A paradigm for post-embryonic Oct4 re-expression: E7-induced hydroxymethylation regulates Oct4 expression in cervical cancer.

The Octamer-binding transcription factor-4 (Oct4) is upregulated in different malignancies, yet a paradigm for mechanisms of Oct4 post-embryonic re-expression is inadequately understood. In cervical cancer, Oct4 expression is higher in human papillomavirus (HPV)-related than HPV-unrelated cervical cancers and this upregulation correlates with the expression of the E7 oncogene. We have reported that E7 affects the Oct4-transcriptional output and Oct4-related phenotypes in cervical cancer, however, the underlying mechanism remains elusive. Here, we characterize the Oct4-protein interactions in cervical cancer cells via computational analyses and Mass Spectrometry and reveal that Methyl-binding proteins (MBD2 and MBD3), are determinants of Oct4-driven transcription. E7 triggers MBD2 downregulation and TET1 upregulation, thereby disrupting the methylation status of the Oct4 gene. This coincides with an increase in the total DNA hydroxymethylation leading to the re-expression of Oct4 in cervical cancer and likely affecting broader transcriptional patterns. Our findings reveal a previously unreported mechanism by which the E7 oncogene can regulate Oct4 re-expression and global transcriptional patterns by increasing DNA hydroxymethylation and lowering the barrier to cellular plasticity during carcinogenesis.

CGG toolkit: Software components for computational genomics.

Public-domain availability for bioinformatics software resources is a key requirement that ensures long-term permanence and methodological reproducibility for research and development across the life sciences. These issues are particularly critical for widely used, efficient, and well-proven methods, especially those developed in research settings that often face funding discontinuities. We re-launch a range of established software components for computational genomics, as legacy version 1.0.1, suitable for sequence matching, masking, searching, clustering and visualization for protein family discovery, annotation and functional characterization on a genome scale. These applications are made available online as open source and include MagicMatch, GeneCAST, support scripts for CoGenT-like sequence collections, GeneRAGE and DifFuse, supported by centrally administered bioinformatics infrastructure funding. The toolkit may also be conceived as a flexible genome comparison software pipeline that supports research in this domain. We illustrate basic use by examples and pictorial representations of the registered tools, which are further described with appropriate documentation files in the corresponding GitHub release.

LIRcentral: a manually curated online database of experimentally validated functional LIR motifs.

Several selective macroautophagy receptor and adaptor proteins bind members of the Atg8 (autophagy related 8) family using short linear motifs (SLiMs), most often referred to as Atg8-family interacting motifs (AIMs) or LC3-interacting regions (LIRs). AIM/LIR motifs have been extensively studied during the last fifteen years, since they can uncover the underlying biological mechanisms and possible substrates for this key catabolic process of eukaryotic cells. Prompted by the fact that experimental information regarding LIR motifs can be found scattered across heterogeneous literature resources, we have developed LIRcentral (https://lircentral.eu), a freely available online repository for user-friendly access to comprehensive, high-quality information regarding LIR motifs from manually curated publications. Herein, we describe the development of LIRcentral and showcase currently available data and features, along with our plans for the expansion of this resource. Information incorporated in LIRcentral is useful for accomplishing a variety of research tasks, including: (i) guiding wet biology researchers for the characterization of novel instances of LIR motifs, (ii) giving bioinformaticians/computational biologists access to high-quality LIR motifs for building novel prediction methods for LIR motifs and LIR containing proteins (LIRCPs) and (iii) performing analyses to better understand the biological importance/features of functional LIR motifs. We welcome feedback on the LIRcentral content and functionality by all interested researchers and anticipate this work to spearhead a community effort for sustaining this resource which will further promote progress in studying LIR motifs/LIRCPs.Abbreviations: AIM, Atg8-family interacting motif; Atg8, autophagy related 8; GABARAP, GABA type A receptor-associated protein; LIR, LC3-interacting region; LIRCP, LIR-containing protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PMID, PubMed identifier; PPI, protein-protein interaction; SLiM, short linear motif.

