451 results on '"Fuyuhiko Inagaki"'
Search Results
52. Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance
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James Nyirenda, Daisuke Kohda, Takashi Saitoh, Nobuo Maita, Fuyuhiko Inagaki, Nobuo N. Noda, and Shunsuke Matsumoto
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Models, Molecular ,Stereochemistry ,Archaeal Proteins ,Molecular Sequence Data ,Crystal structure ,Crystallography, X-Ray ,Protein Structure, Secondary ,Residue (chemistry) ,Structural Biology ,Catalytic Domain ,Transferase ,Amino Acid Sequence ,Asparagine ,Nuclear Magnetic Resonance, Biomolecular ,Molecular Biology ,chemistry.chemical_classification ,Chemistry ,Oligosaccharyltransferase ,Membrane Proteins ,Oligosaccharide ,Solutions ,Crystallography ,Enzyme ,Amino Acid Substitution ,Hexosyltransferases ,Biocatalysis ,Mutagenesis, Site-Directed ,Cystine ,Pyrococcus horikoshii ,Glycoprotein - Abstract
Summary Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by 15 N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.
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- 2013
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53. Noncanonical recognition and UBL loading of distinct E2s by autophagy-essential Atg7
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Yoshihiro Kobashigawa, Masaya Yamaguchi, Nobuo N. Noda, Yoshinori Ohsumi, Kazuaki Matoba, Yuko Fujioka, Hayashi Yamamoto, Hisashi Hoshida, Rinji Akada, Fuyuhiko Inagaki, Hitoshi Nakatogawa, and Ryoko Sawada
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chemistry.chemical_classification ,Models, Molecular ,Esterification ,Mechanism (biology) ,ATG8 ,Autophagy ,Arabidopsis ,Ubiquitin-Activating Enzymes ,Crystallography, X-Ray ,Cell biology ,ATG12 ,Enzyme ,chemistry ,Structural Biology ,Molecular Biology - Abstract
Autophagy requires ubiquitin-like Atg8 and Atg12 conjugation systems, where Atg7 has a critical role as the sole E1 enzyme. Although Atg7 recognizes two distinct E2s, Atg3 and Atg10, it is not understood how Atg7 correctly loads these E2s with their cognate ubiquitin-like proteins, Atg8 and Atg12. Here, we report the crystal structures of the N-terminal domain of Atg7 bound to Atg10 or Atg3 of thermotolerant yeast and plant homologs. The observed Atg7-Atg10 and Atg7-Atg3 interactions, which resemble each other but are quite distinct from the canonical E1-E2 interaction, makes Atg7 suitable for transferring Atg12 to Atg10 and Atg8 to Atg3 by a trans mechanism. Notably, in vitro experiments showed that Atg7 loads Atg3 and Atg10 with Atg8 and Atg12 in a nonspecific manner, which suggests that cognate conjugate formation in vivo is not an intrinsic quality of Atg7.
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- 2012
54. Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions
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Nobuo N. Noda, Mariko Yokogawa, Shizuo Akira, Takashi Tsushima, Fuyuhiko Inagaki, Osamu Takeuchi, Yoshiaki Enokizono, Daron M. Standley, Hiroyuki Kumeta, and Kazuo Yamashita
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Models, Molecular ,0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,Protein Conformation ,RNase P ,Protein domain ,Plasma protein binding ,Biology ,Crystallography, X-Ray ,Protein Engineering ,urologic and male genital diseases ,Article ,Mice ,Structure-Activity Relationship ,03 medical and health sciences ,Ribonucleases ,0302 clinical medicine ,Protein structure ,Protein Domains ,Animals ,Binding site ,Inflammation ,Zinc finger ,Binding Sites ,Multidisciplinary ,Protein engineering ,Cell biology ,030104 developmental biology ,Biochemistry ,Mutation ,Protein Multimerization ,PIN domain ,030217 neurology & neurosurgery ,Protein Binding - Abstract
Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus—the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.
