Akiyasu C. Yoshizawa

KAAS: an automatic genome annotation and pathway reconstruction server

The number of complete and draft genomes is rapidly growing in recent years, and it has become increasingly important to automate the identification of functional properties and biological roles of genes in these genomes. In the KEGG database, genes in complete genomes are annotated with the KEGG orthology (KO) identifiers, or the K numbers, based on the best hit information using Smith-Waterman scores as well as by the manual curation. Each K number represents an ortholog group of genes, and it is directly linked to an object in the KEGG pathway map or the BRITE functional hierarchy. Here, we have developed a web-based server called KAAS (KEGG Automatic Annotation Server: http://www.genome.jp/kegg/kaas/) i.e. an implementation of a rapid method to automatically assign K numbers to genes in the genome, enabling reconstruction of KEGG pathways and BRITE hierarchies. The method is based on sequence similarities, bi-directional best hit information and some heuristics, and has achieved a high degree of accuracy when compared with the manually curated KEGG GENES database.The number of complete and draft genomes is rapidly growing in recent years, and it has become increasingly important to automate the identification of functional properties and biological roles of genes in these genomes. In the KEGG database, genes in complete genomes are annotated with the KEGG orthology (KO) identifiers, or the K numbers, based on the best hit information using Smith–Waterman scores as well as by the manual curation. Each K number represents an ortholog group of genes, and it is directly linked to an object in the KEGG pathway map or the BRITE functional hierarchy. Here, we have developed a web-based server called KAAS (KEGG Automatic Annotation Server: http://www.genome.jp/kegg/kaas/) i.e. an implementation of a rapid method to automatically assign K numbers to genes in the genome, enabling reconstruction of KEGG pathways and BRITE hierarchies. The method is based on sequence similarities, bi-directional best hit information and some heuristics, and has achieved a high degree of accuracy when compared with the manually curated KEGG GENES database.

KEGG OC: a large-scale automatic construction of taxonomy-based ortholog clusters

The identification of orthologous genes in an increasing number of fully sequenced genomes is a challenging issue in recent genome science. Here we present KEGG OC (http://www.genome.jp/tools/oc/), a novel database of ortholog clusters (OCs). The current version of KEGG OC contains 1 176 030 OCs, obtained by clustering 8 357 175 genes in 2112 complete genomes (153 eukaryotes, 1830 bacteria and 129 archaea). The OCs were constructed by applying the quasi-clique-based clustering method to all possible protein coding genes in all complete genomes, based on their amino acid sequence similarities. It is computationally efficient to calculate OCs, which enables to regularly update the contents. KEGG OC has the following two features: (i) It consists of all complete genomes of a wide variety of organisms from three domains of life, and the number of organisms is the largest among the existing databases; and (ii) It is compatible with the KEGG database by sharing the same sets of genes and identifiers, which leads to seamless integration of OCs with useful components in KEGG such as biological pathways, pathway modules, functional hierarchy, diseases and drugs. The KEGG OC resources are accessible via OC Viewer that provides an interactive visualization of OCs at different taxonomic levels.

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic.

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane‐related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum‐related transport pathways throughout eukaryotic evolution.

jPOSTrepo: an international standard data repository for proteomes

Major advancements have recently been made in mass spectrometry-based proteomics, yielding an increasing number of datasets from various proteomics projects worldwide. In order to facilitate the sharing and reuse of promising datasets, it is important to construct appropriate, high-quality public data repositories. jPOSTrepo (https://repository.jpostdb.org/) has successfully implemented several unique features, including high-speed file uploading, flexible file management and easy-to-use interfaces. This repository has been launched as a public repository containing various proteomic datasets and is available for researchers worldwide. In addition, our repository has joined the ProteomeXchange consortium, which includes the most popular public repositories such as PRIDE in Europe for MS/MS datasets and PASSEL for SRM datasets in the USA. Later MassIVE was introduced in the USA and accepted into the ProteomeXchange, as was our repository in July 2016, providing important datasets from Asia/Oceania. Accordingly, this repository thus contributes to a global alliance to share and store all datasets from a wide variety of proteomics experiments. Thus, the repository is expected to become a major repository, particularly for data collected in the Asia/Oceania region.

Comprehensive analysis of glycosyltransferases in eukaryotic genomes for structural and functional characterization of glycans.

Glycosyltransferases comprise highly divergent groups of enzymes, which play a central role in the synthesis of complex glycans. Because the repertoire of glycosyltransferases in the genome determines the range of synthesizable glycans, and because the increasing amount of genome sequence data is now available, it is essential to examine these enzymes across organisms to explore possible structures and functions of the glycoconjugates. In this study, we systematically investigated 36 eukaryotic genomes and obtained 3426 glycosyltransferase homologs for biosynthesis of major glycans, classified into 53 families based on sequence similarity. The families were further grouped into six functional categories based on the biosynthetic pathways, which revealed characteristic patterns among organism groups in the degree of conservation and in the number of paralogs. The results also revealed a strong correlation between the number of glycosyltransferases and the number of coding genes in each genome. We then predicted the ability to synthesize major glycan structures including N-glycan precursors and GPI-anchors in each organism from the combination of the glycosyltransferase families. This indicates that not only parasitic protists but also some algae are likely to synthesize smaller structures than the structures known to be conserved among a wide range of eukaryotes. Finally we discuss the functions of two large families, sialyltransferases and beta 4-glycosyltransferases, by performing finer classifications into subfamilies. Our findings suggest that universality and diversity of glycans originate from two types of evolution of glycosyltransferase families, namely conserved families with few paralogs and diverged families with many paralogs.

CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.

ODB: a database for operon organizations, 2011 update

ODB (Operon DataBase) aims to collect data of all known and conserved operons in completely sequenced genomes. Three newly updated features of this database have been added as follows: (i) Data from included operons were updated. The genome-wide analysis of transcription and transcriptional units has become popular recently and ODB successfully integrates these high-throughput operon data, including genome-wide transcriptional units of five prokaryotes and two eukaryotes. The current version of our database contains information from about 10 000 known operons in more than 50 genomes, and more than 400 000 conserved operons obtained from more than 1000 bacterial genomes. (ii) ODB proposes the idea of reference operons as a new operon prediction tool. A reference operon, a set of possible orthologous genes that organize operons, is defined by clustering all known operons. A large number of known operons, including the recently added genome-wide analysis of operons, allowed us to define more reliable reference operons. (iii) ODB also provides new graphical interfaces. One is for comparative analyses of operon structures in multiple genomes. The other is for visualization of possible operons in multiple genomes obtained from the reference operons. The 2011 updated version of ODB is now available at http://operondb.jp/.

An age-related homeostasis mechanism is essential for spontaneous amelioration of hemophilia B Leyden

Regulation of age-related changes in gene expression underlies many diseases. We previously discovered the first puberty-onset gene switch, the age-related stability element (ASE)/age-related increase element (AIE)-mediated genetic mechanism for age-related gene regulation. Here, we report that this mechanism underlies the mysterious puberty-onset amelioration of abnormal bleeding seen in hemophilia B Leyden. Transgenic mice robustly mimicking the Leyden phenotype were constructed. Analysis of these animals indicated that ASE plays a central role in the puberty-onset amelioration of the disease. Human factor IX expression in these animals was reproducibly nullified by hypophysectomy, but nearly fully restored by administration of growth hormone, being consistent with the observed sex-independent recovery of factor IX expression. Ets1 was identified as the specific liver nuclear protein binding only to the functional ASE, G/CAGGAAG, and not to other Ets consensus elements. This study demonstrates the clinical relevance of the first discovered puberty-onset gene switch, the ASE/AIE-mediated regulatory mechanism.

Integrated proteomics identified novel activation of dynein IC2-GR-COX-1 signaling in neurofibromatosis type I (NF1) disease model cells.

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, and though its loss is implicated in the neuronal abnormality of NF1 patients, its precise cellular function remains unclear. To study the molecular mechanism of NF1 pathogenesis, we prepared NF1 gene knockdown (KD) PC12 cells, as a NF1 disease model, and analyzed their molecular (gene and protein) expression profiles with a unique integrated proteomics approach, comprising iTRAQ, 2D-DIGE, and DNA microarrays, using an integrated protein and gene expression analysis chart (iPEACH). In NF1-KD PC12 cells showing abnormal neuronal differentiation after NGF treatment, of 3198 molecules quantitatively identified and listed in iPEACH, 97 molecules continuously up- or down-regulated over time were extracted. Pathway and network analysis further revealed overrepresentation of calcium signaling and transcriptional regulation by glucocorticoid receptor (GR) in the up-regulated protein set, whereas nerve system development was overrepresented in the down-regulated protein set. The novel up-regulated network we discovered, “dynein IC2-GR-COX-1 signaling,” was then examined in NF1-KD cells. Validation studies confirmed that NF1 knockdown induces altered splicing and phosphorylation patterns of dynein IC2 isomers, up-regulation and accumulation of nuclear GR, and increased COX-1 expression in NGF-treated cells. Moreover, the neurite retraction phenotype observed in NF1-KD cells was significantly recovered by knockdown of the dynein IC2-C isoform and COX-1. In addition, dynein IC2 siRNA significantly inhibited nuclear translocation and accumulation of GR and up-regulation of COX-1 expression. These results suggest that dynein IC2 up-regulates GR nuclear translocation and accumulation, and subsequently causes increased COX-1 expression, in this NF1 disease model. Our integrated proteomics strategy, which combines multiple approaches, demonstrates that NF1-related neural abnormalities are, in part, caused by up-regulation of dynein IC2-GR-COX-1 signaling, which may be a novel therapeutic target for NF1.

Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: Implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism

Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate. Several mutations in the human T-protein gene are known to cause nonketotic hyperglycinemia. Here, we report the crystal structure of Escherichia coli T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction. The structure of the complex shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Invariant Arg292 of the T-protein is essential for complex assembly. The structure also provides novel insights in understanding the disease-causing mutations, in addition to the disease-related impairment in the cofactor-enzyme interactions reported previously. Furthermore, structural and mutational analyses suggest that the reversible transfer of the methylene group between the lipoate and tetrahydrofolate should proceed through the electron relay-assisted iminium intermediate formation.