A library of sensitive position-specific scoring matrices for high-throughput identification of nuclear pore complex subunits.

The nuclear pore complex exhibits different manifestations across eukaryotes, with certain components being restricted to specific clades. Several studies have been conducted to delineate the nuclear pore complex composition in various model organisms. Due to its pivotal role in cell viability, traditional lab experiments, such as gene knockdowns, can prove inconclusive and need to be complemented by a high-quality computational process. Here, using an extensive data collection, we create a robust library of nucleoporin protein sequences and their respective family-specific position-specific scoring matrices. By extensively validating each profile in different settings, we propose that the created profiles can be used to detect nucleoporins in proteomes with high sensitivity and specificity compared to existing methods. This library of profiles and the underlying sequence data can be used for the detection of nucleoporins in target proteomes.

Functional Tuning of Intrinsically Disordered Regions in Human Proteins by Composition Bias.

Intrinsically disordered regions (IDRs) in protein sequences are flexible, have low structural constraints and as a result have faster rates of evolution. This lack of evolutionary conservation greatly limits the use of sequence homology for the classification and functional assessment of IDRs, as opposed to globular domains. The study of IDRs requires other properties for their classification and functional prediction. While composition bias is not a necessary property of IDRs, compositionally biased regions (CBRs) have been noted as frequent part of IDRs. We hypothesized that to characterize IDRs, it could be helpful to study their overlap with particular types of CBRs. Here, we evaluate this overlap in the human proteome. A total of 2/3 of residues in IDRs overlap CBRs. Considering CBRs enriched in one type of amino acid, we can distinguish CBRs that tend to be fully included within long IDRs (R, H, N, D, P, G), from those that partially overlap shorter IDRs (S, E, K, T), and others that tend to overlap IDR terminals (Q, A). CBRs overlap more often IDRs in nuclear proteins and in proteins involved in liquid-liquid phase separation (LLPS). Study of protein interaction networks reveals the enrichment of CBRs in IDRs by tandem repetition of short linear motifs (rich in S or P), and the existence of E-rich polar regions that could support specific protein interactions with non-specific interactions. Our results open ways to pin down the function of IDRs from their partial compositional biases.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.

Statistical and Machine Learning Techniques in Human Microbiome Studies: Contemporary Challenges and Solutions.

The human microbiome has emerged as a central research topic in human biology and biomedicine. Current microbiome studies generate high-throughput omics data across different body sites, populations, and life stages. Many of the challenges in microbiome research are similar to other high-throughput studies, the quantitative analyses need to address the heterogeneity of data, specific statistical properties, and the remarkable variation in microbiome composition across individuals and body sites. This has led to a broad spectrum of statistical and machine learning challenges that range from study design, data processing, and standardization to analysis, modeling, cross-study comparison, prediction, data science ecosystems, and reproducible reporting. Nevertheless, although many statistics and machine learning approaches and tools have been developed, new techniques are needed to deal with emerging applications and the vast heterogeneity of microbiome data. We review and discuss emerging applications of statistical and machine learning techniques in human microbiome studies and introduce the COST Action CA18131 "ML4Microbiome" that brings together microbiome researchers and machine learning experts to address current challenges such as standardization of analysis pipelines for reproducibility of data analysis results, benchmarking, improvement, or development of existing and new tools and ontologies.

Evolutionary Study of Disorder in Protein Sequences.

