UniTmp: unified resources for transmembrane proteins.

The UNIfied database of TransMembrane Proteins (UniTmp) is a comprehensive and freely accessible resource of transmembrane protein structural information at different levels, from localization of protein segments, through the topology of the protein to the membrane-embedded 3D structure. We not only annotated tens of thousands of new structures and experiments, but we also developed a new system that can serve these resources in parallel. UniTmp is a unified platform that merges TOPDB (Topology Data Bank of Transmembrane Proteins), TOPDOM (database of conservatively located domains and motifs in proteins), PDBTM (Protein Data Bank of Transmembrane Proteins) and HTP (Human Transmembrane Proteome) databases and provides interoperability between the incorporated resources and an easy way to keep them regularly updated. The current update contains 9235 membrane-embedded structures, 9088 sequences with 536 035 topology-annotated segments and 8692 conservatively localized protein domains or motifs as well as 5466 annotated human transmembrane proteins. The UniTmp database can be accessed at https://www.unitmp.org.

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.

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.

TmAlphaFold database: membrane localization and evaluation of AlphaFold2 predicted alpha-helical transmembrane protein structures.

AI-driven protein structure prediction, most notably AlphaFold2 (AF2) opens new frontiers for almost all fields of structural biology. As traditional structure prediction methods for transmembrane proteins were both complicated and error prone, AF2 is a great help to the community. Complementing the relatively meager number of experimental structures, AF2 provides 3D predictions for thousands of new alpha-helical membrane proteins. However, the lack of reliable structural templates and the fact that AF2 was not trained to handle phase boundaries also necessitates a delicate assessment of structural correctness. In our new database, Transmembrane AlphaFold database (TmAlphaFold database), we apply TMDET, a simple geometry-based method to visualize the likeliest position of the membrane plane. In addition, we calculate several parameters to evaluate the location of the protein into the membrane. This also allows TmAlphaFold database to show whether the predicted 3D structure is realistic or not. The TmAlphaFold database is available at https://tmalphafold.ttk.hu/.

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.

Discovery and Characterization of Linear Motif Mediated Protein-Protein Complexes.

There are myriads of protein-protein complexes that form within the cell. In addition to classical binding events between globular domains, many protein-protein interactions involve short disordered protein regions. The latter contain so-called linear motifs binding specifically to ordered protein domain surfaces. Linear binding motifs are classified based on their consensus sequence, where only a few amino acids are conserved. In this chapter we will review experimental and in silico techniques that can be used for the discovery and characterization of linear motif mediated protein-protein complexes involved in cellular signaling, protein level and gene expression regulation.

In Vivo and In Vitro Characterization of the RNA Binding Capacity of SETD1A (KMT2F).

For several histone lysine methyltransferases (HKMTs), RNA binding has been already shown to be a functionally relevant feature, but detailed information on the RNA interactome of these proteins is not always known. Of the six human KMT2 proteins responsible for the methylation of the H3K4 residue, two-SETD1A and SETD1B-contain RNA recognition domains (RRMs). Here we investigated the RNA binding capacity of SETD1A and identified a broad range of interacting RNAs within HEK293T cells. Our analysis revealed that similar to yeast Set1, SETD1A is also capable of binding several coding and non-coding RNAs, including RNA species related to RNA processing. We also show direct RNA binding activity of the individual RRM domain in vitro, which is in contrast with the RRM domain found in yeast Set1. Structural modeling revealed important details on the possible RNA recognition mode of SETD1A and highlighted some fundamental differences between SETD1A and Set1, explaining the differences in the RNA binding capacity of their respective RRMs.

PolarProtPred: predicting apical and basolateral localization of transmembrane proteins using putative short linear motifs and deep learning.

