Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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Cancer metabolomics: Toward the development of novel anticancer therapeutics targeting the energy metabolism in tumor microenvironment

Kami K., Tomitsuka E., Kita K., Tomita M., Soga T., Esumi H.

Japanese Journal of Clinical Chemistry 37 ( 4 ) 361 - 367 2008.12

ISSN 03705633

Role of Na<sup>+</sup>-Ca<sup>2+</sup> exchange in neonatal and adult ventricular cells

Sano H., Naito Y., Tomita M.

WMSCI 2008 - The 12th World Multi-Conference on Systemics, Cybernetics and Informatics, Jointly with the 14th International Conference on Information Systems Analysis and Synthesis, ISAS 2008 - Proc. 2 142 - 144 2008.12

The present paper expanded an integrated mathematical model, which successfully simulated developmental changes in electrophysiological activities of rodent ventricular cells, to contrive models that reflect one of the hypothesis on the role of Na+-Ca"+ exchange in neonatal ventricular cell: the Na+-Ca+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR. Our simulation with the expanded model showed that neonatal ventricular cell consumed larger amount of adenosine triphosphate (ATP) than adult ventricular cell did, owing to long relaxation time. The amount of ATP consumed per beat was reduced to the amount equivalent to adult ventricular cell, as increasing the relative current density of Na+-Ca2+ exchange (F NCx) by 4-fold, which is consistent with the observed current density in neonatal rabbit ventricular cell. To further validate the hypothesis, the relative functional activity of SR (FSR) was incrementally increased from neonatal to adult levels and subsequently increased FNCX, and found that increasing FNcx had negative impact on sarcomere relaxation velocity in ventricular cell model with large FSR. All the results thus theoretically supported the hypothesis that the Na+-Ca2+ exchange assumes a relatively greater role in neonatal cells with a morphologically sparse SR.

Signaling flux redistribution at toll-like receptor pathway junctions

Selvarajoo K., Takada Y., Gohda J., Helmy M., Akira S., Tomita M., Tsuchiya M., Inoue J.I., Matsuo K.

PLoS ONE 3 ( 10 ) e3430 2008.10

Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus, Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR) 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS) stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR) at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and lfit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxl10 and lfit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed. © 2008 Selvarajoo et al.

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Design of Peptide Inhibitors for the Importin α/β Nuclear Import Pathway by Activity-Based Profiling

Kosugi S., Hasebe M., Entani T., Takayama S., Tomita M., Yanagawa H.

Chemistry and Biology 15 ( 9 ) 940 - 949 2008.09

ISSN 10745521

Despite the current availability of selective inhibitors for the classical nuclear export pathway, no inhibitor for the classical nuclear import pathway has been developed. Here we describe the development of specific inhibitors for the importin α/β pathway using a novel method of peptide inhibitor design. An activity-based profile was created via systematic mutational analysis of a peptide template of a nuclear localization signal. An additivity-based design using the activity-based profile generated two peptides with affinities for importin α that were approximately 5 million times higher than that of the starting template sequence. The high affinity of these peptides resulted in specific inhibition of the importin α/β pathway. These peptide inhibitors provide a useful tool for studying nuclear import events. Moreover, our inhibitor design method should enable the development of potent inhibitors from a peptide seed. © 2008 Elsevier Ltd. All rights reserved.

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Stabilizing synthetic data in the DNA of living organisms

Yachie N, Ohashi Y, Tomita M

Syst Synth Biol. 2 ( 1-2 ) 19-25 - 25 2008.06

Research paper (scientific journal), Joint Work, ISSN 18725325

Data-encoding synthetic DNA, inserted into the genome of a living organism, is thought to be more robust than the current media. Because the living genome is duplicated and copied into new generations, one of the merits of using DNA material is long-term data storage within heritable media. A disadvantage of this approach is that encoded data can be unexpectedly broken by mutation, deletion, and insertion of DNA, which occurs naturally during evolution and prolongation, or laboratory experiments. For this reason, several information theory-based approaches have been developed as an error check of broken DNA data in order to achieve data durability. These approaches cannot efficiently recover badly damaged data- encoding DNA. We recently developed a DNA data-storage approach based on the multiple sequence alignment method to achieve a high level of data durability. In this paper, we overview this technology and discuss strategies for optimal application of this approach.