Intrinsically disordered proteins (IDPs) contain regions lacking intrinsic globular structure (intrinsically disordered regions, IDRs). IDPs are present across the tree of life, with great variability of IDR type and frequency even between closely related taxa. To investigate the function of IDRs, we evaluated and compared the distribution of disorder content in 10,695 reference proteomes, confirming its high variability and finding certain correlation along the Euteleostomi (bony vertebrates) lineage to number of cell types. We used the comparison of orthologs to study the function of disorder related to increase in cell types, observing that multiple interacting subunits of protein complexes might gain IDRs in evolution, thus stressing the function of IDRs in modulating protein-protein interactions, particularly in the cell nucleus. Interestingly, the conservation of local compositional biases of IDPs follows residue-type specific patterns, with E- and K-rich regions being evolutionarily stable and Q- and A-rich regions being more dynamic. We provide a framework for targeted evolutionary studies of the emergence of IDRs. We believe that, given the large variability of IDR distributions in different species, studies using this evolutionary perspective are required.

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline-rich tyrosine kinase 2-beta: Insights from molecular dynamics simulations.

The focal adhesion kinase (FAK) and the proline-rich tyrosine kinase 2-beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic-5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK-Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase-dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all-atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK-FAT and PYK2-FAT domains. Our simulations and free-energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK-FAT and PYK2-FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK-FAT: Paxillin and PYK2-FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small-molecule inhibitors of the FAK-FAT and PYK2-FAT focal adhesion based functions.

Pseudomonas aeruginosa core metabolism exerts a widespread growth-independent control on virulence.

To assess the role of core metabolism genes in bacterial virulence - independently of their effect on growth - we correlated the genome, the transcriptome and the pathogenicity in flies and mice of 30 fully sequenced Pseudomonas strains. Gene presence correlates robustly with pathogenicity differences among all Pseudomonas species, but not among the P. aeruginosa strains. However, gene expression differences are evident between highly and lowly pathogenic P. aeruginosa strains in multiple virulence factors and a few metabolism genes. Moreover, 16.5%, a noticeable fraction of the core metabolism genes of P. aeruginosa strain PA14 (compared to 8.5% of the non-metabolic genes tested), appear necessary for full virulence when mutated. Most of these virulence-defective core metabolism mutants are compromised in at least one key virulence mechanism independently of auxotrophy. A pathway level analysis of PA14 core metabolism, uncovers beta-oxidation and the biosynthesis of amino-acids, succinate, citramalate, and chorismate to be important for full virulence. Strikingly, the relative expression among P. aeruginosa strains of genes belonging in these metabolic pathways is indicative of their pathogenicity. Thus, P. aeruginosa strain-to-strain virulence variation, remains largely obscure at the genome level, but can be dissected at the pathway level via functional transcriptomics of core metabolism.

PlaToLoCo: the first web meta-server for visualization and annotation of low complexity regions in proteins.

Low complexity regions (LCRs) in protein sequences are characterized by a less diverse amino acid composition compared to typically observed sequence diversity. Recent studies have shown that LCRs may co-occur with intrinsically disordered regions, are highly conserved in many organisms, and often play important roles in protein functions and in diseases. In previous decades, several methods have been developed to identify regions with LCRs or amino acid bias, but most of them as stand-alone applications and currently there is no web-based tool which allows users to explore LCRs in protein sequences with additional functional annotations. We aim to fill this gap by providing PlaToLoCo - PLAtform of TOols for LOw COmplexity-a meta-server that integrates and collects the output of five different state-of-the-art tools for discovering LCRs and provides functional annotations such as domain detection, transmembrane segment prediction, and calculation of amino acid frequencies. In addition, the union or intersection of the results of the search on a query sequence can be obtained. By developing the PlaToLoCo meta-server, we provide the community with a fast and easily accessible tool for the analysis of LCRs with additional information included to aid the interpretation of the results. The PlaToLoCo platform is available at: http://platoloco.aei.polsl.pl/.

Disentangling the complexity of low complexity proteins.

