Details of a Researcher - Tomita, Masaru

Papers - Tomita, Masaru

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G-language genome analysis environment with REST and SOAP web service interfaces

Arakawa, K., Kido, N., Oshita, K. and Tomita, M.

Nucleic Acids Res. (Nucleic Acids Research) 38 ( SUPPL. 2 ) W700–W705 - 5 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 03051048

G-language genome analysis environment (G-language GAE) contains more than 100 programs that focus on the analysis of bacterial genomes, including programs for the identification of binding sites by means of information theory, analysis of nucleotide composition bias and the distribution of particular oligonucleotides, calculation of codon bias and prediction of expression levels, and visualization of genomic information. We have provided a collection of web services for these programs by utilizing REST and SOAP technologies. The REST interface, available at http://rest.glanguage. org/, provides access to all 145 functions of the G-language GAE. These functions can be accessed from other online resources. All analysis functions are represented by unique universal resource identifiers. Users can access the functions directly via the corresponding universe resource locators (URLs), and biological web sites can readily embed the functions by simply linking to these URLs. The SOAP services, available at http://www.g-language.org/wiki/soap/, provide language-independent programmatic access to 77 analysis programs. The SOAP service Web Services Definition Language file can be readily loaded into graphical clients such as the Taverna workbench to integrate the programs with other services and workflows. © The Author(s) 2010. Published by Oxford University Press.

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Collective Dynamics of Specific Gene Ensembles Crucial for Neutrophil Differentiation: The Existence of Genome Vehicles Revealed

Tsuchiya, M., Piras, V., Giuliani, A., Tomita, M. and Selvarajoo

PLoS One (PLoS ONE) 5 ( 8 ) e12116 2010

Research paper (scientific journal), Joint Work, Accepted

Cell fate decision remarkably generates specific cell differentiation path among the multiple possibilities that can arise through the complex interplay of high-dimensional genome activities. The coordinated action of thousands of genes to switch cell fate decision has indicated the existence of stable attractors guiding the process. However, origins of the intracellular mechanisms that create "cellular attractor" still remain unknown. Here, we examined the collective behavior of genome-wide expressions for neutrophil differentiation through two different stimuli, dimethyl sulfoxide (DMSO) and alltrans- retinoic acid (atRA). To overcome the difficulties of dealing with single gene expression noises, we grouped genes into ensembles and analyzed their expression dynamics in correlation space defined by Pearson correlation and mutual information. The standard deviation of correlation distributions of gene ensembles reduces when the ensemble size is increased following the inverse square root law, for both ensembles chosen randomly from whole genome and ranked according to expression variances across time. Choosing the ensemble size of 200 genes, we show the two probability distributions of correlations of randomly selected genes for atRA and DMSO responses overlapped after 48 hours, defining the neutrophil attractor. Next, tracking the ranked ensembles' trajectories, we noticed that only certain, not all, fall into the attractor in a fractal-like manner. The removal of these genome elements from the whole genomes, for both atRA and DMSO responses, destroys the attractor providing evidence for the existence of specific genome elements (named "genome vehicle") responsible for the neutrophil attractor. Notably, within the genome vehicles, genes with low or moderate expression changes, which are often considered noisy and insignificant, are essential components for the creation of the neutrophil attractor. Further investigations along with our findings might provide a comprehensive mechanistic view of cell fate decision. © 2010 Tsuchiya et al.

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Large-scale tRNA intron transposition in the archaeal order Thermoproteales represents a novel mechanism of intron gain

Fujishima, K., Sugahara, J., Tomita, M. and Kanai, A.