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GANDivAWeb: A web server for detecting early folding units ("foldons") from protein 3D structures

Laborde T., Tomita M., Krishnan A.

BMC Structural Biology 8 2008.05

Background. It has long been known that small regions of proteins tend to fold independently and are then stabilized by interactions between these distinct subunits or modules. Such units, also known as autonomous folding units (AFUs) or"foldons" play a key role in protein folding. A knowledge of such early folding units has diverse applications in protein engineering as well as in developing an understanding of the protein folding process. Such AFUs can also be used as model systems in order to study the structural organization of proteins. Results. In an earlier work, we had utilized a global network partitioning algorithm to identify modules in proteins. We had shown that these modules correlate well with AFUs. In this work, we have developed a webserver, GANDivAWeb, to identify early folding units or "foldons" in networks using the algorithm described earlier. The website has three functionalities: (a) It is able to display information on the modularity of a database of 1420 proteins used in the original work, (b) It can take as input an uploaded PDB file, identify the modules using the GANDivA algorithm and email the results back to the user and (c) It can take as input an uploaded PDB file and a results file (obtained from functionality (b)) and display the results using the embedded viewer. The results include the module decomposition of the protein, plots of cartoon representations of the protein colored by module identity and connectivity as well as contour plots of the hydrophobicity and relative accessible surface area (RASA) distributions. Conclusion. We believe that the GANDivAWeb server, will be a useful tool for scientists interested in the phenomena of protein folding as well as in protein engineering. Our tool not only provides a knowledge of the AFUs through a natural graph partitioning approach but is also able to identify residues that are critical during folding. It is our intention to use this tool to study the topological determinants of protein folding by analyzing the topological changes in proteins over the unfolding/folding pathways. © 2008 Laborde et al; licensee BioMed Central Ltd.

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Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Sugiyama,N., Nakagami,H., Mochida,K., Daudi,A., Tomita,M., Shirasu,K., and Ishihama,Y.

Mol Syst Biol. 4 193 2008.05

Research paper (scientific journal), Joint Work, ISSN 17444292

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome-wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine-specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho-residues in Arabidopsis is similar to that in humans, where over 90 tyrosine-specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants. © 2008 EMBO and Nature Publishing Group.

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Developing Bottom-fermenting Yeast Strains That Produce High SO2 Levels by Integrated Metabolome and Transcriptome Analysis

Yoshida S., Imoto J., Minato T., Oouchi R., Sugihara M., Imai T., Ishiguro T., Mizutani S., Tomita M., Soga T., Yoshimoto H.

Applied and Environmental Microbiology 74 ( 9 ) 2787 - 2796 2008.05

ISSN 00992240

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO 2 is desirable, it is complicated by the fact that undesirable H 2S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO2-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO2 and H2S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO 2, resulting in high SO2 and low H2S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO2 than the parent but produced parental levels of H2S. Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis

Masuda T., Tomita M., Ishihama Y.

Journal of Proteome Research 7 ( 2 ) 731 - 740 2008.02

ISSN 15353893

We have developed a new protocol for digesting hydrophobic proteins using trypsin with the aid of phase-transfer surfactants (PTS), such as sodium deoxycholate (SDC). SDC increases the solubility of hydrophobic proteins, enhances the activity of trypsin, and improves the accessibility to trypsin of proteins denatured during the extraction process. After digestion, SDC was successfully removed from the acidified solution containing tryptic peptides by adding a water-immiscible organic solvent, into which SDC was predominantly transferred, while the digested peptides remained in the aqueous phase. Compared with a protocol using an acid-labile surfactant, this PTS protocol increased the number of identified proteins and the recovery of hydrophobic peptides in the analysis of 400 ng of a membrane-enriched fraction of Escherichia coli. Application of the PTS protocol to 9.0 μg of a membrane-enriched pellet from human cervical cancer HeLa cells resulted in identification of a total of 1450 proteins, of which 764 (53%) were membrane proteins, by two-dimensional strong cation exchange (SCX)-C18 LC-MSMS with 5 SCX fractions. The distribution of the number of transmembrane domains in proteins identified in this study was in agreement with that in the IPI human database, suggesting that the PTS protocol can provide unbiased digestion of the membrane proteome. © 2008 American Chemical Society.

