MobiDB: 10 years of intrinsically disordered proteins.

The MobiDB database (URL: https://mobidb.org/) is a knowledge base of intrinsically disordered proteins. MobiDB aggregates disorder annotations derived from the literature and from experimental evidence along with predictions for all known protein sequences. MobiDB generates new knowledge and captures the functional significance of disordered regions by processing and combining complementary sources of information. Since its first release 10 years ago, the MobiDB database has evolved in order to improve the quality and coverage of protein disorder annotations and its accessibility. MobiDB has now reached its maturity in terms of data standardization and visualization. Here, we present a new release which focuses on the optimization of user experience and database content. The major advances compared to the previous version are the integration of AlphaFoldDB predictions and the re-implementation of the homology transfer pipeline, which expands manually curated annotations by two orders of magnitude. Finally, the entry page has been restyled in order to provide an overview of the available annotations along with two separate views that highlight structural disorder evidence and functions associated with different binding modes.

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.

BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

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.

orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control.

BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.

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.

On the dynamical incompleteness of the Protein Data Bank.

Major scientific challenges that are beyond the capability of individuals need to be addressed by multi-disciplinary and multi-institutional consortia. Examples of these endeavours include the Human Genome Project, and more recently, the Structural Genomics (SG) initiative. The SG initiative pursues the expansion of structural coverage to include at least one structural representative for each protein family to derive the remaining structures using homology modelling. However, biological function is inherently connected with protein dynamics that can be studied by knowing different structures of the same protein. This ensemble of structures provides snapshots of protein conformational diversity under native conditions. Thus, sequence redundancy in the Protein Data Bank (PDB) (i.e. crystallization of the same protein under different conditions) is therefore an essential input contributing to experimentally based studies of protein dynamics and providing insights into protein function. In this work, we show that sequence redundancy, a key concept for exploring protein dynamics, is highly biased and fundamentally incomplete in the PDB. Additionally, our results show that dynamical behaviour of proteins cannot be inferred using homologous proteins. Minor to moderate changes in sequence can produce great differences in dynamical behaviour. Nonetheless, the structural and dynamical incompleteness of the PDB is apparently unrelated concepts in SG. While the first could be reversed by promoting the extension of the structural coverage, we would like to emphasize that further focused efforts will be needed to amend the incompleteness of the PDB in terms of dynamical information content, essential to fully understand protein function.

The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth.

As sessile organisms, plants have evolved mechanisms to adapt to variable and rapidly fluctuating environmental conditions. Calcium (Ca2+) in plant cells is a versatile intracellular second messenger that is essential for stimulating short- and long-term responses to environmental stresses through changes in its concentration in the cytosol ([Ca2+]cyt). Increases in [Ca2+]cyt direct the strength and length of these stimuli. In order to terminate them, the cells must then remove the cytosolic Ca2+ against a concentration gradient, either taking it away from the cell or storing it in organelles such as the endoplasmic reticulum (ER) and/or vacuoles. Here, we review current knowledge about the biological roles of plant P-type Ca2+-ATPases as potential actors in the regulation of this cytosolic Ca2+ efflux, with a focus the IIA ER-type Ca2+-ATPases (ECAs) and the IIB autoinhibited Ca2+-ATPases (ACAs). While ECAs are analogous proteins to animal sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), ACAs are equivalent to animal plasma membrane-type ATPases (PMCAs). We examine their expression patterns in cells exhibiting polar growth and consider their appearance during the evolution of the plant lineage. Full details of the functions and coordination of ECAs and ACAs during plant growth and development have not yet been elucidated. Our current understanding of the regulation of fluctuations in Ca2+ gradients in the cytoplasm and organelles during growth is in its infancy, but recent technological advances in Ca2+ imaging are expected to shed light on this subject.

MISTIC2: comprehensive server to study coevolution in protein families.

Correlated mutations between residue pairs in evolutionarily related proteins arise from constraints needed to maintain a functional and stable protein. Identifying these inter-related positions narrows down the search for structurally or functionally important sites. MISTIC is a server designed to assist users to calculate covariation in protein families and provide them with an interactive tool to visualize the results. Here, we present MISTIC2, an update to the previous server, that allows to calculate four covariation methods (MIp, mfDCA, plmDCA and gaussianDCA). The results visualization framework has been reworked for improved performance, compatibility and user experience. It includes a circos representation of the information contained in the alignment, an interactive covariation network, a 3D structure viewer and a sequence logo. Others components provide additional information such as residue annotations, a roc curve for assessing contact prediction, data tables and different ways of filtering the data and exporting figures. Comparison of different methods is easily done and scores combination is also possible. A newly implemented web service allows users to access MISTIC2 programmatically using an API to calculate covariation and retrieve results. MISTIC2 is available at: https://mistic2.leloir.org.ar.

Comutation and exclusion analysis in human tumors: A tool for cancer biology studies and for rational selection of multitargeted therapeutic approaches.

