Michael Caudy

Reactome: a database of reactions, pathways and biological processes

Reactome (http://www.reactome.org) is a collaboration among groups at the Ontario Institute for Cancer Research, Cold Spring Harbor Laboratory, New York University School of Medicine and The European Bioinformatics Institute, to develop an open source curated bioinformatics database of human pathways and reactions. Recently, we developed a new web site with improved tools for pathway browsing and data analysis. The Pathway Browser is an Systems Biology Graphical Notation (SBGN)-based visualization system that supports zooming, scrolling and event highlighting. It exploits PSIQUIC web services to overlay our curated pathways with molecular interaction data from the Reactome Functional Interaction Network and external interaction databases such as IntAct, BioGRID, ChEMBL, iRefIndex, MINT and STRING. Our Pathway and Expression Analysis tools enable ID mapping, pathway assignment and overrepresentation analysis of user-supplied data sets. To support pathway annotation and analysis in other species, we continue to make orthology-based inferences of pathways in non-human species, applying Ensembl Compara to identify orthologs of curated human proteins in each of 20 other species. The resulting inferred pathway sets can be browsed and analyzed with our Species Comparison tool. Collaborations are also underway to create manually curated data sets on the Reactome framework for chicken, Drosophila and rice.

Reactome knowledgebase of human biological pathways and processes.

Reactome (http://www.reactome.org) is an expert-authored, peer-reviewed knowledgebase of human reactions and pathways that functions as a data mining resource and electronic textbook. Its current release includes 2975 human proteins, 2907 reactions and 4455 literature citations. A new entity-level pathway viewer and improved search and data mining tools facilitate searching and visualizing pathway data and the analysis of user-supplied high-throughput data sets. Reactome has increased its utility to the model organism communities with improved orthology prediction methods allowing pathway inference for 22 species and through collaborations to create manually curated Reactome pathway datasets for species including Arabidopsis, Oryza sativa (rice), Drosophila and Gallus gallus (chicken). Reactomes data content and software can all be freely used and redistributed under open source terms.

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

The function of hairy-related bHLH repressor proteins in cell fate decisions

Hairy‐related proteins are a distinct subfamily of basic helix‐loop‐helix (bHLH) proteins that generally function as DNA‐binding transcriptional repressors. These proteins act in opposition to bHLH transcriptional activator proteins such as the proneural and myogenic proteins; together, the activator and repressor genes that encode these proteins have co‐evolved as a regulatory gene “cassette” or “module” for controlling cell fate decisions. In the development of the Drosophila peripheral nervous system, Hairy‐related genes function at multiple steps during neurogenesis, for example, as positional information genes that establish the “prepattern” that controls where “proneural cluster” equivalence groups will form, and later as nuclear effectors of the Notch signaling pathway to “single out” individual precursor cells within the equivalence group. Hairy‐related genes also function in the establishment and restriction of other types of equivalence groups, such as those for muscle and Malphigian tubule precursors. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis. BioEssays 20:298‐306, 1998.© 1998 John Wiley & Sons, Inc.

HES-1 repression of differentiation and proliferation in PC12 cells: role for the helix 3-helix 4 domain in transcription repression.

ABSTRACT HES-1 is a Hairy-related basic helix-loop-helix protein with three evolutionarily conserved regions known to define its function as a transcription repressor. The basic region, helix-loop-helix domain, and WRPW motif have been characterized for their molecular function in DNA binding, dimer formation, and corepressor recruitment, respectively. In contrast, the function conferred by a fourth conserved region, the helix 3-helix 4 (H-3/4) domain, is not known. To better understand H-3/4 domain function, we expressed HES-1 variants under tetracycline-inducible control in PC12 cells. As expected, the induced expression of moderate levels of wild-type HES-1 in PC12 cells strongly inhibited nerve growth factor-induced differentiation. This repression was dependent on the H-3/4 domain. Unexpectedly, expression of HES-1 also arrested cell growth, an effect that could be reversed upon down regulation of HES-1. Concomitant with growth arrest, there was a strong reduction in bromodeoxyuridine incorporation and PCNA protein levels, although not in cyclin D1 expression. Expression of a HES-1 protein carrying the H-3/4 domain, but not the WRPW domain, still partially inhibited both proliferation and differentiation. Transcription assays in PC12 cells directly demonstrated that the H-3/4 domain can mediate DNA-binding-dependent transcription repression, even in the absence of corepressor recruitment by the WRPW motif. HES-1 expression strongly repressed transcription of the p21 cip1promoter, a cyclin–cyclin-dependent kinase inhibitor up regulated during NGF-induced differentiation, and the H-3/4 domain is necessary for this repression. Thus, the H-3/4 domain of HES-1 contributes to transcription repression independently of WRPW function, inhibits neurite formation, and facilitates two distinct and previously uncharacterized roles for HES-1: the inhibition of cell proliferation and the direct transcriptional repression of the NGF-induced gene,p21.