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Perspective in the evolution of human microRNAs: Copy number expansion and acquisition of target gene specialization

Watanabe Y., Tomita M., Kanai A.

Progress of Theoretical Physics Supplement ( 173 ) 219 - 228 2008

ISSN 03759687

A novel class of small, noncoding RNAs called microRNAs (miRNAs) was recently identified, and up to 20%-30% of human genes are said to be regulated by the members of this class of RNA. miRNAs regulate their target genes by imperfect binding to the 3′ untranslated region (UTR) of the gene transcript, and computational predictions of the binding between miRNAs and target gene transcripts have been performed in order to determine which genes are regulated by these RNAs. Phylogenetic analysis has also been used as a powerful tool to predict miRNA target genes. Much emphasis has therefore been placed on studying the phylogenetic conservation and evolution of this novel type of gene regulator, although there still is much that is not known. Here, we propose a hypothesis of how human miRNAs optimized their gene regulation by adjusting their transcript levels, and how they evolved through specific selection of their target genes. We analyzed the correlation between the conservation of miRNAs among species and three features: the number of transcripts, the formation of duplicates, and the number of target genes. The number of miRNA transcripts and the formation of duplicates increased as the conservation rate increased. In contrast, the number of target genes decreased as the conservation rate increased. Therefore, we propose that miRNAs gradually gain an ability to regulate specific target genes when such regulation has a positive effect on the organism. As its pool of target genes is refined, the ability of an miRNA to regulate the genes may be stabilized by an increase in the miRNA transcript number and the formation of duplicates.

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Nuclear export signal consensus sequences defined using a localization-based yeast selection system

Kosugi, S., Hasebe, M., Tomita, M. and Yanagawa, H.

Traffic 9 ( 12 ) 2053 - 2062 2008

Joint Work, ISSN 13989219

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export-dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Δtys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1-dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments. © 2008 The Authors Journal compilation © 2008 Blackwell Munksgaard.

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Computational Genome Analysis Using The G-language System

Arakawa,K., Suzuki,H. and Tomita,M.

Genges,Genomes and Genomics 2 1-13 2008

Research paper (scientific journal), Joint Work

Visualizing Conplex Omics Information-Scientific Visualization for Genomics and Systems Biology

Arakawa, K., Yachie, N. and Tomita M.

BIOforum Europe 6 27-29 2008

Research paper (scientific journal), Joint Work

Junction ribonuclease activity specified in RNases HII/2

Ohtani, N., Tomita, M. and Itaya, M.

FEBS J. 275 ( 21 ) 5444-55 - 5455 2008

Research paper (scientific journal), Joint Work, ISSN 1742464X

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5′ terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA//RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction. © 2008 The Authors.

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Versatile architecture of a bacterial aconitase B and its catalytic performance in the sequential reaction coupled with isocitrate dehydrogenase

Tsuchiya D., Shimizu N., and Tomita M.

BBA - Proteins and Proteomics. 1784 ( 11 ) 1847-56. - 1856 2008

Research paper (scientific journal), Joint Work, ISSN 15709639

Aconitase B (AcnB) and isocitrate dehydrogenase (ICDH) catalyze the sequential reaction in the Krebs cycle. Since each enzyme was characterized as an independent protein in a diluted condition, the catalytic performance within the cellular metabolism remains unclear. In particular, high macromolecular concentration in the cytosol promotes weak interactions, which affects structure and function of the proteins. We found that the two bacterial enzymes exhibit variable catalytic performance of the sequential reaction, depending on the oligomerization state. The small-angle solution X-ray scattering and the chemical crosslinking analyses revealed that not only the two enzymes but also the fusion protein of the two enzymes assume homodimers in solution. Interestingly, the fusion protein maintains the homodimeric architecture of ICDH, but not AcnB. Instead, one of the two monomeric AcnB regions associates with the homodimeric ICDH region. The fusion protein displayed different catalytic performance of the sequential reaction from that observed in the mixture of the AcnB and ICDH proteins in an equimolar ratio. Connecting the two proteins by a flexible linker yielded a locally high concentration to promote the weak protein-protein interaction. The versatile architecture of AcnB may alter the metabolic process involving the Krebs cycle. © 2008 Elsevier B.V. All rights reserved.