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- 2016
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55. NMR Structural Biology Using Paramagnetic Lanthanide Probe
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Tomohide Saio and Fuyuhiko Inagaki
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Lanthanide ,Paramagnetism ,Structural biology ,Chemistry ,Analytical chemistry ,Physical chemistry - Published
- 2016
56. Structural insights into Atg10-mediated formation of the autophagy-essential Atg12-Atg5 conjugate
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Masaya Yamaguchi, Yoshihiro Kobashigawa, Nobuo N. Noda, Rinji Akada, Yoshinori Ohsumi, Hiroyuki Kumeta, Takayuki Shima, Fuyuhiko Inagaki, and Hayashi Yamamoto
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Models, Molecular ,Magnetic Resonance Spectroscopy ,Stereochemistry ,Ubiquitin-Protein Ligases ,ATG5 ,Molecular Sequence Data ,Crystallography, X-Ray ,Protein Structure, Secondary ,ATG12 ,Fungal Proteins ,Kluyveromyces ,Kluyveromyces marxianus ,Structural Biology ,Side chain ,Autophagy ,Escherichia coli ,Amino Acid Sequence ,Molecular Biology ,Binding Sites ,biology ,biology.organism_classification ,Yeast ,Phosphoric Monoester Hydrolases ,Recombinant Proteins ,Transport protein ,Protein Structure, Tertiary ,Kinetics ,Mutation ,Sequence Alignment ,Conjugate ,Plasmids ,Protein Binding - Abstract
SummaryThe Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 of KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme.
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- 2012
57. Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy
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Nobuo N. Noda, Yoshinori Ohsumi, Yuko Fujioka, Fuyuhiko Inagaki, Takafumi Kobayashi, and Wakana Adachi
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Protein Folding ,Saccharomyces cerevisiae Proteins ,Protein family ,Protein domain ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,BAG3 ,Biochemistry ,Protein Structure, Secondary ,Vps30 ,SeqA protein domain ,Structural Biology ,Autophagy ,Molecular Biology ,Vacuolar protein sorting ,Crystallography ,Vacuolar Protein Sorting ,Membrane Proteins ,Cell Biology ,Autophagy-related protein 13 ,Beclin 1 ,Protein tertiary structure ,Protein Structure, Tertiary ,Cell biology ,VPS25 ,Protein Transport ,Protein Structure and Folding ,Crystal Structure ,Atg6 ,Phosphatidylinositol 3-Kinase ,Microtubule-Associated Proteins - Abstract
Background: Vps30/Atg6 is responsible for both autophagy and vacuolar protein sorting. Results: Structure of the Vps30 BARA domain was determined and its function was characterized. Conclusion: BARA domain has a unique fold and is specifically required for autophagy. Significance: This study will be a basis for elucidating the various functions of Vps30 homologs., Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.
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- 2012
58. Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein–protein complex structure determination
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Tomohide Saio, Fuyuhiko Inagaki, Masahiro Ushio, Masashi Yokochi, Kenji Ogura, Mitsuhiro Sekiguchi, and Yoshihiro Kobashigawa
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Models, Molecular ,Lanthanide ,Lanthanide binding tag ,Tacrolimus Binding Protein 1A ,Lanthanoid Series Elements ,Biochemistry ,Article ,Ion ,Magnetics ,Paramagnetism ,Humans ,Transition Temperature ,Peptide bond ,Fluorometry ,Protein Interaction Domains and Motifs ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Chemistry ,TOR Serine-Threonine Kinases ,FRB ,Rigid body ,Magnetic susceptibility ,Recombinant Proteins ,Crystallography ,Differential scanning fluorometry ,Chemical physics ,Docking (molecular) ,Multiprotein Complexes ,FKBP12 ,mTOR ,Target protein ,Pseudo contact shift ,Protein Binding - Abstract
Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein–protein and protein–ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination. Electronic supplementary material The online version of this article (doi:10.1007/s10858-012-9623-8) contains supplementary material, which is available to authorized users.