There are multiple definitions for low complexity regions (LCRs) in protein sequences, with all of them broadly considering LCRs as regions with fewer amino acid types compared to an average composition. Following this view, LCRs can also be defined as regions showing composition bias. In this critical review, we focus on the definition of sequence complexity of LCRs and their connection with structure. We present statistics and methodological approaches that measure low complexity (LC) and related sequence properties. Composition bias is often associated with LC and disorder, but repeats, while compositionally biased, might also induce ordered structures. We illustrate this dichotomy, and more generally the overlaps between different properties related to LCRs, using examples. We argue that statistical measures alone cannot capture all structural aspects of LCRs and recommend the combined usage of a variety of predictive tools and measurements. While the methodologies available to study LCRs are already very advanced, we foresee that a more comprehensive annotation of sequences in the databases will enable the improvement of predictions and a better understanding of the evolution and the connection between structure and function of LCRs. This will require the use of standards for the generation and exchange of data describing all aspects of LCRs. SHORT ABSTRACT: There are multiple definitions for low complexity regions (LCRs) in protein sequences. In this critical review, we focus on the definition of sequence complexity of LCRs and their connection with structure. We present statistics and methodological approaches that measure low complexity (LC) and related sequence properties. Composition bias is often associated with LC and disorder, but repeats, while compositionally biased, might also induce ordered structures. We illustrate this dichotomy, plus overlaps between different properties related to LCRs, using examples.

DisProt: intrinsic protein disorder annotation in 2020.

The Database of Protein Disorder (DisProt, URL: https://disprot.org) provides manually curated annotations of intrinsically disordered proteins from the literature. Here we report recent developments with DisProt (version 8), including the doubling of protein entries, a new disorder ontology, improvements of the annotation format and a completely new website. The website includes a redesigned graphical interface, a better search engine, a clearer API for programmatic access and a new annotation interface that integrates text mining technologies. The new entry format provides a greater flexibility, simplifies maintenance and allows the capture of more information from the literature. The new disorder ontology has been formalized and made interoperable by adopting the OWL format, as well as its structure and term definitions have been improved. The new annotation interface has made the curation process faster and more effective. We recently showed that new DisProt annotations can be effectively used to train and validate disorder predictors. We believe the growth of DisProt will accelerate, contributing to the improvement of function and disorder predictors and therefore to illuminate the 'dark' proteome.

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.

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with 'ready-to-use' deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others.

An updated view of the oligosaccharyltransferase complex in Plasmodium.

Despite the controversy regarding the importance of protein N-linked glycosylation in species of the genus Plasmodium, genes potentially encoding core subunits of the oligosaccharyltransferase (OST) complex have already been characterized in completely sequenced genomes of malaria parasites. Nevertheless, the currently established notion is that only four out of eight subunits of the OST complex-which is considered conserved across eukaryotes-are present in Plasmodium species. In this study, we carefully conduct computational analysis to provide unequivocal evidence that all components of the OST complex, with the exception of Swp1/Ribophorin II, can be reliably identified within completely sequenced plasmodial genomes. In fact, most of the subunits currently considered as absent from Plasmodium refer to uncharacterized protein sequences already existing in sequence databases. Interestingly, the main reason why the unusually short Ost4 subunit (36 residues long in yeast) has not been identified so far in plasmodia (and possibly other species) is the failure of gene-prediction pipelines to detect such a short coding sequence. We further identify elusive OST subunits in select protist species with completely sequenced genomes. Thus, our work highlights the necessity of a systematic approach towards the characterization of OST subunits across eukaryotes. This is necessary both for obtaining a concrete picture of the evolution of the OST complex but also for elucidating its possible role in eukaryotic pathogens.

Hypothesis, analysis and synthesis, it's all Greek to me.

The linguistic foundations of science and technology include many terms that have been borrowed from ancient languages. In the case of terms with origins in the Greek language, the modern meaning can often differ significantly from the original one. Here we use the PubMed database to demonstrate the prevalence of words of Greek origin in the language of modern science, and call for scientists to exercise care when coining new terms.

A fragment of the alarmin prothymosin α as a novel biomarker in murine models of bacteria-induced sepsis.

Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.