記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09168451.2015.1058699

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure-function relationships of this multifaceted primordial ribozyme.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1006/jsbi.1998.4053

記述言語：その他   掲載種別：研究論文（学術雑誌）   出版者・発行元：Aging Cell  

Mechanical perturbation triggers activation of resident myogenic stem cells to enter the cell cycle through a cascade of events including hepatocyte growth factor (HGF) release from its extracellular tethering and the subsequent presentation to signaling-receptor c-met. Here, we show that with aging, extracellular HGF undergoes tyrosine-residue (Y) nitration and loses c-met binding, thereby disturbing muscle homeostasis. Biochemical studies demonstrated that nitration/dysfunction is specific to HGF among other major growth factors and is characterized by its locations at Y198 and Y250 in c-met-binding domains. Direct-immunofluorescence microscopy of lower hind limb muscles from three age groups of rat, provided direct in vivo evidence for age-related increases in nitration of ECM-bound HGF, preferentially stained for anti-nitrated Y198 and Y250-HGF mAbs (raised in-house) in fast IIa and IIx myofibers. Overall, findings highlight inhibitory impacts of HGF nitration on myogenic stem cell dynamics, pioneering a cogent discussion for better understanding age-related muscle atrophy and impaired regeneration with fibrosis (including sarcopenia and frailty).

DOI： 10.1111/acel.14041

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記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/acel.14117

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrep.2022.101295

記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrep.2022.101295

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Many biological features of Chattonella species display diurnal oscillations, but little is known about the corresponding cellular responses at the proteome level. In the present study, we hypothesized that the abundance of certain proteins would periodically fluctuate to suit the diurnal changes of biological processes in Chattonella cells. To test this hypothesis, we undertook proteomic analysis over 7 time points covering a 24-h period in C. marina var. antiqua cells at early stationary phase (12:00, 17:00, 20:00, 24:00 on day 8, and 05:00, 08:00, 12:00 on the following day). The protein profiles of C. marina var. antiqua cells collected from the diurnal cycle shared a majority of common protein spots. However, the expression of 16 out of 124 detected spots exhibited significant differences between light and dark periods and/or over the 7 time points of the diurnal cycle. The proteins of OEE, Cyt c553, and AtpB showed significantly higher expression levels in the light period, and generally were maximally expressed during the period of 12:00–17:00. The proteins of GAPDH and RPL12 generally showed higher expression levels in the dark period, and were maximally expressed during the period of 20:00–24:00. These findings support our hypothesis, and suggest that the periodic expressions of these proteins may be critical either for optimal cell utilization of resources or to maintain proper cell function throughout diurnal cycles.

DOI： 10.1016/j.jembe.2020.151361

記述言語：英語   掲載種別：研究論文（学術雑誌）  

A characteristic feature of archaeal ribonuclease P (RNase P) RNAs is that they have extended helices P12.1 and P12.2 containing kink-turn (K-turn) motifs to which the archaeal RNase P protein Rpp38, a homologue of the human RNase P protein Rpp38, specifically binds. PhoRpp38 from the hyperthermophilic archaeon Pyrococcus horikoshii is involved in the elevation of the optimum temperature of the reconstituted RNase P by binding the K-turns in P12.1 and P12.2. Previously, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was determined at 3.4 Å resolution. In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å. Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G·A and A·G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10-P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRpp29, which are the archaeal homologues of the human proteins Rpp21 and Rpp29, respectively, was affinity-purified and crystallized. The crystals thus grown diffracted to a resolution of 6.35 Å. Structure determination of the crystals will demonstrate the previously proposed secondary structure of stem-loops including helices P12.1 and P12.2 and will also provide insight into the structural organization of the specificity domain in P. horikoshii RNase P RNA.