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Aligning LC Peaks by Converting Gradient Retention Times to Retention Index of Peptides in Proteomic Experiments

Shinoda, K., Tomita, M., Ishihama, Y.

Bioinformatics. 24 ( 14 ) 1590-5 - 1595 2008

Research paper (scientific journal), Joint Work, ISSN 13674803

Motivation: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. Results: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k0 (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (tg) to log k0. Using the optimized function, tg values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale. © The Author 2008. Published by Oxford University Press. All rights reserved.

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Restriction on conjugational transfer of pLS20 in Bacillus subtilis 168

Ohtani, N., Sato, M., Tomita, M. and Itaya, M ,

Biosci. Biotechnol. Biochem. , 72 ( 9 ) 2472-5 - 2475 2008

Research paper (scientific journal), Joint Work, ISSN 09168451

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Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex

Ohtani, N., Tomita, M. and Itaya, M.

Biochem. J. 412 ( 3 ) 517-26 - 526 2008

Research paper (scientific journal), Joint Work, ISSN 02646021

The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA-DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5′ side of the last ribonucleotide at the RNA-DNA junction. In fact, the TTHA0198 protein prefers the RNA-DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA-DNA junction of the RNA-DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5′ terminus of the RNA-DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA-DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity. © The Authors.

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GeNESiS: Gene Network Evolution SImulation Software

Anton Kratz, Masaru Tomita and Arun Krishnan

BMC Bioinformatics 9 541 2008

Research paper (scientific journal), Joint Work

Background: There has been a lot of interest in recent years focusing on the modeling and simulation of Gene Regulatory Networks (GRNs). However, the evolutionary mechanisms that give rise to GRNs in the first place are still largely unknown. In an earlier work, we developed a framework to analyze the effect of objective functions, input types and starting populations on the evolution of GRNs with a specific emphasis on the robustness of evolved GRNs. Results: In this work, we present a parallel software package, GeNESiS for the modeling and simulation of the evolution of gene regulatory networks (GRNs). The software models the process of gene regulation through a combination of finite-state and stochastic models. The evolution of GRNs is then simulated by means of a genetic algorithm with the network connections represented as binary strings. The software allows users to simulate the evolution under varying selective pressures and starting conditions. We believe that the software provides a way for researchers to understand the evolutionary behavior of populations of GRNs. Conclusion: We believe that GeNESiS will serve as a useful tool for scientists interested in understanding the evolution of gene regulatory networks under a range of different conditions and selective pressures. Such modeling efforts can lead to a greater understanding of the network characteristics of GRNs. © 2008 Kratz et al; licensee BioMed Central Ltd.

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Successive and Selective Release of Phosphorylated Peptides Captured by hydroxy Acid-Modified Metal Oxide Chromatography

Kyono, Y.,Sugiyama, N., Imami, K., Tomita, M. and Ishihama, Y.

Journal of Proteome Research 7 ( 10 ) 4585-4593 - 4593 2008

Research paper (scientific journal), Joint Work, ISSN 15353893

We developed a novel approach to enlarge phosphoproteome coverage using successive elution of phosphopeptides with various buffers in series from a single microcolumn packed with hydroxy acid-modified metal oxides, such as titania and zirconia. Elution conditions were investigated to maximize the recovery of phosphopeptides from three standard phosphoproteins. Secondary amines, such as piperidine and pyrrolidine, provided better efficiency than the conventional conditions using ammonium hydroxide and phosphate buffers. Furthermore, elution with these secondary amines provided unique phosphopeptides that were not eluted under the conventional conditions in the analysis of HeLa cell lysates. On the basis of these results, we fractionated phosphopeptides captured by a single metal oxide microcolumn using successive elution with 5% ammonium hydroxide solution, 5% piperidine solution and 5% pyrrolidine solution in series. We identified 1803 nonredun- dant phosphopeptides from 100 μg of HeLa cells, which represented a 1.6-fold increase in phosphopeptide number and a 1.9-fold increase in total peak area of phosphopeptides in comparison with the results obtained under the conventional conditions. Since this approach is applicable to any single tip-based protocol without coupling with other enrichment methods, this simple strategy can be easily incorporated as an option into existing protocols for phosphopeptide enrichment, and would be suitable for high-throughput analysis in a parallel format. © 2008 American Chemical Society.

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