multiple scoring methods are available to build a position-specific scoring matrix (PSSM) describing the motif specificity determinant model. This model can then be modified by a user to add prior knowledge of specificity determinants through an interactive PSSM heatmap. PSSMSearch includes a statistical framework to calculate the significance of specificity determinant model matches against a proteome of interest. PSSMSearch also includes the SLiMSearch framework's annotation, motif functional analysis and filtering tools to highlight relevant discriminatory information. Additional tools to annotate statistically significant shared keywords and GO terms, or experimental evidence of interaction with a motif-recognizing protein have been added. Finally, PSSM-based conservation metrics have been created for taxonomic range analyses. The PSSMSearch web server is available at http://slim.ucd.ie/pssmsearch/.
Specific protein–protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein–protein ...interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type‐specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide‐phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. Finally, we show how the amino acid resolution‐binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome‐wide discovery of SLiM‐based interactions.
Synopsis
An optimized phage peptidome that tiles the disordered regions of the human proteome is presented, allowing the field of motif‐based interactions to transition into high‐throughput. Guidelines and tools for data analysis are provided.
An optimized second generation human disorderome (HD2) phage library tiles all disordered regions from the human proteome.
Different peptide display parameters are tested, including display on the major or minor coat proteins of the M13 phage, and splitting the library design based sub‐cellular localization of the peptide containing proteins.
PepTools is a dedicated toolkit to annotate peptides and to identify consensus motifs.
> 2,000 motif‐based interactions are presented, together with information on potential disease mutations or phosphorylation sites that might affect the interactions.
An optimized phage peptidome that tiles the disordered regions of the human proteome is presented, allowing the field of motif‐based interactions to transition into high‐throughput. Guidelines and tools for data analysis are provided.
Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here ...we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.
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•The Ebola virus NP recruits the PP2A-B56 phosphatase through an LxxIxE binding motif•The LxxIxE motif in NP is required for VP30 dephosphorylation and viral transcription•A specific PP2A-B56 inhibitor impairs recombinant Ebola virus transcription and infection
Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form. Kruse et al. show that the Ebola nucleoprotein recruits the host PP2A-B56 phosphatase through a B56-binding LxxIxE motif, leading to VP30 dephosphorylation and viral transcription.
Short linear motifs (SLiMs) are compact interaction interfaces indispensable for a variety of cellular processes.Accumulated evidence points at SLiM-binding pockets as a common target for viral ...hijacking via viral mimicry of host motifs.Recent studies have revealed that blocking SLiM-binding pockets can significantly disrupt the viral life cycle.Targeting human SLiM-binding pockets hijacked by multiple viruses spanning many viral families could allow the development of broad-spectrum antiviral drugs.
Short linear motif (SLiM)-mediated interactions offer a unique strategy for viral intervention due to their compact interfaces, ease of convergent evolution, and key functional roles. Consequently, many viruses extensively mimic host SLiMs to hijack or deregulate cellular pathways and the same motif-binding pocket is often targeted by numerous unrelated viruses. A toolkit of therapeutics targeting commonly mimicked SLiMs could provide prophylactic and therapeutic broad-spectrum antivirals and vastly improve our ability to treat ongoing and future viral outbreaks. In this opinion article, we discuss the therapeutic relevance of SLiMs, advocating their suitability as targets for broad-spectrum antiviral inhibitors.
The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic ...Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.
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•The N-terminal half of BubR1 contains two ABBA motifs that bind Cdc20•The motifs are required for the MCC to bind and inhibit active APC/C-Cdc20•These ABBA motifs are essential for the spindle assembly checkpoint•The ABBA motifs flank a KEN box to form a cassette highly conserved in evolution
Di Fiore et al. show that two ABBA motifs in BubR1 are crucial for the Spindle Assembly Checkpoint because they are needed for the Mitotic Checkpoint Complex (MCC) to bind and inhibit the Anaphase Promoting Complex/Cyclosome (APC/C). Their results show how the MCC inhibits active APC/C.
Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of ...protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents.
The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate ...interactions by providing binding sites for peptide‐binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large‐scale identification. We present a novel proteomic peptide phage display (ProP‐PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large‐scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP‐PD library was validated through selections against SLiM‐binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM‐binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non‐C‐terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein–protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40–130 μm through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full‐length proteins. Taken together, we outline a general pipeline for the design and construction of ProP‐PD libraries and the analysis of ProP‐PD‐derived, SLiM‐based PPIs. We demonstrated the methods potential to identify low affinity motif‐mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.
The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs. These compact interfaces provide binding sites for peptide‐binding domains. We present a novel proteomic peptide phage display library that displays peptides directly mapping to disordered regions of the human proteome and validate its performance. This opens for large‐scale analysis of motif‐based interactions.
Phosphorylation is a ubiquitous post‐translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of ...phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide‐phage display library to screen for phosphosites that modulate short linear motif‐based interactions. The peptidome covers ~13,500 phospho‐serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild‐type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif‐mediated interactions. Affinity measurements confirmed the phospho‐modulation of 14 out of 18 tested interactions. We performed a detailed follow‐up on a phospho‐dependent interaction between clathrin and the mitotic spindle protein hepatoma‐upregulated protein (HURP), demonstrating the essentiality of the phospho‐dependency to the mitotic function of HURP. Structural characterisation of the clathrin‐HURP complex elucidated the molecular basis for the phospho‐dependency. Our work showcases the power of phosphomimetic ProP‐PD to discover novel phospho‐modulated interactions required for cellular function.
Synopsis
A new phosphomimetic proteomic peptide phage library is used to screen for phospho‐modulated interactions between short linear motifs and protein domains. Follow‐up analyses show that S839 phosphorylation of HURP is required for interaction with clathrin and mitotic function.
A novel phosphomimetic ProP‐PD library is generated, displaying intrinsically disordered regions of the proteome with functionally prioritised phosphosites.
Phosphomimetic ProP‐PD selections provide binding preferences for wild‐type, phosphomimetic and phosphorylated peptides at large‐scale.
S839 HURP phosphorylation is required for clathrin binding and its role in mitosis.
SLiM‐based interactions from ProP‐PD selections are available in a web‐based resource (http://slim.icr.ac.uk/proppd/).
A new phosphomimetic proteomic peptide phage library is used to screen for phospho‐modulated interactions between short linear motifs and protein domains. Follow‐up analyses show that S839 phosphorylation of HURP is required for interaction with clathrin and mitotic function.
Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and ...modulate host factors through the use of short linear motifs (SLiMs) embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues), and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively). Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses), except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel instances of SLiMs subverting host functions, such as innate and acquired immunity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Short Linear Motifs (SLiMs) are the smallest structural and functional components of modular eukaryotic proteins. They are also the most abundant, especially when considering ...post-translational modifications. As well as being found throughout the cell as part of regulatory processes, SLiMs are extensively mimicked by intracellular pathogens. At the heart of the Eukaryotic Linear Motif (ELM) Resource is a representative (not comprehensive) database. The ELM entries are created by a growing community of skilled annotators and provide an introduction to linear motif functionality for biomedical researchers. The 2024 ELM update includes 346 novel motif instances in areas ranging from innate immunity to both protein and RNA degradation systems. In total, 39 classes of newly annotated motifs have been added, and another 17 existing entries have been updated in the database. The 2024 ELM release now includes 356 motif classes incorporating 4283 individual motif instances manually curated from 4274 scientific publications and including >700 links to experimentally determined 3D structures. In a recent development, the InterPro protein module resource now also includes ELM data. ELM is available at: http://elm.eu.org.
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