The Protein Ontology: a structured representation of protein forms and complexes

The Protein Ontology (PRO) provides a formal, logically-based classification of specific protein classes including structured representations of protein isoforms, variants and modified forms. Initially focused on proteins found in human, mouse and Escherichia coli, PRO now includes representations of protein complexes. The PRO Consortium works in concert with the developers of other biomedical ontologies and protein knowledge bases to provide the ability to formally organize and integrate representations of precise protein forms so as to enhance accessibility to results of protein research. PRO (http://pir.georgetown.edu/pro) is part of the Open Biomedical Ontology Foundry.

Regulation of hippocampal neuronal differentiation by the basic helix-loop-helix transcription factors HES-1 and MASH-1.

HES‐1 is a vertebrate homologue of the Drosophila basic helix‐loop‐helix (bHLH) protein Hairy, a transcriptional repressor that negatively regulates neuronal differentiation. HES‐1 expression in neuronal precursors precedes and represses the expression of the neuronal commitment gene MASH‐1, a bHLH activator homologous to the proneural Achaete‐Scute genes in Drosophila. Down‐regulation of HES‐1 expression in developing neuroblasts may be necessary for the induction of a regulatory cascade of bHLH activator proteins that controls the commitment and progression of neuronal differentiation. Here we show that the differentiation of embryonic day‐17 rat hippocampal neurons in culture was coincident with a decline in HES‐1 expression and DNA binding. Therefore, we examined the effect of forced expression of HES‐1 and MASH‐1 upon nerve growth factor (NGF) ‐induced differentiation in TrkA transfected hippocampal neurons. Expression of HES‐1 inhibited both the intrinsic and NGF‐induced neurite outgrowth, whereas MASH‐1 expression increased neurite outgrowth. Strikingly, the increased hippocampal differentiation observed with MASH‐1 expression is completely blocked by coexpression of HES‐1. Furthermore, both wild‐type HES‐1 and a non‐DNA binding mutant of HES‐1 repressed MASH‐1‐dependent transcription activation. These results suggest that down‐regulation of HES‐1 is necessary for autonomous, growth facto‐ induced and MASH‐1‐activated hippocampal differentiation. J. Neurosci. Res. 56:229–240, 1999.

Association of the dihydrolipoamide dehydrogenase gene with Alzheimer's disease in an Ashkenazi Jewish population

Abundant biochemical evidence links deficient activity of mitochondrial α‐ketoglutarate dehydrogenase with neuropathologically confirmed Alzheimers disease (AD). Reduced α‐ketoglutarate dehydrogenase activity has also been associated with anti‐mortem measures of clinical disability. One of the genes encoding this complex, namely, DLD, lies within a chromosome 7 region that is in linkage disequilibrium with AD. We therefore examined the hypothesis that variation in DLD is associated with AD risk. Denaturing HPLC was used to search for sequence variations in the coding and flanking regions of all exons of DLD, but no abundant variants that alter protein sequence were found. However, four common SNPs were identified and genotyped in a case‐control series of 297 Caucasians from New York City, including 229 residents of a Jewish nursing home. Logistic regression analysis was performed for the four‐locus DLD genotype, sex, and ApoE4 status to determine the association of these independent variables with AD. Significant associations with AD were observed for ApoE4 (P < 10−6) and sex combined with DLD genotype (P = 0.013). The association with the DLD genotypes appears only in the male population in both the Caucasian series (P = 0.0009, n = 83) and the Ashkenazi Jewish subseries (P = 0.017, n = 49). The DLD genotype appears to operate independently of APOE in conferring AD risk.

The systematic annotation of the three main GPCR families in Reactome

Reactome is an open-source, freely available database of human biological pathways and processes. A major goal of our work is to provide an integrated view of cellular signalling processes that spans from ligand–receptor interactions to molecular readouts at the level of metabolic and transcriptional events. To this end, we have built the first catalogue of all human G protein-coupled receptors (GPCRs) known to bind endogenous or natural ligands. The UniProt database has records for 797 proteins classified as GPCRs and sorted into families A/1, B/2 and C/3 on the basis of amino accid sequence. To these records we have added details from the IUPHAR database and our own manual curation of relevant literature to create reactions in which 563 GPCRs bind ligands and also interact with specific G-proteins to initiate signalling cascades. We believe the remaining 234 GPCRs are true orphans. The Reactome GPCR pathway can be viewed as a detailed interactive diagram and can be exported in many forms. It provides a template for the orthology-based inference of GPCR reactions for diverse model organism species, and can be overlaid with protein–protein interaction and gene expression datasets to facilitate overrepresentation studies and other forms of pathway analysis. Database URL: http://www.reactome.org

Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters.

ABSTRACT In Drosophila melanogaster, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a “combinatorial” and “architectural” DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. Here we show the novel result that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters.