Rhea, the reaction knowledgebase in 2022.

Rhea (https://www.rhea-db.org) is an expert-curated knowledgebase of biochemical reactions based on the chemical ontology ChEBI (Chemical Entities of Biological Interest) (https://www.ebi.ac.uk/chebi). In this paper, we describe a number of key developments in Rhea since our last report in the database issue of Nucleic Acids Research in 2019. These include improved reaction coverage in Rhea, the adoption of Rhea as the reference vocabulary for enzyme annotation in the UniProt knowledgebase UniProtKB (https://www.uniprot.org), the development of a new Rhea website, and the designation of Rhea as an ELIXIR Core Data Resource. We hope that these and other developments will enhance the utility of Rhea as a reference resource to study and engineer enzymes and the metabolic systems in which they function.

A Coordinated Approach by Public Domain Bioinformatics Resources to Aid the Fight Against Alzheimer's Disease Through Expert Curation of Key Protein Targets.

BACKGROUND: The analysis and interpretation of data generated from patient-derived clinical samples relies on access to high-quality bioinformatics resources. These are maintained and updated by expert curators extracting knowledge from unstructured biological data described in free-text journal articles and converting this into more structured, computationally-accessible forms. This enables analyses such as functional enrichment of sets of genes/proteins using the Gene Ontology, and makes the searching of data more productive by managing issues such as gene/protein name synonyms, identifier mapping, and data quality. OBJECTIVE: To undertake a coordinated annotation update of key public-domain resources to better support Alzheimer's disease research. METHODS: We have systematically identified target proteins critical to disease process, in part by accessing informed input from the clinical research community. RESULTS: Data from 954 papers have been added to the UniProtKB, Gene Ontology, and the International Molecular Exchange Consortium (IMEx) databases, with 299 human proteins and 279 orthologs updated in UniProtKB. 745 binary interactions were added to the IMEx human molecular interaction dataset. CONCLUSION: This represents a significant enhancement in the expert curated data pertinent to Alzheimer's disease available in a number of biomedical databases. Relevant protein entries have been updated in UniProtKB and concomitantly in the Gene Ontology. Molecular interaction networks have been significantly extended in the IMEx Consortium dataset and a set of reference protein complexes created. All the resources described are open-source and freely available to the research community and we provide examples of how these data could be exploited by researchers.

Updates in Rhea: SPARQLing biochemical reaction data.

Rhea (http://www.rhea-db.org) is a comprehensive and non-redundant resource of over 11 000 expert-curated biochemical reactions that uses chemical entities from the ChEBI ontology to represent reaction participants. Originally designed as an annotation vocabulary for the UniProt Knowledgebase (UniProtKB), Rhea also provides reaction data for a range of other core knowledgebases and data repositories including ChEBI and MetaboLights. Here we describe recent developments in Rhea, focusing on a new resource description framework representation of Rhea reaction data and an SPARQL endpoint (https://sparql.rhea-db.org/sparql) that provides access to it. We demonstrate how federated queries that combine the Rhea SPARQL endpoint and other SPARQL endpoints such as that of UniProt can provide improved metabolite annotation and support integrative analyses that link the metabolome through the proteome to the transcriptome and genome. These developments will significantly boost the utility of Rhea as a means to link chemistry and biology for a more holistic understanding of biological systems and their function in health and disease.

Updates in Rhea - an expert curated resource of biochemical reactions.

Rhea (http://www.rhea-db.org) is a comprehensive and non-redundant resource of expert-curated biochemical reactions designed for the functional annotation of enzymes and the description of metabolic networks. Rhea describes enzyme-catalyzed reactions covering the IUBMB Enzyme Nomenclature list as well as additional reactions, including spontaneously occurring reactions, using entities from the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Here we describe developments in Rhea since our last report in the database issue of Nucleic Acids Research. These include the first implementation of a simple hierarchical classification of reactions, improved coverage of the IUBMB Enzyme Nomenclature list and additional reactions through continuing expert curation, and the development of a new website to serve this improved dataset.

The SwissLipids knowledgebase for lipid biology.

MOTIVATION: Lipids are a large and diverse group of biological molecules with roles in membrane formation, energy storage and signaling. Cellular lipidomes may contain tens of thousands of structures, a staggering degree of complexity whose significance is not yet fully understood. High-throughput mass spectrometry-based platforms provide a means to study this complexity, but the interpretation of lipidomic data and its integration with prior knowledge of lipid biology suffers from a lack of appropriate tools to manage the data and extract knowledge from it. RESULTS: To facilitate the description and exploration of lipidomic data and its integration with prior biological knowledge, we have developed a knowledge resource for lipids and their biology-SwissLipids. SwissLipids provides curated knowledge of lipid structures and metabolism which is used to generate an in silico library of feasible lipid structures. These are arranged in a hierarchical classification that links mass spectrometry analytical outputs to all possible lipid structures, metabolic reactions and enzymes. SwissLipids provides a reference namespace for lipidomic data publication, data exploration and hypothesis generation. The current version of SwissLipids includes over 244 000 known and theoretically possible lipid structures, over 800 proteins, and curated links to published knowledge from over 620 peer-reviewed publications. We are continually updating the SwissLipids hierarchy with new lipid categories and new expert curated knowledge. AVAILABILITY: SwissLipids is freely available at http://www.swisslipids.org/. CONTACT: alan.bridge@isb-sib.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Updates in Rhea--a manually curated resource of biochemical reactions.