Mol. Biol. Evol. (Molecular Biology and Evolution) 27 ( 10 ) 2233–2243 - 2243 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 07374038

Recently, diverse arrangements of transfer RNA (tRNA) genes have been found in the domain Archaea, in which the tRNA is interrupted by a maximum of three introns or is even fragmented into two or three genes. Whereas most of the eukaryotic tRNA introns are inserted strictly at the canonical nucleotide position (37/38), archaeal intron-containing tRNAs have a wide diversity of small tRNA introns, which differ in their numbers and locations. This feature is especially pronounced in the archaeal order Thermoproteales. In this study, we performed a comprehensive sequence comparison of 286 tRNA introns and their genes in seven Thermoproteales species to clarify how these introns have emerged and diversified during tRNA gene evolution. We identified 46 intron groups containing sets of highly similar sequences (>70%) and showed that 16 of them contain sequences from evolutionarily distinct tRNA genes. The phylogeny of these 16 intron groups indicates that transposition events have occurred at least seven times throughout the evolution of Thermoproteales. These findings suggest that frequent intron transposition occurs among the tRNA genes of Thermoproteales. Further computational analysis revealed limited insertion positions and corresponding amino acid types of tRNA genes. This has arisen because the bulge-helix-bulge splicing motif is required at the newly transposed position if the pre-tRNA is to be correctly processed. These results clearly demonstrate a newly identified mechanism that facilitates the late gain of short introns at various noncanonical positions in archaeal tRNAs. © 2010 The Author.

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A Metabolic Model of Human Erythrocytes: Practical Application of the E-Cell Simulation Environment

Yachie, A. K., Nishino, T., Shimo, H., Suematsu, M., and Tomita, M.

J. Biomed. Biotechnol. (Journal of Biomedicine and Biotechnology) 2010 642420 2010

Research paper (scientific journal), Joint Work, Accepted, ISSN 11107243

The human red blood cell (RBC) has long been used for modeling of complex biological networks, for elucidation of a wide variety of dynamic phenomena, and for understanding the fundamental topology of metabolic pathways. Here, we introduce our recent work on an RBC metabolic model using the E-Cell Simulation Environment. The model is sufficiently detailed to predict the temporal hypoxic response of each metabolite and, at the same time, successfully integrates modulation of metabolism and of the oxygen transporting capacity of hemoglobin. The model includes the mechanisms of RBC maintenance as a single cell system and the functioning of RBCs as components of a higher order system. Modeling of RBC metabolism is now approaching a fully mature stage of realistic predictions at the molecular level and will be useful for predicting conditions in biotechnological applications such as long-term cold storage of RBCs. Copyright © 2010 Ayako Yachie-Kinoshita et al.

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Unbiased quantitation of Escherichia coli membrane proteome using phase-transfer surfactants

Masuda,T., Saito, N., Tomita, M. and Ishihama, M.

Mol Cell Proteomics. (Molecular and Cellular Proteomics) 8 ( 12 ) 2770-2777 - 2777 2009.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of DNA-dependent protein kinase as a cofactor for the forkhead transcription factor FoxA2

Nock A., Ascano J.M., Jones T., Barrero M.J., Sugiyama N., Tomita M., Ishihama Y., Malik S.

Journal of Biological Chemistry (Journal of Biological Chemistry) 284 ( 30 ) 19915 - 19926 2009.07

ISSN 00219258

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2.Weshow that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Degradation of ppGpp by nudix pyrophosphatase modulates the transition of growth phase in the bacterium Thermus thermophilus

Ooga T., Ohashi Y., Kuramitsu S., Koyama Y., Tomita M., Soga T., Masui R.

Journal of Biological Chemistry (Journal of Biological Chemistry) 284 ( 23 ) 15549 - 15556 2009.06

ISSN 00219258

A major bacterial alarmone, guanosine 3′,5′-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3′,5′-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Benzodiazepine and antidepressant use in elderly patients with anxiety disorders: a survey of 796 outpatients in Japan.

Uchida H, Suzuki T, Mamo DC, Mulsant BH, Kikuchi T, Takeuchi H, Tomita M, Watanabe K, Yagi G, Kashima H

Journal of anxiety disorders 23 ( 4 ) 477 - 81 2009.05

ISSN 0887-6185

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Characterization of a heat-stable enzyme possessing GTP-dependent RNA ligase activity from a hyperthermophilic archaeon, Pyrococcus furiosus

Kanai A., Sato A., Fukuda Y., Okada K., Matsuda T., Sakamoto T., Muto Y., Yokoyama S., Kawai G., Tomita M.