DOI： 10.1107/S2053230X17018039

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1107/S2053230X17018039

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) is an endoribonuclease involved in maturation of the 5'-end of tRNA. We found previously that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. The crystal structures of the five proteins have been determined, a three-dimensional (3-D) model of PhopRNA has been constructed, and biochemical data, including protein-RNA interaction sites, have become available. Here, this information was combined to orient the crystallographic structures of the proteins relative to their RNA binding sites in the PhopRNA model. Some alterations were made to the PhopRNA model to improve the fit. In the resulting structure, a heterotetramer composed of PhoPop5 and PhoRpp30 bridges helices P3 and P16 in the PhopRNA C-domain, thereby probably stabilizing a double-stranded RNA structure (helix P4) containing catalytic Mg2+ ions, while a heterodimer of PhoRpp21 and PhoRpp29 locates on a single-stranded loop connecting helices P11 and P12 in the specificity domain (S-domain) in PhopRNA, probably forming an appropriate conformation of the precursor tRNA (pre-tRNA) binding site. The fifth protein PhoRpp38 binds each kink-turn (K-turn) motif in helices P12.1, P12.2, and P16 in PhopRNA. Comparison of the structure of the resulting 3-D model with that of bacterial RNase P suggests transition from RNA-RNA interactions in bacterial RNase P to protein-RNA interactions in archaeal RNase P. The proposed 3-D model of P. horikoshii RNase P will serve as a framework for further structural and functional studies on archaeal, as well as eukaryotic, RNase Ps.

DOI： 10.1016/j.bbrc.2017.09.085

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Time series variations in protein expression profiles in field Chattonella marina cells were investigated during a HAB occurred in the inner part of Ariake Sea, Japan (5-14 September, 2012). This study aimed to gather information on the molecular mechanisms underlying the physiological responses in field C. marina population during HAB. Proteomic analysis showed that the abundance of -37% protein spots (40 out of 108 detected from 2-DE gel images) significantly varied with the sampling date. Significant decreases in the abundances of proteins involved in photosystem II (LHCP 4), electron transfer chain (Cyt c553), Calvin cycle (GAPDH), and chloroplast antioxidant system (2-Cys Prx) were observed as the bloom progressed, suggesting the efficiencies of those photosynthetic pathways declined during the bloom. In addition, the abundances of the above proteins showed significant positive correlations with the F_v/F_m ratio and growth rate of C. marina and with DIN concentrations (except LHCP 4). Our findings suggested that declined expressions of those photosynthesis-related proteins presented some molecular foundation of the decreases in F_v/F_m ratio and growth rate of C. marina during the bloom, and also provided insight into mechanistic links between the external/internal factors and physiological responses of C. marina that may ultimately dictate the ecology of the bloom.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PhoRpp38 in the hyperthermophilic archaeon Pyrococcus horikoshii, a homologue of human ribonuclease P (RNase P) protein Rpp38, belongs to the ribosomal protein L7Ae family that specifically recognizes a kink-turn (K-turn) motif. A previous biochemical study showed that PhoRpp38 specifically binds to two stem-loops, SL12 and SL16, containing helices P12.1/12.2 and P15/16 respectively, in P. horikoshii RNase P RNA (PhopRNA). In order to gain insight into the PhoRpp38 binding mode to PhopRNA, we determined the crystal structure of PhoRpp38 in complex with the SL12 mutant (SL12M) at a resolution of 3.4 angstrom. The structure revealed that Lys35 on the beta-strand (beta 1) and Asn38, Glu39, and Lys42 on the alpha-helix (alpha 2) in PhoRpp38 interact with characteristic G.A and A.G pairs in SL12M, where Ile93, Glu94, and Val95, on a loop between alpha 4 and beta 4 in PhoRpp38, interact with the 3-nucleotide bulge (G-G-U) in the SL12M. The structure demonstrates the previously proposed secondary structure of SL12, including helix P12.2. Structure-based mutational analysis indicated that amino acid residues involved in the binding to SL12 are also responsible for the binding to SL16. This result suggested that each PhoRpp38 binds to the K-turns in SL12 and SL16 in PhopRNA. A pull-down assay further suggested the presence of a second K-turn in SL12. Based on the present results, together with available data, we discuss a structural basis for recognition of K-turn motifs in PhopRNA by PhoRpp38. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2016.04.118

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp30-(PhoPop5)2-PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two C-terminal helices (α4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA.

