Shin Kawano

Structural basis of yeast Tim40/Mia40 as an oxidative translocator in the mitochondrial intermembrane space

The mitochondrial intermembrane space (IMS) contains many small cysteine-bearing proteins, and their passage across the outer membrane and subsequent folding require recognition and disulfide bond transfer by an oxidative translocator Tim40/Mia40 in the inner membrane facing the IMS. Here we determined the crystal structure of the core domain of yeast Mia40 (Mia40C4) as a fusion protein with maltose-binding protein at a resolution of 3 Å. The overall structure of Mia40C4 is a fruit-dish-like shape with a hydrophobic concave region, which accommodates a linker segment of the fusion protein in a helical conformation, likely mimicking a bound substrate. Replacement of the hydrophobic residues in this region resulted in growth defects and impaired assembly of a substrate protein. The Cys296-Cys298 disulfide bond is close to the hydrophobic concave region or possible substrate-binding site, so that it can mediate disulfide bond transfer to substrate proteins. These results are consistent with the growth phenotypes of Mia40 mutant cells containing Ser replacement of the conserved cysteine residues.

The ProteomeXchange consortium in 2017: supporting the cultural change in proteomics public data deposition

The ProteomeXchange (PX) Consortium of proteomics resources (http://www.proteomexchange.org) was formally started in 2011 to standardize data submission and dissemination of mass spectrometry proteomics data worldwide. We give an overview of the current consortium activities and describe the advances of the past few years. Augmenting the PX founding members (PRIDE and PeptideAtlas, including the PASSEL resource), two new members have joined the consortium: MassIVE and jPOST. ProteomeCentral remains as the common data access portal, providing the ability to search for data sets in all participating PX resources, now with enhanced data visualization components. We describe the updated submission guidelines, now expanded to include four members instead of two. As demonstrated by data submission statistics, PX is supporting a change in culture of the proteomics field: public data sharing is now an accepted standard, supported by requirements for journal submissions resulting in public data release becoming the norm. More than 4500 data sets have been submitted to the various PX resources since 2012. Human is the most represented species with approximately half of the data sets, followed by some of the main model organisms and a growing list of more than 900 diverse species. Data reprocessing activities are becoming more prominent, with both MassIVE and PeptideAtlas releasing the results of reprocessed data sets. Finally, we outline the upcoming advances for ProteomeXchange.

Structure of bacterial cellulose synthase subunit D octamer with four inner passageways

The cellulose synthesizing terminal complex consisting of subunits A, B, C, and D in Acetobacter xylinum spans the outer and inner cell membranes to synthesize and extrude glucan chains, which are assembled into subelementary fibrils and further into a ribbon. We determined the structures of subunit D (AxCeSD/AxBcsD) with both N- and C-terminal His6 tags, and in complex with cellopentaose. The structure of AxCeSD shows an exquisite cylinder shape (height: ∼65 Å, outer diameter: ∼90 Å, and inner diameter: ∼25 Å) with a right-hand twisted dimer interface on the cylinder wall, formed by octamer as a functional unit. All N termini of the octamer are positioned inside the AxCeSD cylinder and create four passageways. The location of cellopentaoses in the complex structure suggests that four glucan chains are extruded individually through their own passageway along the dimer interface in a twisted manner. The complex structure also shows that the N-terminal loop, especially residue Lys6, seems to be important for cellulose production, as confirmed by in vivo assay using mutant cells with axcesD gene disruption and N-terminus truncation. Taking all results together, a model of the bacterial terminal complex is discussed.

Prediction of glycan structures from gene expression data based on glycosyltransferase reactions

MOTIVATION Glycan chains are synthesized by a combination of several kinds of glycosyltransferases (GTs). Thus, once we know the repertoire of GTs in the genome, in the transcriptome or in the proteome, it should in principle be possible to predict the repertoire of possible glycan structures in an organism or at a specific stage of the cell. Here, we show that a repertoire of glycan structures can be predicted from the set of GTs in the transcriptome. That is, using knowledge about glycan structure characteristics, we can predict glycan structures from incomplete or noisy data such as DNA microarray data. RESULTS First, we constructed a reaction pattern library consisting of bond-formation patterns of GT reactions and investigated the co-occurrence frequencies of all reaction patterns in the glycan database. This was followed by the prediction of glycan structures using this library and a co-occurrence score. A penalty score was also implemented in the prediction method. Then we examined the performance of prediction by the leave-one-out cross validation method using individual reaction pattern profiles in the KEGG GLYCAN database as virtual expression profiles. The accuracy of prediction was 81%. Finally, we applied the prediction method to real expression data. Using expression profiles from the human carcinoma cell, glycan structures with sialic acid and sialyl Lewis X epitope were predicted, which corresponded well with experimental results.

DBTSS as an integrative platform for transcriptome, epigenome and genome sequence variation data

DBTSS (http://dbtss.hgc.jp/) was originally constructed as a collection of uniquely determined transcriptional start sites (TSSs) in humans and some other species in 2002. Since then, it has been regularly updated and in recent updates epigenetic information has also been incorporated because such information is useful for characterizing the biological relevance of these TSSs/downstream genes. In the newest release, Release 9, we further integrated public and original single nucleotide variation (SNV) data into our database. For our original data, we generated SNV data from genomic analyses of various cancer types, including 97 lung adenocarcinomas and 57 lung small cell carcinomas from Japanese patients as well as 26 cell lines of lung cancer origin. In addition, we obtained publically available SNV data from other cancer types and germline variations in total of 11,322 individuals. With these updates, users can examine the association between sequence variation pattern in clinical lung cancers with its corresponding TSS-seq, RNA-seq, ChIP-seq and BS-seq data. Consequently, DBTSS is no longer a mere storage site for TSS information but has evolved into an integrative platform of a variety of genome activity data.