RNA 15 ( 3 ) 420 - 431 2009.03

ISSN 13558382

Using an expression protein library of a hyperthermophilic archaeon, Pyrococcus furiosus, we identified a gene (PF0027) that encodes a protein with heat-stable cyclic nucleotide phosphodiesterase (CPDase) activity. The PF0027 gene encoded a 21-kDa protein and an amino acid sequence that showed ∼27% identity to that of the 2′-5′ tRNA ligase protein, ligT (20 kDa), from Escherichia coli. We found that the purified PF0027 protein possessed GTP-dependent RNA ligase activity and that synthetic tRNA halves bearing 2′,3′-cyclic phosphate and 5′-OH termini were substrates for the ligation reaction in vitro. GTP hydrolysis was not required for the reaction, and GTPγS enhanced the tRNA ligation activity of PF0027 protein, suggesting that the ligation step is regulated by a novel mechanism. In comparison to the strong CPDase activity of the PF0027 protein, the RNA ligase activity itself was quite weak, and the ligation product was unstable during in vitro reaction. Finally, we used NMR to determine the solution structure of the PF0027 protein and discuss the implications of our results in understanding the role of the PF0027 protein. Copyright © 2009 RNA Society.

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Six classes of nuclear localization signals specific to different binding grooves of importin α

Kosugi S., Hasebe M., Matsumura N., Takashima H., Miyamoto-Sato E., Tomita M., Yanagawa H.

Journal of Biological Chemistry 284 ( 1 ) 478 - 485 2009.01

ISSN 00219258

The importin α/β pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin α. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin α and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin α, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin α-dependent NLSs provide useful information for identifying NLSs. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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emPAI Calc -- for the estimation of protein abundance from largescale identification data by liquid chromatography-tandem massspectrometry

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

Bioinformatics (Bioinformatics) 26 ( 4 ) 576-577 - 577 2009

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

Summary: emPAI Calc is an open-source web application for the estimation of protein abundance. It uses the correlation between the number of identified peptides and protein abundance in mass spectrometry-based proteomic experiments. The program is the first implementation of our previously reported emPAI algorithm; it calculates the emPAI from the protein identification results obtained by database search engines such as Mascot.™ Availability: http://empai.iab.keio.ac.jp/; http://empai.iab.keio.ac.jp/supplement.php Source codes are available under Mozilla Public License. Contact: y-ishi@ttck.keio.ac.jp Supplementary Information: Supplementary data are available at Bioinformatics online. © The Author 2009. Published by Oxford University Press.

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Cooperativity of two active sites in bacterial homodimeric aconitases

Tsuchiya D, Shimizu N, Tomita M

Biochem Biophys Res Commun 379 ( 2 ) 485-488 - 488 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

Aconitase catalyzes a reversible isomerization of citrate into isocitrate in the Krebs cycle. Escherichia coli possesses two kinds of aconitases, aconitase A (AcnA) and B (AcnB), whose structural organizations are different. We analyzed the structural state of AcnA by the chemical crosslinking and small-angle X-ray scattering. The protein adopts a homodimer in solution, as AcnB does. The catalytic assay of the two aconitases revealed that the isomerization of isocitrate displayed a negative cooperativity of the two active sites within each homodimer. On the other hand, insignificant cooperativity was observed in the reverse reaction. Therefore, the homodimerization of AcnAB yields a substrate-dependent cooperative effect. In conjunction with the dissociable homodimer of AcnB, the catalytic property could affect the intracellular metabolic process involving the Krebs cycle. © 2008 Elsevier Inc. All rights reserved.

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Predicting novel features of toll-like receptor 3 signaling in macrophages

Helmy, M., Gohda, J, Inoue, J., Tomita, M. Tsuchiya, M. and Selvarajoo, K.