DOI： 10.1093/jb/mvv067

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood. To gain insight into the biological function of archaeal RNase Ps in vivo, we prepared archaeal mutants KUWΔP3, KUWΔP8, and KUWΔP16, in which the gene segments encoding stem-loops containing helices, respectively, P3, P8 and P16 in RNase P RNA (TkopRNA) of the hyperthermophilic archaeon Thermococcus kodakarensis were deleted. Phenotypic analysis showed that KUWΔP3 and KUWΔP16 grew slowly compared with wild-type T. kodakarensis KUW1, while KUWΔP8 displayed no difference from T. kodakarensis KUW1. RNase P isolated using an affinity-tag from KUWΔP3 had reduced pre-tRNA cleavage activity compared with that from T. kodakarensis KUW1. Moreover, quantitative RT-PCR (qRT-PCR) and Northern blots analyses of KUWΔP3 showed greater accumulation of unprocessed transcripts for pre-tRNAs than that of T. kodakarensis KUW1. The current study represents the first attempt to prepare mutant T. kodakarensis with impaired RNase P for functional investigation. Comparative whole-transcriptome analysis of T. kodakarensis KUW1 and KUWΔP3 should allow for the comprehensive identification of RNA substrates for archaeal RNase Ps.

DOI： 10.1016/j.bbrc.2015.11.012

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Fluorescence resonance energy transfer-based assay showed that archaeal ribonuclease P (RNase P) proteins significantly promoted DNA annealing and strand displacement. Moreover, we found that archaeal RNase P proteins could discriminate nucleotide exchanges in DNA chains via their activity accelerating DNA strand displacement, suggesting that they have potential for biotechnological application to genetic diagnosis.

DOI： 10.1080/09168451.2015.1058699

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ribonuclease P (RNase P) proteins TkoPop5 and TkoRpp30, homologs of human Pop5 and Rpp30, respectively, in the hyperthermophilic archaeon Thermococcus kodakarensis were prepared and characterized with respect to pre-tRNA cleavage activity using the reconstitution system of the well-studied Pyrococcus horikoshii RNase P. The reconstituted particle containing TkoPop5 in place of the P. horikoshii counterpart PhoPop5 retained pre-tRNA cleavage activity comparable to that of the reconstituted P. horikoshii RNase P, while that containing TkoRpp30 instead of its corresponding protein PhoRpp30 had slightly lower activity than the P. horikoshii RNase P. Moreover, we determined crystal structures of TkoRpp30 alone and in complex with TkoPop5. Like their P. horikoshii counterparts, whose structures were solved previously, TkoRpp30 and TkoPop5 fold into TIM barrel and RRM-like fold, respectively. This finding demonstrates that RNase P proteins in T. kodakarensis and P. horikoshii are interchangeable and that their three-dimensional structures are highly conserved.

DOI： 10.1080/09168451.2014.1003130

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Proteinaceous RNase P (PRORP1) in Arabidopsis thaliana is an endoribonuclease that catalyzes hydrolysis to remove the 5'-leader sequence of precursor tRNAs (pre-tRNAs). PRORP1 is composed of pentatricopeptide repeat (PPR) motifs, a central linker region, and a metal nuclease domain, the NYN domain. The PPR motifs are single-stranded RNA-binding motifs that recognize bases in a modular fashion. To obtain insight into the mechanism by which the PPR motifs in PRORP1 recognize a target sequence in catalysis, N-terminal successive deletion mutants were overproduced in Escherichia coli, and the resulting proteins were characterized in terms of enzymatic activity using chloroplast pre-tRNA(Phe) as a substrate. Although Δ89, in which all PPR motifs are present, retained the pre-tRNA cleavage activity, Δ129 devoid of the first PPR motif (PPR1) had significantly reduced cleavage activity. Likewise, deletions of the second (PPR2) or third PPR (PPR3) motif abolished the cleavage activity, suggesting that PPR motifs play a crucial role in catalysis. A proposed recognition code for PPR motifs predicted that PPR2-PPR5 in PRORP1 recognize C, A/U, A, and U, respectively, whose sequence is in good agreement with C56-A57-A58-A59 in the TψC loop in pre-tRNA(Phe). Mutational analyses of nucleotide residues in the TψC loop as well as nucleotide-specifying residues (NSRs) in PPR motifs further suggested that PPR2 and PPR3 in PRORP1 favorably recognize nucleotide bases C56 and A57 at the TψC loop in pre-tRNA(Phe), respectively. This prediction and previous biochemical data were combined to construct a fitting model of tRNA onto PRORP1, showing that the mechanism by which PRORP1 recognizes pre-tRNAs appears to be distinct from that by bacterial RNase P.