Rhea (http://www.ebi.ac.uk/rhea) is a comprehensive and non-redundant resource of expert-curated biochemical reactions described using species from the ChEBI (Chemical Entities of Biological Interest) ontology of small molecules. Rhea has been designed for the functional annotation of enzymes and the description of genome-scale metabolic networks, providing stoichiometrically balanced enzyme-catalyzed reactions (covering the IUBMB Enzyme Nomenclature list and additional reactions), transport reactions and spontaneously occurring reactions. Rhea reactions are extensively curated with links to source literature and are mapped to other publicly available enzyme and pathway databases such as Reactome, BioCyc, KEGG and UniPathway, through manual curation and computational methods. Here we describe developments in Rhea since our last report in the 2012 database issue of Nucleic Acids Research. These include significant growth in the number of Rhea reactions and the inclusion of reactions involving complex macromolecules such as proteins, nucleic acids and other polymers that lie outside the scope of ChEBI. Together these developments will significantly increase the utility of Rhea as a tool for the description, analysis and reconciliation of genome-scale metabolic models.

Deficient DNA damage response and cell cycle checkpoints lead to accumulation of point mutations in human embryonic stem cells.

Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study, we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that, due to the absence of p21/Waf1/Cip1, cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage, the majority of hESCs succumb to apoptosis; however, a subpopulation continues to proliferate, carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that, due to deficient DNA damage response, the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs, paving the way for further major genetic changes.

Nucleotide excision repair and B lymphoma: somatic hypermutation is not the only culprit.

Different mechanisms account for the development of B lymphoma. Malignant transformation of B lymphocytes arises from progressive loss of genome integrity combined with uncontrolled cell proliferation, often triggered by foreign or self antigens. It is well established that somatic hypermutation, the pathway responsible for introducing high levels of mutations in immunoglobulin genes, also targets several other genes, contributing mainly to germinal center-derived B-cell lymphoma. We have recently discovered that a major DNA repair pathway, nucleotide excision repair (NER), is downregulated in quiescent B lymphocytes. Upon B-cell stimulation, unrepaired DNA damage results in the accumulation of mutations in a different and likely larger set of genes, including normally silent genes (e.g., oncogenes) as well as cell cycle and activation-induced genes. This mechanism potentially produces a transforming event relevant to a wider palette of B lymphomas. Here we discuss the relative contribution of both mechanisms to lymphomagenesis and possible implications of NER downregulation for other types of malignancies and for B cell-mediated immunity. Given that hematopoietic cancer stem cells remain quiescent for long periods of time, we propose that downregulation of NER during quiescence, in an environment that causes both genotoxic stress and proliferation, could be a general mechanism for carcinogenesis.

Circulating human B lymphocytes are deficient in nucleotide excision repair and accumulate mutations upon proliferation.

Faithful repair of DNA lesions is a crucial task that dividing cells must actively perform to maintain genome integrity. Strikingly, nucleotide excision repair (NER), the most versatile DNA repair system, is specifically down-regulated in terminally differentiated cells. This prompted us to examine whether NER attenuation might be a common feature of all G0-arrested cells, and in particular of those that retain the capacity to reenter cell cycle and might thus convert unrepaired DNA lesions into mutations, a prerequisite for malignant transformation. Here we report that quiescent primary human B lymphocytes down-regulate NER at the global genome level while maintaining proficient repair of constitutively expressed genes. Quiescent B cells exposed to an environment that causes both DNA damage and proliferation accumulate point mutations in silent and inducible genes crucial for cell replication and differentiation, such as BCL6 and Cyclin D2. Similar to differentiated cells, NER attenuation in quiescent cells is associated with incomplete phosphorylation of the ubiquitin activating enzyme Ube1, which is required for proficient NER. Our data establish a mechanistic link between NER attenuation during quiescence and cell mutagenesis and also support the concept that oncogenic events targeting cell cycle- or activation-induced genes might initiate genomic instability and lymphomagenesis.

DAP10 deficiency breaks the immune tolerance against transplantable syngeneic melanoma.