MOTIVATION: Cell polarity refers to the asymmetric organization of cellular components in various cells. Epithelial cells are the best-known examples of polarized cells, featuring apical and basolateral membrane domains. Mounting evidence suggests that short linear motifs play a major role in protein trafficking to these domains, although the exact rules governing them are still elusive. RESULTS: In this study we prepared neural networks that capture recurrent patterns to classify transmembrane proteins localizing into apical and basolateral membranes. Asymmetric expression of drug transporters results in vectorial drug transport, governing the pharmacokinetics of numerous substances, yet the data on how proteins are sorted in epithelial cells is very scattered. The provided method may offer help to experimentalists to identify or better characterize molecular networks regulating the distribution of transporters or surface receptors (including viral entry receptors like that of COVID-19). AVAILABILITY AND IMPLEMENTATION: The prediction server PolarProtPred is available at http://polarprotpred.ttk.hu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Systematic Discovery of FBXW7-Binding Phosphodegrons Highlights Mitogen-Activated Protein Kinases as Important Regulators of Intracellular Protein Levels.

A FBXW7 is an F-box E3 ubiquitin-ligase affecting cell growth by controlling protein degradation. Mechanistically, its effect on its substrates depends on the phosphorylation of degron motifs, but the abundance of these phosphodegrons has not been systematically explored. We used a ratiometric protein degradation assay geared towards the identification of FBXW7-binding degron motifs phosphorylated by mitogen-activated protein kinases (MAPKs). Most of the known FBXW7 targets are localized in the nucleus and function as transcription factors. Here, in addition to more transcription affecting factors (ETV5, KLF4, SP5, JAZF1, and ZMIZ1 CAMTA2), we identified phosphodegrons located in proteins involved in chromatin regulation (ARID4B, KMT2E, KMT2D, and KAT6B) or cytoskeletal regulation (MAP2, Myozenin-2, SMTL2, and AKAP11), and some other proteins with miscellaneous functions (EIF4G3, CDT1, and CCAR2). We show that the protein level of full-length ARID4B, ETV5, JAZF1, and ZMIZ1 are affected by different MAPKs since their FBXW7-mediated degradation was diminished in the presence of MAPK-specific inhibitors. Our results suggest that MAPK and FBXW7 partnership plays an important cellular role by directly affecting the level of key regulatory proteins. The data also suggest that the p38α-controlled phosphodegron in JAZF1 may be responsible for the pathological regulation of the cancer-related JAZF1-SUZ12 fusion construct implicated in endometrial stromal sarcoma.

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.

A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes.

One of the most popular means to follow interactions between bio(macro)molecules is Förster resonance energy transfer (FRET). There is large interest in widening the selection of fluorescent FRET pairs especially in the region of the red/far red range, where minimal autofluorescence is encountered. A set of bioorthogonally applicable fluorescent dyes, synthesized recently in our lab, were paired (Cy3T/Cy5T; Cy1A/Cy3T and Cy1A/CBRD1A) based on their spectral characteristics in order to test their potential in FRET applications. For fast elaboration of the selected pairs we have created a bioorthogonalized platform based on complementary 17-mer DNA oligomers. The cyclooctynylated strands were modified nearly quantitatively with the fluorophores via bioorthogonal chemistry steps, using azide- (Cy1; CBRD1) or tetrazine-modified (Cy3; Cy5) dyes. Reactions were followed by capillary electrophoresis using a method specifically developed for this project. FRET efficiencies of the fluorescent dye pairs were compared both in close proximity (5' and 3' matched) and at larger distance (5' and 5' matched). The specificity of FRET signals was further elaborated by denaturation and competition studies. Cy1A/Cy3T and Cy1A/CBRD1A introduced here as novel FRET pairs are highly recommended for FRET applications based on the significant changes in fluorescence intensities of the donor and acceptor peaks. Application of one of the FRET pairs was demonstrated in live cells, transfected with labeled oligos. Furthermore, the concise installation of the dyes allows for efficient fluorescence modification of any selected DNA strands as was demonstrated in the construction of Cy3T labeled oligomers, which were used in the FISH-based detection of Helicobacter pylori.

Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

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/.

Deep structural insights into RNA-binding disordered protein regions.