Malignant tumors originate from somatic mutations and other genomic and epigenomic alterations, which lead to loss of control of the cellular circuitry. These alterations present patterns of co-occurrence and mutual exclusivity that can influence prognosis and modify response to drugs, highlighting the need for multitargeted therapies. Studies in this area have generally focused in particular malignancies and considered whole genes instead of specific mutations, ignoring the fact that different alterations in the same gene can have widely different effects. Here, we present a comprehensive analysis of co-dependencies of individual somatic mutations in the whole spectrum of human tumors. Combining multitesting with conditional and expected mutational probabilities, we have discovered rules governing the codependencies of driver and nondriver mutations. We also uncovered pairs and networks of comutations and exclusions, some of them restricted to certain cancer types and others widespread. These pairs and networks are not only of basic but also of clinical interest, and can be of help in the selection of multitargeted antitumor therapies. In this respect, recurrent driver comutations suggest combinations of drugs that might be effective in the clinical setting, while recurrent exclusions indicate combinations unlikely to be useful.

Characterization of intellectual disability and autism comorbidity through gene panel sequencing.

Intellectual disability (ID) and autism spectrum disorder (ASD) are clinically and genetically heterogeneous diseases. Recent whole exome sequencing studies indicated that genes associated with different neurological diseases are shared across disorders and converge on common functional pathways. Using the Ion Torrent platform, we developed a low-cost next-generation sequencing gene panel that has been transferred into clinical practice, replacing single disease-gene analyses for the early diagnosis of individuals with ID/ASD. The gene panel was designed using an innovative in silico approach based on disease networks and mining data from public resources to score disease-gene associations. We analyzed 150 unrelated individuals with ID and/or ASD and a confident diagnosis has been reached in 26 cases (17%). Likely pathogenic mutations have been identified in another 15 patients, reaching a total diagnostic yield of 27%. Our data also support the pathogenic role of genes recently proposed to be involved in ASD. Although many of the identified variants need further investigation to be considered disease-causing, our results indicate the efficiency of the targeted gene panel on the identification of novel and rare variants in patients with ID and ASD.

MIToS.jl: mutual information tools for protein sequence analysis in the Julia language.

Motivation: MIToS is an environment for mutual information analysis and a framework for protein multiple sequence alignments (MSAs) and protein structures (PDB) management in Julia language. It integrates sequence and structural information through SIFTS, making Pfam MSAs analysis straightforward. MIToS streamlines the implementation of any measure calculated from residue contingency tables and its optimization and testing in terms of protein contact prediction. As an example, we implemented and tested a BLOSUM62-based pseudo-count strategy in mutual information analysis. Availability and Implementation: The software is totally implemented in Julia and supported for Linux, OS X and Windows. It's freely available on GitHub under MIT license: http://mitos.leloir.org.ar . Contacts: diegozea@gmail.com or cmb@leloir.org.ar. Supplementary information: Supplementary data are available at Bioinformatics online.

How is structural divergence related to evolutionary information?

The analysis of evolutionary information in a protein family, such as conservation and covariation, is often linked to its structural information. Multiple sequence alignments of distant homologous sequences are used to measure evolutionary variables. Although high structural differences between proteins can be expected in such divergent alignments, most works linking evolutionary and structural information use a single structure ignoring the structural variability within protein families. The goal of this work is to elucidate the relevance of structural divergence when sequence-based measures are integrated with structural information. We found that inter-residue contacts and solvent accessibility undergo large variations in protein families. Our results show that high covariation scores tend to reveal residue contacts that are conserved in the family, instead of protein or conformer specific contacts. We also found that residue accessible surface area shows a high variability between structures of the same family. As a consequence, the mean relative solvent accessibility of multiple structures correlates better with the conservation pattern than the relative solvent accessibility of a single structure. We conclude that the use of comprehensive structural information allows a more accurate interpretation of the information computed from sequence alignments. Therefore, considering structural divergence would lead to a better understanding of protein function, dynamics, and evolution.

The arabidopsis DNA polymerase δ has a role in the deposition of transcriptionally active epigenetic marks, development and flowering.

DNA replication is a key process in living organisms. DNA polymerase α (Polα) initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3) locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT) expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.

The conundrum of UDP-Glc entrance into the yeast ER lumen.