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- 2012
59. Autophagy-related protein 32 as autophagic degron and directly initiates mitophagy
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Nobuo N. Noda, Fuyuhiko Inagaki, Yoshinori Ohsumi, Sho W. Suzuki, Ayako Hashimoto, Miou Matsunami, Ikuko Takahashi, Noriko Kondo-Okamoto, Hitoshi Nakatogawa, and Koji Okamoto
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Autophagosome ,Saccharomyces cerevisiae Proteins ,Mitochondrial intermembrane space ,ATG8 ,Vesicular Transport Proteins ,Mitochondrial Degradation ,Autophagy-Related Proteins ,Receptors, Cytoplasmic and Nuclear ,Saccharomyces cerevisiae ,Biochemistry ,Mitochondrial Proteins ,Mitochondrial membrane transport protein ,Phagosomes ,Mitophagy ,Autophagy ,Molecular Biology ,Mitochondrial transport ,biology ,Autophagy-Related Protein 8 Family ,Cell Biology ,Mitochondria ,Protein Structure, Tertiary ,Cell biology ,Multiprotein Complexes ,Mitochondrial Membranes ,Mutation ,biology.protein ,Microtubule-Associated Proteins - Abstract
Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.
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- 2012
60. Solution structures of yeast Saccharomyces cerevisiae calmodulin in calcium- and target peptide-bound states reveal similarities and differences to vertebrate calmodulin
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Hiroyuki Itoh, Hiroyuki Kumeta, Michio Yazawa, Yoshihiro Kobashigawa, Kenji Ogura, Ryosuke Yoshida, Kiyohiro Takahasi, and Fuyuhiko Inagaki
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chemistry.chemical_classification ,Calmodulin ,biology ,Saccharomyces cerevisiae ,Target peptide ,Peptide ,Peptide binding ,Cell Biology ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,Protein structure ,chemistry ,Biochemistry ,Genetics ,Biophysics ,biology.protein ,Peptide sequence - Abstract
We determined the solution structures of the calmodulin (CaM) isoform from yeast Saccharomyces cerevisiae (yCaM) in the calcium-bound form and in complex with a target peptide using NMR spectroscopy and small-angle X-ray scattering (SAXS). yCaM shows a number of unique features distinct from the vertebrate CaM isoforms: (i) it has only approximately 60% sequence identity to vertebrate CaM; (ii) its fourth Ca(2+)-binding domain is inactivated by amino acid substitution. As NMR analyses of Ca(2+)-bound full-length yCaM implied that the fourth EF-hand motif region (EF4) presents a disordered conformation, we determined the solution structure of an EF4-deletion mutant of Ca(2+)-bound yCaM. The deletion mutant showed a compact globular structure, with the target recognition sites of the N-terminal domain and the third EF-hand region bound to each other. Furthermore, we determined the solution structure of Ca(2+)-bound yCaM complexed with a calcineurin-derived peptide. Interestingly, the structure closely resembled that of the vertebrate CaM-calcineurin complex, with the EF4 region in cooperation with the peptide binding. Moreover, the results of SAXS analyses were consistent with the NMR solution structures and showed the conformational changes of yCaM in three functional stages. These unique structural characteristics of yCaM are closely related to Ca(2+)-mediated signal transduction in yeast.
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- 2012
61. Regioselective Intramolecular [3+2] Annulation of Allene-Nitrones
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Harumi Kobayashi, Fuyuhiko Inagaki, and Chisato Mukai
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nitrone ,chemistry.chemical_classification ,Annulation ,Double bond ,Bicyclic molecule ,Stereochemistry ,allene ,Allene ,Hydrocarbons, Cyclic ,General Chemistry ,General Medicine ,Cycloaddition ,Nitrone ,Alkadienes ,chemistry.chemical_compound ,chemistry ,Cyclization ,1,3-dipolar cycloaddition ,Intramolecular force ,Drug Discovery ,1,3-Dipolar cycloaddition ,Nitrogen Oxides ,Sulfones - Abstract
The regioselective intramolecular 1,3-dipolar cycloaddition of the phenylsulfonylallene-nitrone derivatives has been developed. This reaction showed that the distal double bond of the allene exclusively reacted with the nitrone group to produce the bicyclic isoxazolidine derivatives regardless of the substitution pattern on the allenyl moiety.
