Unveiling the Complexity of cis-Regulation Mechanisms in Kinases: A Comprehensive Analysis.

Protein cis-regulatory elements (CREs) are regions that modulate the activity of a protein through intramolecular interactions. Kinases, pivotal enzymes in numerous biological processes, often undergo regulatory control via inhibitory interactions in cis. This study delves into the mechanisms of cis regulation in kinases mediated by CREs, employing a combined structural and sequence analysis. To accomplish this, we curated an extensive dataset of kinases featuring annotated CREs, organized into homolog families through multiple sequence alignments. Key molecular attributes, including disorder and secondary structure content, active and ATP-binding sites, post-translational modifications, and disease-associated mutations, were systematically mapped onto all sequences. Additionally, we explored the potential for conformational changes between active and inactive states. Finally, we explored the presence of these kinases within membraneless organelles and elucidated their functional roles therein. CREs display a continuum of structures, ranging from short disordered stretches to fully folded domains. The adaptability demonstrated by CREs in achieving the common goal of kinase inhibition spans from direct autoinhibitory interaction with the active site within the kinase domain, to CREs binding to an alternative site, inducing allosteric regulation revealing distinct types of inhibitory mechanisms, which we exemplify by archetypical representative systems. While this study provides a systematic approach to comprehend kinase CREs, further experimental investigations are imperative to unravel the complexity within distinct kinase families. The insights gleaned from this research lay the foundation for future studies aiming to decipher the molecular basis of kinase dysregulation, and explore potential therapeutic interventions.

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.

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.

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.

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.

DisPhaseDB: An integrative database of diseases related variations in liquid-liquid phase separation proteins.

Motivation: Proteins involved in liquid-liquid phase separation (LLPS) and membraneless organelles (MLOs) are recognized to be decisive for many biological processes and also responsible for several diseases. The recent explosion of research in the area still lacks tools for the analysis and data integration among different repositories. Currently, there is not a comprehensive and dedicated database that collects all disease-related variations in combination with the protein location, biological role in the MLO, and all the metadata available for each protein and disease. Disease-related protein variants and additional features are dispersed and the user has to navigate many databases, with a different focus, formats, and often not user friendly. Results: We present DisPhaseDB, a database dedicated to disease-related variants of liquid-liquid phase separation proteins. It integrates 10 databases, contains 5,741 proteins, 1,660,059 variants, and 4,051 disease terms. It also offers intuitive navigation and an informative display. It constitutes a pivotal starting point for further analysis, encouraging the development of new computational tools.The database is freely available at http://disphasedb.leloir.org.ar.

Panels and models for accurate prediction of tumor mutation burden in tumor samples.

Immune checkpoint blockade (ICB) is becoming standard-of-care in many types of human malignancies, but patient selection is still imperfect. Tumor mutation burden (TMB) is being evaluated as a biomarker for ICB in clinical trials, but most of the sequencing panels used to estimate it are inadequately designed. Here, we present a bioinformatics-based method to select panels and mathematical models for accurate TMB prediction. Our method is based on tumor-specific, forward-step selection of genes, generation of panels using a linear regression algorithm, and rigorous internal and external validation comparing predicted with experimental TMB. As a result, we propose cancer-specific panels for 14 malignancies which can offer reliable, clinically relevant estimates of TMBs. Our work facilitates a better prediction of TMB that can improve the selection of patients for ICB therapy.

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.