PLoS ONE. 4 ( 11 ) e4661 2009

Research paper (scientific journal), Joint Work, Accepted

The Toll-like receptor (TLR) 3 plays a critical role in mammalian innate immune response against viral attacks by recognizing double-stranded RNA (dsRNA) or its synthetic analog polyinosinic-polycytidylic acid (poly (I:C)). This leads to the activation of MAP kinases and NF-κB which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. To understand the complex interplay of the various intracellular signaling molecules in the regulation of NF-κB and MAP kinases, we developed a computational TLR3 model based upon perturbation-response approach. We curated literature and databases to determine the TLR3 signaling topology specifically for murine macrophages. For initial model creation, we used wildtype temporal activation profiles of MAP kinases and NF-κB and, for model testing, used TRAF6 KO and TRADD KO data. From dynamic simulations we predict i) the existence of missing intermediary steps between extracellular poly (I:C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation. Our work shows activation dynamics of signaling molecules can be used in conjunction with perturbation-response models to decipher novel signaling features of complicated immune pathways. © 2009 Helmy et al.

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Tri-split tRNA is a transfer RNA made from 3 transcripts that provides insight into the evolution of fragmented tRNAs in archaea

Fujishima, K., Sugahara, J., Kikuta, K. Hirano, R.,Sato, A., Tomita, M. and Kanai

Proc Natl Acad Sci U S A. 106 ( 8 ) 2683-7 - 2687 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00278424

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNAGly's with synonymous codons are created from 1 constitutive 3′ half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a "tri-split tRNA." Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in ifntron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs. © 2009 by The National Academy of Sciences of the USA.

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Genome Projector: zoomable genome map with multiple views

Arakawa, K., Tamaki, S., Kono, N., Kido, N., Ikegami, K., Ogawa, R.and Tomita, M

BMC Bioinformatics 10 31 2009

Research paper (scientific journal), Joint Work, Accepted

BACKGROUND: Molecular biology data exist on diverse scales, from the level of molecules to -omics. At the same time, the data at each scale can be categorised into multiple layers, such as the genome, transcriptome, proteome, metabolome, and biochemical pathways. Due to the highly multi-layer and multi-dimensional nature of biological information, software interfaces for database browsing should provide an intuitive interface that allows for rapid migration across different views and scales. The Zoomable User Interface (ZUI) and tabbed browsing have proven successful for this purpose in other areas, especially to navigate the vast information in the World Wide Web. RESULTS: This paper presents Genome Projector, a Web-based gateway for genomics information with a zoomable user interface using Google Maps API, equipped with four seamlessly accessible and searchable views: a circular genome map, a traditional genome map, a biochemical pathways map, and a DNA walk map. The Web application for 320 bacterial genomes is available at http://www.g-language.org/GenomeProjector/. All data and software including the source code, documentations, and development API are freely available under the GNU General Public License. Zoomable maps can be easily created from any image file using the development API, and an online data mapping service for Genome Projector is also available at our Web site. CONCLUSION: Genome Projector is an intuitive Web application for browsing genomics information, implemented with a zoomable user interface and tabbed browsing utilising Google Maps API and Asynchronous JavaScript and XML (AJAX) technology.

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Local and Global responses in complex gene regulation networks

Tsuchiya, M., Selvarajoo, K., Piras, V., Tomita, M. and Giuliani, A.

Physica A 388 ( 8 ) 1738-1746 - 1746 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 03784371

An exacerbated sensitivity to apparently minor stimuli and a general resilience of the entire system stay together side-by-side in biological systems. This apparent paradox can be explained by the consideration of biological systems as very strongly interconnected network systems. Some nodes of these networks, thanks to their peculiar location in the network architecture, are responsible for the sensitivity aspects, while the large degree of interconnection is at the basis of the resilience properties of the system. One relevant feature of the high degree of connectivity of gene regulation networks is the emergence of collective ordered phenomena influencing the entire genome and not only a specific portion of transcripts. The great majority of existing gene regulation models give the impression of purely local 'hard-wired' mechanisms disregarding the emergence of global ordered behavior encompassing thousands of genes while the general, genome wide, aspects are less known. Here we address, on a data analysis perspective, the discrimination between local and global scale regulations, this goal was achieved by means of the examination of two biological systems: innate immune response in macrophages and oscillating growth dynamics in yeast. Our aim was to reconcile the 'hard-wired' local view of gene regulation with a global continuous and scalable one borrowed from statistical physics. This reconciliation is based on the network paradigm in which the local 'hard-wired' activities correspond to the activation of specific crucial nodes in the regulation network, while the scalable continuous responses can be equated to the collective oscillations of the network after a perturbation. © 2008 Elsevier B.V. All rights reserved.