BioHackathon series in 2011 and 2012: penetration of ontology and linked data in life science domains

The application of semantic technologies to the integration of biological data and the interoperability of bioinformatics analysis and visualization tools has been the common theme of a series of annual BioHackathons hosted in Japan for the past five years. Here we provide a review of the activities and outcomes from the BioHackathons held in 2011 in Kyoto and 2012 in Toyama. In order to efficiently implement semantic technologies in the life sciences, participants formed various sub-groups and worked on the following topics: Resource Description Framework (RDF) models for specific domains, text mining of the literature, ontology development, essential metadata for biological databases, platforms to enable efficient Semantic Web technology development and interoperability, and the development of applications for Semantic Web data. In this review, we briefly introduce the themes covered by these sub-groups. The observations made, conclusions drawn, and software development projects that emerged from these activities are discussed.

Structural characterization of the Acetobacter xylinum endo-β-1,4-glucanase CMCax required for cellulose biosynthesis

Previous studies have demonstrated that endoglucanase is required for cellulose biosynthesis both in bacteria and plants. However, it has yet to be elucidated how the endoglucanases function in the mechanism of cellulose biosynthesis. Here we describe the crystal structure of the cellulose biosynthesis‐related endo‐β‐1,47‐glucanase (CMCax; EC 3.2.1.4) from the cellulose‐producing Gramnegative bacterium, Acetobacter xylinum (= Gluconacetobacter xylinus), determined at 1.65‐Å resolution. CMCax falls into the glycoside hydrolase family 8 (GH‐8), and the structure showed that the overall fold of the CMCax is similar to those of other glycoside hydrolases belonging to GH‐8. Structure comparison with Clostridium thermocellum CelA, the best characterized GH‐8 endoglucanase, revealed that sugar recognition subsite +3 is completely missing in CMCax. The absence of the subsite +3 leads to significant broadness of the cleft at the cellooligosaccharide reducing‐end side. CMCax is known to be a secreted enzyme and is present in the culture medium. However, electron microscopic analysis using immunostaining clearly demonstrated that a portion of CMCax is localized to the cell surface, suggesting a link with other known membrane‐anchored endoglucanases that are required for cellulose biosynthesis. Proteins 2006.

Effects of endogenous endo-β-1,4-glucanase on cellulose biosynthesis in Acetobacter xylinum ATCC23769

Endo-beta-1,4-glucanase (CMCax; EC 3.2.1.4) from Acetobacter xylinum ATCC23769 was expressed as a 6 x His-tagged fusion protein in Escherichia coli. The optimal temperature, pH, K(m) and V(max) of the purified His-tagged CMCax toward carboxymethyl cellulose were 50 degrees C, 4.5, 20 mg/ml and 37.2 microM/min, respectively. The number of recognition residues of cello-oligosaccharide by this enzyme were five (cellopentaose) or longer, and the stereochemical course of hydrolysis was of the inverting type. Addition of a small amount (1.5 mg/l) of His-tagged CMCax into a culture medium enhanced cellulose production 1.2-fold. CMCax overproduction in A. xylinum also enhanced the yield of cellulose production. Transmission electron microscopic analysis revealed that the cellulose ribbons secreted from the CMCax overproducing strain were dispersed compared with those from the wild type strain in the same manner as by carboxymethyl cellulose addition. These results could suggest that CMCax from A. xylinum influences in cellulose ribbon assembly, which is considered to be a rate-determined process in cellulose synthesis.

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.

Introducing glycomics data into the Semantic Web

BackgroundGlycoscience is a research field focusing on complex carbohydrates (otherwise known as glycans)a, which can, for example, serve as “switches” that toggle between different functions of a glycoprotein or glycolipid. Due to the advancement of glycomics technologies that are used to characterize glycan structures, many glycomics databases are now publicly available and provide useful information for glycoscience research. However, these databases have almost no link to other life science databases.ResultsIn order to implement support for the Semantic Web most efficiently for glycomics research, the developers of major glycomics databases agreed on a minimal standard for representing glycan structure and annotation information using RDF (Resource Description Framework). Moreover, all of the participants implemented this standard prototype and generated preliminary RDF versions of their data. To test the utility of the converted data, all of the data sets were uploaded into a Virtuoso triple store, and several SPARQL queries were tested as “proofs-of-concept” to illustrate the utility of the Semantic Web in querying across databases which were originally difficult to implement.ConclusionsWe were able to successfully retrieve information by linking UniCarbKB, GlycomeDB and JCGGDB in a single SPARQL query to obtain our target information. We also tested queries linking UniProt with GlycoEpitope as well as lectin data with GlycomeDB through PDB. As a result, we have been able to link proteomics data with glycomics data through the implementation of Semantic Web technologies, allowing for more flexible queries across these domains.