Linear motifs regulating protein secretion, sorting and autophagy in Leishmania parasites are diverged with respect to their host equivalents.

The pathogenic, tropical Leishmania flagellates belong to an early-branching eukaryotic lineage (Kinetoplastida) with several unique features. Unfortunately, they are poorly understood from a molecular biology perspective, making development of mechanistically novel and selective drugs difficult. Here, we explore three functionally critical targeting short linear motif systems as well as their receptors in depth, using a combination of structural modeling, evolutionary sequence divergence and deep learning. Secretory signal peptides, endoplasmic reticulum (ER) retention motifs (KDEL motifs), and autophagy signals (motifs interacting with ATG8 family members) are ancient and essential components of cellular life. Although expected to be conserved amongst the kinetoplastids, we observe that all three systems show a varying degree of divergence from their better studied equivalents in animals, plants, or fungi. We not only describe their behaviour, but also build models that allow the prediction of localization and potential functions for several uncharacterized Leishmania proteins. The unusually Ala/Val-rich secretory signal peptides, endoplasmic reticulum resident proteins ending in Asp-Leu-COOH and atypical ATG8-like proteins are all unique molecular features of kinetoplastid parasites. Several of their critical protein-protein interactions could serve as targets of selective antimicrobial agents against Leishmaniasis due to their systematic divergence from the host.

Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.

Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.

ELM-the Eukaryotic Linear Motif resource-2024 update.

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.

LeishMANIAdb: a comparative resource for Leishmania proteins.

Leishmaniasis is a detrimental disease causing serious changes in quality of life and some forms can lead to death. The disease is spread by the parasite Leishmania transmitted by sandfly vectors and their primary hosts are vertebrates including humans. The pathogen penetrates host cells and secretes proteins (the secretome) to repurpose cells for pathogen growth and to alter cell signaling via host-pathogen protein-protein interactions). Here, we present LeishMANIAdb, a database specifically designed to investigate how Leishmania virulence factors may interfere with host proteins. Since the secretomes of different Leishmania species are only partially characterized, we collated various experimental evidence and used computational predictions to identify Leishmania secreted proteins to generate a user-friendly unified web resource allowing users to access all information available on experimental and predicted secretomes. In addition, we manually annotated host-pathogen interactions of 211 proteins and the localization/function of 3764 transmembrane (TM) proteins of different Leishmania species. We also enriched all proteins with automatic structural and functional predictions that can provide new insights in the molecular mechanisms of infection. Our database may provide novel insights into Leishmania host-pathogen interactions and help to identify new therapeutic targets for this neglected disease. Database URL  https://leishmaniadb.ttk.hu/.

Using Linear Motif Database Resources to Identify SH2 Domain Binders.

The SH2-binding phosphotyrosine class of short linear motifs (SLiMs) are key conditional regulatory elements, particularly in signaling protein complexes beneath the cell's plasma membrane. In addition to transmitting cellular signaling information, they can also play roles in cellular hijack by invasive pathogens. Researchers can take advantage of bioinformatics tools and resources to predict the motifs at conserved phosphotyrosine residues in regions of intrinsically disordered protein. A candidate SH2-binding motif can be established and assigned to one or more of the SH2 domain subgroups. It is, however, not so straightforward to predict which SH2 domains are capable of binding the given candidate. This is largely due to the cooperative nature of the binding amino acids which enables poorer binding residues to be tolerated when the other residues are optimal. High-throughput peptide arrays are powerful tools used to derive SH2 domain-binding specificity, but they are unable to capture these cooperative effects and also suffer from other shortcomings. Tissue and cell type expression can help to restrict the list of available interactors: for example, some well-studied SH2 domain proteins are only present in the immune cell lineages. In this article, we provide a table of motif patterns and four bioinformatics strategies that introduce a range of tools that can be used in motif hunting in cellular and pathogen proteins. Experimental followup is essential to determine which SH2 domain/motif-containing proteins are the actual functional partners.

Pipeline for transferring annotations between proteins beyond globular domains.