DAP10, an activating adaptor protein, associates with the NKG2D protein to form a multisubunit receptor complex that is expressed in lymphoid and myeloid cells. The ligands for NKG2D-DAP10 receptor are expressed in both normal and tumor cells, suggesting distinct roles for this receptor in autoimmunity and cancer. In this study, we report that constitutive DAP10 activating signaling is part of regulatory mechanisms that control immunity against tumors. Mice lacking DAP10 (DAP10KO), showed enhanced immunity against melanoma malignancies due to hyperactive functioning of NK1.1+CD3+ NKT cells. DAP10 deficiency resulted in substantially increased NKT cell functions, including cytokine production and cytotoxicity, leading to efficient killing of melanoma tumors. Moreover, the antitumor phenotype of DAP10KO mice correlated with impaired activation status of CD4+CD25+ T regulatory cells (Tregs). Upon activation, DAP10KO Tregs maintained higher levels of IL-2 and produced significantly lower amounts of IL-10 and IFN-gamma cytokines when compared with wild-type Tregs. Our data suggest that DAP10 signaling is involved in adjusting the activation threshold and generation of NKT cells and Tregs to avoid autoreactivity, but also modulates antitumor mechanisms.

Physiological roles of murine DAP10 adapter protein in tumor immunity and autoimmunity.

The immune system has evolved to tolerate what is self and reject what is foreign. The recognition of self from non-self is performed by activating and inhibitory receptors, which signal immune cells via adapter molecules, determining the outcome of the immune response. DAP10, a transmembrane adapter protein expressed broadly in hematopoietic cells, associates with NKG2D activating receptor forming a multisubunit complex, which recognizes self-proteins upregulated during tumorigenesis, infection, and autoimmune response. Analysis of immune reactions against syngeneic tumors, as well as autoimmune responses in the DAP10-deficient mice, revealed an important physiological role of DAP10 signaling in maintaining tolerance to self, probably by controlling the development and activation threshold of autoreactive T cells.

Transcription domain-associated repair in human cells.

Nucleotide excision repair (NER), which is arguably the most versatile DNA repair system, is strongly attenuated in human cells of the monocytic lineage when they differentiate into macrophages. Within active genes, however, both DNA strands continue to be proficiently repaired. The proficient repair of the nontranscribed strand cannot be explained by the dedicated subpathway of transcription-coupled repair (TCR), which is targeted to the transcribed strand in expressed genes. We now report that the previously termed differentiation-associated repair (DAR) depends upon transcription, but not simply upon RNA polymerase II (RNAPII) encountering a lesion: proficient repair of both DNA strands can occur in a part of a gene that the polymerase never reaches, and even if the translocation of RNAPII is blocked with transcription inhibitors. This suggests that DAR may be a subset of global NER, restricted to the subnuclear compartments or chromatin domains within which transcription occurs. Downregulation of selected NER genes with small interfering RNA has confirmed that DAR relies upon the same genes as global genome repair, rather than upon TCR-specific genes. Our findings support the general view that the genomic domains within which transcription is active are more accessible than the bulk of the genome to the recognition and repair of lesions through the global pathway and that TCR is superimposed upon that pathway of NER.

The production of IL-1 receptor antagonist in IFN-beta-stimulated human monocytes depends on the activation of phosphatidylinositol 3-kinase but not of STAT1.

IFN-beta induces the production of secreted IL-1R antagonist (sIL-1Ra) without triggering synthesis of the agonist IL-1beta in human monocytes. This might account for its anti-inflammatory properties. Canonically, IFN-beta signals through activation of JAK/STAT pathway, although PI3K and MAPK have also been involved. In this study, the role of PI3K, MEK1, and STAT1 in IFN-beta-induced sIL-1Ra production is investigated in freshly isolated human blood monocytes. PI3K, but not MEK1 activation is essential for sIL-1Ra production in monocytes treated with IFN-beta, as demonstrated by using the respective inhibitors of PI3K and MEK1, Ly294002 and PD98059. The use of cycloheximide and actinomycin D shows that sIL-1Ra was an immediate early gene induced by IFN-beta and that PI3K was controlling sIL-1Ra gene transcription. Although both inhibitors of PI3K and MEK1 diminished the Ser(727) phosphorylation of STAT1 induced by IFN-beta, only Ly294002 inhibited sIL-1Ra production. Furthermore, the inhibition of STAT1-Ser(727) phosphorylation by Ly294002 did not affect STAT1 translocation, suggesting that STAT1 was not involved in sIL-1Ra gene induction. This was confirmed in monocytes that were transfected with small interfering RNA specifically targeting STAT1. Indeed, monocytes in which effective STAT1 gene knockdown was achieved were fully responsive to IFN-beta in terms of sIL-1Ra production. Taken together, the present data demonstrate that the induction of sIL-1Ra transcription and production by IFN-beta in human monocytes involved PI3K, but not STAT1 activation.