DOI： 10.1016/j.bbrc.2014.07.030

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The secondary structure of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 has several peripheral stem-loops. In order to investigate their functional role, we prepared six mutants, ΔP1, ΔP3, ΔP8, ΔP9, ΔP12 and ΔP15, in which the stem-loops including helices P1, P3, P8, P9, P12/12.1/12.2 and P15/16 in PhopRNA were individually deleted, respectively, and characterized them with respect to pre-tRNA cleavage activity in the presence of five proteins and also to the ability to form a complex with the proteins. The reconstituted particles containing ΔP3, ΔP8 or ΔP9 retained considerable levels of activity (35-65%), while those containing ΔP1, ΔP12 or ΔP15 had markedly reduced activity (13%). It was further found that the reconstituted particles comprising ΔP3 or ΔP15 lacked PhoPop5 and PhoRpp30, whereas those containing ΔP1, ΔP8, ΔP9 or ΔP12 bound to all five proteins. Since it is known that PhoPop5 functions in a complex with PhoRpp30, the present result suggests that the peripheral stem-loops containing P3 or P15/16 are involved in the structural formation of a catalytic site by interacting with the protein complex PhoPop5-PhoRpp30.

DOI： 10.1093/jb/mvt092

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) is a ribonucleoprotein complex essential for the processing of 5' leader sequences of precursor tRNAs (pre-tRNA). PhoPop5 is an archaeal homolog of human RNase P protein hPop5 involved in the activation of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii, probably by promoting RNA annealing (AN) and RNA strand displacement (SD). Although PhoPop5 folds into the RNA recognition motif (RRM), it is distinct from the typical RRM in that it has an insertion of α-helix (α2) between α1 and β2. Biochemical and structural data have shown that the dimerization of PhoPop5 through the loop between α1 and α2 is required for the activation of PhopRNA. In addition, PhoPop5 has additional helices (α4 and α5) at the C-terminus, which pack against one face of the β-sheet. In this study, we examined the contribution of the C-terminal helices to the activation of PhopRNA using mutation analyses. Reconstitution experiments and fluorescence resonance energy transfer (FRET)-based assays indicated that deletion of the C-terminal helices α4 and α5 significantly influenced on the pre-tRNA cleavage activity and abolished AN and SD activities, while that of α5 had little effect on these activities. Moreover, the FRET assay showed that deletion of the loop between α1 and α2 had no influence on the AN and SD activity. Further mutational analyses suggested that basic residues at α4 are involved in interaction with PhopRNA, while hydrophobic residues at α4 participate in interaction with hydrophobic residues at the β-sheet, thereby stabilizing an appropriate orientation of the helix α4. Together, these results indicate that extra-structural elements in the RRM in PhoPop5 play a crucial role in the activation of PhopRNA.

DOI： 10.1016/j.bbrc.2013.09.140

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv /Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6 f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb.

DOI： 10.1002/jbt.21505

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues. METHODS: The structure of wild-type bmGSTu was determined with a resolution of 2.1Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu. RESULTS: We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features. CONCLUSIONS: This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function. GENERAL SIGNIFICANCE: Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.

DOI： 10.1016/j.bbagen.2011.06.022

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5' leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure-function relationships of this multifaceted primordial ribozyme.