DisProt is the primary repository of Intrinsically Disordered Proteins (IDPs). This database is manually curated and the annotations there have strong experimental support. Currently, DisProt contains a relatively small number of proteins highlighting the importance of transferring annotations regarding verified disorder state and corresponding functions to homologous proteins in other species. In such a way, providing them with highly valuable information to better understand their biological roles. While the principles and practicalities of homology transfer are well-established for globular proteins, these are largely lacking for disordered proteins. We used DisProt to evaluate the transferability of the annotation terms to orthologous proteins. For each protein, we looked for their orthologs, with the assumption that they will have a similar function. Then, for each protein and their orthologs, we made multiple sequence alignments (MSAs). Disordered sequences are fast evolving and can be hard to align, therefore, we implemented alignment quality control steps ensuring robust alignments before mapping the annotations. We have designed a pipeline to obtain good-quality MSAs and to transfer annotations from any protein to their orthologs. Applying the pipeline to DisProt proteins, from the 1731 entries with 5623 annotations, we can reach 97,555 orthologs and transfer a total of 301,190 terms by homology. We also provide a web server for consulting the results of DisProt proteins and execute the pipeline for any other protein. The server Homology Transfer IDP (HoTIDP) is accessible at http://hotidp.leloir.org.ar.

A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.

Structural basis for tunable affinity and specificity of LxCxE-dependent protein interactions with the retinoblastoma protein family.

The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity.

PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion.

Retinitis pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (photoreceptor cilium actin regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. On the basis of these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain-binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain-binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications.

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

Divergent Evolution of a Protein-Protein Interaction Revealed through Ancestral Sequence Reconstruction and Resurrection.

The postsynaptic density extends across the postsynaptic dendritic spine with discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. DLG contains three PDZ domains and one important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and a protein called cysteine-rich interactor of PDZ3 (CRIPT). However, little is known regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection, and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was likely present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein-protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

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.

Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity.

Bacterial pathogens have developed complex strategies to successfully survive and proliferate within their hosts. Throughout the infection cycle, direct interaction with host cells occurs. Many bacteria have been found to secrete proteins, such as effectors and toxins, directly into the host cell with the potential to interfere with cell regulatory processes, either enzymatically or through protein-protein interactions (PPIs). Short linear motifs (SLiMs) are abundant peptide modules in cell signaling proteins. Here, we cover the reported examples of eukaryotic-like SLiM mimicry being used by pathogenic bacteria to hijack host cell machinery and discuss how drugs targeting SLiM-regulated cell signaling networks are being evaluated for interference with bacterial infections. This emerging anti-infective opportunity may become an essential contributor to antibiotic replacement strategies.

ELM-the eukaryotic linear motif resource in 2020.

The eukaryotic linear motif (ELM) resource is a repository of manually curated experimentally validated short linear motifs (SLiMs). Since the initial release almost 20 years ago, ELM has become an indispensable resource for the molecular biology community for investigating functional regions in many proteins. In this update, we have added 21 novel motif classes, made major revisions to 12 motif classes and added >400 new instances mostly focused on DNA damage, the cytoskeleton, SH2-binding phosphotyrosine motifs and motif mimicry by pathogenic bacterial effector proteins. The current release of the ELM database contains 289 motif classes and 3523 individual protein motif instances manually curated from 3467 scientific publications. ELM is available at: http://elm.eu.org.

An intrinsically disordered proteins community for ELIXIR.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) are now recognised as major determinants in cellular regulation. This white paper presents a roadmap for future e-infrastructure developments in the field of IDP research within the ELIXIR framework. The goal of these developments is to drive the creation of high-quality tools and resources to support the identification, analysis and functional characterisation of IDPs. The roadmap is the result of a workshop titled "An intrinsically disordered protein user community proposal for ELIXIR" held at the University of Padua. The workshop, and further consultation with the members of the wider IDP community, identified the key priority areas for the roadmap including the development of standards for data annotation, storage and dissemination; integration of IDP data into the ELIXIR Core Data Resources; and the creation of benchmarking criteria for IDP-related software. Here, we discuss these areas of priority, how they can be implemented in cooperation with the ELIXIR platforms, and their connections to existing ELIXIR Communities and international consortia. The article provides a preliminary blueprint for an IDP Community in ELIXIR and is an appeal to identify and involve new stakeholders.

CiliaCarta: An integrated and validated compendium of ciliary genes.

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

Modulation of HIV-1 gene expression by binding of a ULM motif in the Rev protein to UHM-containing splicing factors.

The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

Control of mitotic chromosome condensation by the fission yeast transcription factor Zas1.

Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.