UDP-Glc entrance into the endoplasmic reticulum (ER) of eukaryotic cells is a key step in the quality control of glycoprotein folding, a mechanism requiring transfer of a Glc residue from the nucleotide sugar (NS) to glycoprotein folding intermediates by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). According to a bioinformatics search there are only eight genes in the Schizosaccharomyces pombe genome belonging to the three Pfam families to which all known nucleotide-sugar transporters (NSTs) of the secretory pathway belong. The protein products of two of them (hut1+ and yea4+) localize to the ER, those of genes gms1+, vrg4+, pet1+, pet2+ and pet3+ to the Golgi, whereas that of gms2+ has an unknown location. Here we demonstrate that (1) Δhut1 and Δgpt1 (UGGT null) mutants share several phenotypic features; (2) Δhut1 mutants show a 50% reduction in UDP-Glc transport into ER-derived membranes; (3) in vivo UDP-Glc ER entrance occurred in Δhut1Δyea4Δgms2 mutants and in cells in which Δhut1 disruption was combined with that of each of four of the genes encoding Golgi-located proteins. Therefore, disruption of all genes whose products localize to the ER or have an unknown location did not obliterate UDP-Glc ER entrance. We conclude that the hut1+ gene product is involved in UDP-Glc entrance into the ER, but that at least another as yet unknown NST displaying an unconventional sequence operates in the yeast secretory pathway. This conclusion agrees with our previous results showing that UDP-Glc entrance into the yeast ER does not follow the classical NST antiport mechanism.

I-COMS: Interprotein-COrrelated Mutations Server.

Interprotein contact prediction using multiple sequence alignments (MSAs) is a useful approach to help detect protein-protein interfaces. Different computational methods have been developed in recent years as an approximation to solve this problem. However, as there are discrepancies in the results provided by them, there is still no consensus on which is the best performing methodology. To address this problem, I-COMS (interprotein COrrelated Mutations Server) is presented. I-COMS allows to estimate covariation between residues of different proteins by four different covariation methods. It provides a graphical and interactive output that helps compare results obtained using different methods. I-COMS automatically builds the required MSA for the calculation and produces a rich visualization of either intraprotein and/or interprotein covariating positions in a circos representation. Furthermore, comparison between any two methods is available as well as the overlap between any or all four methodologies. In addition, as a complementary source of information, a matrix visualization of the corresponding scores is made available and the density plot distribution of the inter, intra and inter+intra scores are calculated. Finally, all the results can be downloaded (including MSAs, scores and graphics) for comparison and visualization and/or for further analysis.

Differential contributions of tacaribe arenavirus nucleoprotein N-terminal and C-terminal residues to nucleocapsid functional activity.

UNLABELLED: The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE: The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be determined. In this report, novel findings are provided on critical interactions between the viral ribonucleoprotein components. We identify several amino acid residues in both the N-terminal and C-terminal domains of TCRV NP that differentially contribute to NP-NP and NP-RNA interactions and analyze their relevance for binding of NP to the L polymerase and for nucleocapsid activity. Our results provide insight into the contribution of NP self-interaction to RNP assembly and activity and reveal the involvement of the NP C-terminal domain in RNA binding.

MISTIC: Mutual information server to infer coevolution.

MISTIC (mutual information server to infer coevolution) is a web server for graphical representation of the information contained within a MSA (multiple sequence alignment) and a complete analysis tool for Mutual Information networks in protein families. The server outputs a graphical visualization of several information-related quantities using a circos representation. This provides an integrated view of the MSA in terms of (i) the mutual information (MI) between residue pairs, (ii) sequence conservation and (iii) the residue cumulative and proximity MI scores. Further, an interactive interface to explore and characterize the MI network is provided. Several tools are offered for selecting subsets of nodes from the network for visualization. Node coloring can be set to match different attributes, such as conservation, cumulative MI, proximity MI and secondary structure. Finally, a zip file containing all results can be downloaded. The server is available at http://mistic.leloir.org.ar. In summary, MISTIC allows for a comprehensive, compact, visually rich view of the information contained within an MSA in a manner unique to any other publicly available web server. In particular, the use of circos representation of MI networks and the visualization of the cumulative MI and proximity MI concepts is novel.

Activating mutations cluster in the "molecular brake" regions of protein kinases and do not associate with conserved or catalytic residues.

Mutations leading to activation of proto-oncogenic protein kinases (PKs) are a type of drivers crucial for understanding tumorogenesis and as targets for antitumor drugs. However, bioinformatics tools so far developed to differentiate driver mutations, typically based on conservation considerations, systematically fail to recognize activating mutations in PKs. Here, we present the first comprehensive analysis of the 407 activating mutations described in the literature, which affect 41 PKs. Unexpectedly, we found that these mutations do not associate with conserved positions and do not directly affect ATP binding or catalytic residues. Instead, they cluster around three segments that have been demonstrated to act, in some PKs, as "molecular brakes" of the kinase activity. This finding led us to hypothesize that an auto inhibitory mechanism mediated by such "brakes" is present in all PKs and that the majority of activating mutations act by releasing it. Our results also demonstrate that activating mutations of PKs constitute a distinct group of drivers and that specific bioinformatics tools are needed to identify them in the numerous cancer sequencing projects currently underway. The clustering in three segments should represent the starting point of such tools, a hypothesis that we tested by identifying two somatic mutations in EPHA7 that might be functionally relevant.