DOI： 10.1016/j.bbrc.2011.09.098

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Starch-branching enzyme catalyzes the cleavage of α-1, 4-linkages and the subsequent transfer of α-1,4 glucan to form an α-1,6 branch point in amylopectin. Sequence analysis of the rice-branching enzyme I (BEI) indicated a modular structure in which the central α-amylase domain is flanked on each side by the N-terminal carbohydrate-binding module 48 and the α-amylase C-domain. We determined the crystal structure of BEI at a resolution of 1.9 Å by molecular replacement using the Escherichia coli glycogen BE as a search model. Despite three modular structures, BEI is roughly ellipsoidal in shape with two globular domains that form a prominent groove which is proposed to serve as the α-polyglucan-binding site. Amino acid residues Asp344 and Glu399, which are postulated to play an essential role in catalysis as a nucleophile and a general acid/base, respectively, are located at a central cleft in the groove. Moreover, structural comparison revealed that in BEI, extended loop structures cause a narrowing of the substrate-binding site, whereas shortened loop structures make a larger space at the corresponding subsite in the Klebsiella pneumoniae pullulanase. This structural difference might be attributed to distinct catalytic reactions, transglycosylation and hydrolysis, respectively, by BEI and pullulanase.

DOI： 10.1093/glycob/cwr049

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE: These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori.

DOI： 10.1016/j.bbagen.2011.01.003

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The archaeal toxin, aRelE, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 inhibits protein synthesis, whereas its cognate antitoxin, aRelB, neutralizes aRelE activity by forming a non-toxic complex, aRelB-aRelE. The structural mechanism whereby aRelB neutralizes aRelE activity was examined by biochemical and biophysical analyses. Overexpression of aRelB with an aRelE mutant (ΔC6), in which the C-terminal residues critical for aRelE activity were deleted, in Escherichia coli allowed a stable complex, aRelB-ΔC6, to be purified. Isothermal titration of aRelE or ΔC6 with aRelB indicated that the association constant (Ka) of wild-type aRelB-aRelE is similar to that of aRelB-ΔC6, demonstrating that aRelB makes little contact with the C-terminal active site of aRelE. Overexpression of deletion mutants of aRelB with aRelE indicated that either the N-terminal (pos. 1-27) or C-terminal (pos. 50-67) fragment of aRelB is sufficient to counteract the toxicity of aRelE in E. coli cells and the second α-helix (α2) in aRelB plays a critical role in forming a stable complex with aRelE. The present results demonstrate that aRelB, as expected from its X-ray structure, precludes aRelE from entering the ribosome, wrapping around the molecular surface of aRelE.

DOI： 10.1016/j.bbrc.2010.08.061

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Mutations of amino acids in the C-terminal region of an archaeal toxin, aRelE, from Pyrococcus horikoshii were characterized with respect to protein synthesis inhibitory activity and 70S ribosome-binding activity. The results suggest that basic residues at the C-terminal region in aRelE play a crucial role both in 70S ribosome binding and in protein synthesis inhibition activities.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Starch branching enzyme (SBE) catalyzes the cleavage of alpha-1.4-linkages and the subsequent transfer of alpha-1.4 glucan to form an alpha-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the alpha-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other alpha-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 A on a synchrotron X-ray source.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The crystal structure of the Alba protein (PhoAlba) from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, was determined at a resolution of 2.8 A. PhoAlba structurally belongs to the alpha/beta proteins and is similar not only to archaeal homologues but also to RNA-binding proteins, including the C-terminal half of initiation factor 3 (IF3-C) from Bacillus stearothermophilus, an Esherichia coli protein implicated in cell division (Yhhp), and an Arabidopsis protein of unknown function. We found by gel shift assay that PhoAlba interacts with both ribonuclease P (RNase P) RNA (PhopRNA) and precursor-tRNA(Tyr) (pre-tRNA(Tyr)) in P. horikoshii. However, the addition of PhoAlba to reconstituted particles composed of PhopRNA and four or five protein subunits had little influence on either the pre-tRNA processing activity or the optimum temperature for the processing activity. These results suggest that PhoAlba contributes little to the catalytic activity of P. horikoshii RNase P.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p- like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at; 90 C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 angstrom using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N- terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix- hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex.