Recent efforts to identify RNA binding proteins in various organisms and cellular contexts have yielded a large collection of proteins that are capable of RNA binding in the absence of conventional RNA recognition domains. Many of the recently identified RNA interaction motifs fall into intrinsically disordered protein regions (IDRs). While the recognition mode and specificity of globular RNA binding elements have been thoroughly investigated and described, much less is known about the way IDRs can recognize their RNA partners. Our aim was to summarize the current state of structural knowledge on the RNA binding modes of disordered protein regions and to propose a classification system based on their sequential and structural properties. Through a detailed structural analysis of the complexes that contain disordered protein regions binding to RNA, we found two major binding modes that represent different recognition strategies and, most likely, functions. We compared these examples with DNA binding disordered proteins and found key differences stemming from the nucleic acids as well as similar binding strategies, implying a broader substrate acceptance by these proteins. Due to the very limited number of known structures, we integrated molecular dynamics simulations in our study, whose results support the proposed structural preferences of specific RNA-binding IDRs. To broaden the scope of our review, we included a brief analysis of RNA-binding small molecules and compared their structural characteristics and RNA recognition strategies to the RNA-binding IDRs. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.

Structural insights into the pSer/pThr dependent regulation of the SHP2 tyrosine phosphatase in insulin and CD28 signaling.

Serine/threonine phosphorylation of insulin receptor substrate (IRS) proteins is well known to modulate insulin signaling. However, the molecular details of this process have mostly been elusive. While exploring the role of phosphoserines, we have detected a direct link between Tyr-flanking Ser/Thr phosphorylation sites and regulation of specific phosphotyrosine phosphatases. Here we present a concise structural study on how the activity of SHP2 phosphatase is controlled by an asymmetric, dual phosphorylation of its substrates. The structure of SHP2 has been determined with three different substrate peptides, unveiling the versatile and highly dynamic nature of substrate recruitment. What is more, the relatively stable pre-catalytic state of SHP2 could potentially be useful for inhibitor design. Our findings not only show an unusual dependence of SHP2 catalytic activity on Ser/Thr phosphorylation sites in IRS1 and CD28, but also suggest a negative regulatory mechanism that may also apply to other tyrosine kinase pathways as well.

Quantitative fragmentomics allow affinity mapping of interactomes.

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic target.

The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.

Mutations of Intrinsically Disordered Protein Regions Can Drive Cancer but Lack Therapeutic Strategies.

Many proteins contain intrinsically disordered regions (IDRs) which carry out important functions without relying on a single well-defined conformation. IDRs are increasingly recognized as critical elements of regulatory networks and have been also associated with cancer. However, it is unknown whether mutations targeting IDRs represent a distinct class of driver events associated with specific molecular and system-level properties, cancer types and treatment options. Here, we used an integrative computational approach to explore the direct role of intrinsically disordered protein regions driving cancer. We showed that around 20% of cancer drivers are primarily targeted through a disordered region. These IDRs can function in multiple ways which are distinct from the functional mechanisms of ordered drivers. Disordered drivers play a central role in context-dependent interaction networks and are enriched in specific biological processes such as transcription, gene expression regulation and protein degradation. Furthermore, their modulation represents an alternative mechanism for the emergence of all known cancer hallmarks. Importantly, in certain cancer patients, mutations of disordered drivers represent key driving events. However, treatment options for such patients are currently severely limited. The presented study highlights a largely overlooked class of cancer drivers associated with specific cancer types that need novel therapeutic options.

PolarProtDb: A Database of Transmembrane and Secreted Proteins showing Apical-Basal Polarity.

Most cells in multicellular organisms are somehow asymmetric, polarized: maintaining separate membrane domains. Typical examples are the epithelial cells (apical-basal polarization), neurons (dendritic-axonal domains), or migratory cells (with a leading and a trailing edge). Here we present the most comprehensive database containing experimentally verified mammalian proteins that display polarized sorting or secretion, focusing on epithelial polarity. In addition to the source cells or tissues, homology-based inferences and transmembrane topology (if applicable) are all provided. PolarProtDb also offers a detailed interface displaying all information that may be relevant for trafficking: including post-translational modifications (glycosylations and phosphorylations), known or predicted short linear motifs conserved across orthologs, as well as potential interaction partners. Data on polarized sorting has so far been scattered across myriads of publications, hence difficult to access. This information can help researchers in several areas, such as scanning for potential entry points of viral agents like COVID-19. PolarProtDb shall be a useful resource to design future experiments as well as for comparative analyses. The database is available at http://polarprotdb.enzim.hu.