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Can complex cellular processes be governed by simple linear rules?

Selvarajoo, K., Tomita, M. and Tsuchiya, M.

J Bioinform Comput Biol. (Journal of Bioinformatics and Computational Biology) 7 ( 1 ) 243-268 - 268 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 02197200

Complex living systems have shown remarkably well-orchestrated, self-organized, robust, and stable behavior under a wide range of perturbations. However, despite the recent generation of high-throughput experimental datasets, basic cellular processes such as division, differentiation, and apoptosis still remain elusive. One of the key reasons is the lack of understanding of the governing principles of complex living systems. Here, we have reviewed the success of perturbation-response approaches, where without the requirement of detailed in vivo physiological parameters, the analysis of temporal concentration or activation response unravels biological network features such as causal relationships of reactant species, regulatory motifs, etc. Our review shows that simple linear rules govern the response behavior of biological networks in an ensemble of cells. It is daunting to know why such simplicity could hold in a complex heterogeneous environment. Provided physical reasons can be explained for these phenomena, major advancement in the understanding of basic cellular processes could be achieved. © 2009 Imperial College Press.

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In silico analysis of phosphoproteome data suggests a rich-get-richer process of phosphosite accumulation over evolution

Yachie, N., Saito, R., Sugahara, J., Tomita, M. and Ishihama, Y

Mol Cell Proteomics. (Molecular and Cellular Proteomics) 8 ( 5 ) 1061-71 - 1071 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 15359476

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chemical Cleavage-Assisted Tryptic Digestion for Membrane Proteome Analysis

Iwasaki, M., Masuda, T., Tomita, M. and Ishihama, Y.

J. Proteome Re (Journal of Proteome Research) 8 ( 6 ) 1061-71 - 3175 2009

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

We have developed a simple and unbiased method for membrane proteome analysis using cyanocysteine-mediated cleavage in combination with trypsin digestion. In our previous study, application of the trypsin-based phase-transfer surfactants (PTS) protocol for membrane proteome analysis provided a substantial improvement in the identification of the membrane proteome, but the task remains challenging, because trypsin often generates peptides larger than the observable m/z range. Here, we predict computationally that the combination of Cys cleavage with tryptic digestion would be more effective than trypsin digestion alone for membrane proteome analysis. To validate this prediction, we applied a combined Cys cleavage-trypsin approach to 14 μg of Escherichia coli membrane-enriched pellet. By using two-dimensional LC-MS/MS, we identified a total of 1530 proteins, of which 667 were membrane proteins. This represents a 10% increase over the number identified with the PTS protocol using optimized trypsin-based digestion in our previous study [Masuda, T., et al. J. Proteome Res. 2008, 7 (2), 731-40]. The coverage of the E. coli membrane proteome was approximately 40%, ranging from 37% to 42% in various subcategories. Further, the distribution of the number of transmembrane domains per protein was unbiased compared with that in the GenoBase database. These results indicate that the combination Cys chemical cleavage-assisted trypsin digestion protocol will be a powerful tool for membrane proteome analysis. © 2009 American Chemical Society.

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Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Saito, N., Robert, M., Kochi, H., Matsuo, G., Kakazu, Y., Soga, T. and Tomita, M.

J. Biol. Chem. (Journal of Biological Chemistry) 284 ( 24 ) 16442-51 - 16451 2009

Research paper (scientific journal), Joint Work, Accepted, ISSN 00219258

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU Protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to γ-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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