DOI： 10.1110/ps.073107008

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease NT (RNase NT), induced upon tobacco mosaic virus (TMV) infection in Nicotiana glutinosa leaves, has a broad base specificity. The crystal structures of RNase NT in complex with either 5'-AMP, 5'-GMP, or 2'-UMP were determined at 1.8 A resolutions by molecular replacement. RNase NT consists of seven helices and seven beta strands, and the structure is highly similar to that of RNase NW, a guanylic acid preferential RNase from the N. glutinosa leaves, showing root mean square deviation (rmsd) of 1.1 A over an entire length of two molecules for Calpha atoms. The complex structures revealed that Trp42, Asn44, and Trp50 are involved in interactions with bases at B1 site (primary site), whereas Gln12, Tyr17, Ser78, Leu79, and Phe89 participate in recognition of bases at B2 site (subsite). The 5'-GMP and 5'-AMP bind both B1 and B2 sites in RNase NT, while 2'-UMP predominantly binds B1 site in the complex. The nucleotide binding modes in these complexes would provide a clue to elucidation of structural basis for the broad base specificity for RNase NT.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.

記述言語：英語   掲載種別：研究論文（学術雑誌）  

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1017/S1355838201002345

開催年月日： 2016年11月 - 2016年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神奈川県横浜市　パシフィコ横浜   国名：日本国  

開催年月日： 2016年11月 - 2016年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神奈川県横浜市　パシフィコ横浜   国名：日本国  

開催年月日： 2016年11月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：茨城県水戸市　茨城県立県民文化センター   国名：日本国  

開催年月日： 2016年9月

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開催地：長崎大学   国名：日本国  

開催年月日： 2016年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2016年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2016年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2016年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：長崎大学   国名：日本国  

開催年月日： 2016年6月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡国際会議場   国名：日本国  

開催年月日： 2016年6月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：福岡国際会議場   国名：日本国  

開催年月日： 2016年6月 - 2016年5月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：福岡国際会議場   国名：日本国  

開催年月日： 2015年12月

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開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宮崎県宮崎市　 ANAホリディ・イン リゾート宮崎   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸ポートアイランド   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸ポートアイランド   国名：日本国  

開催年月日： 2015年12月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸ポートアイランド   国名：日本国  

開催年月日： 2015年9月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年9月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年9月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年9月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年9月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年6月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年6月

記述言語：日本語   会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：北九州国際会議場   国名：日本国  

開催年月日： 2015年6月

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開催地：北九州国際会議場   国名：日本国  

開催年月日： 2012年5月

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開催地：京都   国名：日本国  

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開催地：京都女子大学   国名：日本国  

開催年月日： 2011年3月

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開催地：京都女子大学   国名：日本国  

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開催地：東京大学   国名：日本国  

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開催地：湯布院（熊本）   国名：日本国  

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開催地：茨城県，つくば   国名：日本国  

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開催地：山口大学（山口）   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：名古屋国際会議場（名古屋）   国名：日本国  

開催年月日： 2023年10月 - 2024年10月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宇部市文化会館・山口大学常盤キャンパス　山口市   国名：日本国  

開催年月日： 2023年10月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：宇部市文化会館・山口大学常盤キャンパス   国名：日本国  

開催年月日： 2022年11月

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開催地：東洋大学　川越キャンパス   国名：日本国  

開催年月日： 2022年9月

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開催地：長崎大学文教キャンパス 環境科学部・教養教育講義棟（長崎市文教町1-14）   国名：日本国  

開催年月日： 2019年11月

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開催地：米子コンベンションセンター(鳥取県米子市)   国名：日本国  

開催年月日： 2016年5月

記述言語：日本語   会議種別：口頭発表（一般）  

開催地：鹿児島市鹿児島大学郡元キャンパス   国名：日本国  

開催年月日： 2008年8月 - 2008年11月

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：Free Univerisity, berlin, Germany   国名：ドイツ連邦共和国  

会議種別：口頭発表（一般）  

開催地：宮崎県，宮崎県立図書館   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：神戸市   国名：日本国  

Structural analysis of ph1481p-ph1877p complex in ribonuclease P

会議種別：口頭発表（一般）  

開催地：佐賀県，佐賀大学   国名：日本国  

会議種別：シンポジウム・ワークショップ パネル（公募）  

開催地：静岡県，伊豆，大仁ホテル   国名：日本国  

種別：大会・シンポジウム等 

参加者数：50

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

参加者数：100

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導

ティーチングアシスタントの指導