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- 2012
62. Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c
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Takeshi Uchida, Hiroyuki Kumeta, Mizue Imai, Tomohide Saio, Fuyuhiko Inagaki, and Koichiro Ishimori
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0301 basic medicine ,Cytochrome ,Stereochemistry ,Protein Conformation ,Biophysics ,macromolecular substances ,Biochemistry ,Redox ,03 medical and health sciences ,Electron transfer ,Structure-Activity Relationship ,Protein structure ,Cytochrome c oxidase ,Humans ,Binding site ,Molecular Biology ,Binding Sites ,biology ,Chemistry ,Cytochrome c ,Cytochromes c ,Cell Biology ,Electron transport chain ,Enzyme Activation ,Oxygen ,Kinetics ,030104 developmental biology ,biology.protein ,sense organs ,Oxidation-Reduction ,Protein Binding - Abstract
Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the μs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO.
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- 2015
63. Energyless CO2 Absorption, Generation, and Fixation Using Atmospheric CO2
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Fuyuhiko, Inagaki, Yasuhiko, Okada, Chiaki, Matsumoto, Masayuki, Yamada, Kenta, Nakazawa, and Chisato, Mukai
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Molecular Structure ,Atmosphere ,Surface Properties ,Ethanolamine ,Adsorption ,Carbon Dioxide - Abstract
From an economic and ecological perspective, the efficient utilization of atmospheric CO2 as a carbon resource should be a much more important goal than reducing CO2 emissions. However, no strategy to harvest CO2 using atmospheric CO2 at room temperature currently exists, which is presumably due to the extremely low concentration of CO2 in ambient air (approximately 400 ppm=0.04 vol%). We discovered that monoethanolamine (MEA) and its derivatives efficiently absorbed atmospheric CO2 without requiring an energy source. We also found that the absorbed CO2 could be easily liberated with acid. Furthermore, a novel CO2 generator enabled us to synthesize a high value-added material (i.e., 2-oxazolidinone derivatives based on the metal catalyzed CO2-fixation at room temperature) from atmospheric CO2.
- Published
- 2015
64. Ligand-driven conformational changes of MurD visualized by paramagnetic NMR
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Tomohide Saio, Hiroto Yamaguchi, Hideki Tsujishita, Kenji Ogura, Hiroyuki Kumeta, Kazumi Shimizu, Fuyuhiko Inagaki, Yoshihiro Kobashigawa, Masashi Yokochi, and Kota Kodama
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chemistry.chemical_classification ,Ions ,Multidisciplinary ,Magnetic Resonance Spectroscopy ,Ligand ,Protein Conformation ,Nuclear magnetic resonance spectroscopy ,Calorimetry ,Ligands ,Lanthanoid Series Elements ,Article ,Paramagnetism ,Enzyme ,Protein structure ,chemistry ,Biochemistry ,Biophysics ,Peptide Synthases ,Function (biology) - Abstract
Proteins, especially multi-domain proteins, often undergo drastic conformational changes upon binding to ligands or by post-translational modifications, which is a key step to regulate their function. However, the detailed mechanisms of such dynamic regulation of the functional processes are poorly understood because of the lack of an efficient tool. We here demonstrate detailed characterization of conformational changes of MurD, a 47 kDa protein enzyme consisting of three domains, by the use of solution NMR equipped with paramagnetic lanthanide probe. Quantitative analysis of pseudocontact shifts has identified a novel conformational state of MurD, named semi-closed conformation, which is found to be the key to understand how MurD regulates the binding of the ligands. The modulation of the affinity coupled with conformational changes accentuates the importance of conformational state to be evaluated in drug design.
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- 2015
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65. An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR
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Yoshihiro Kobashigawa, Tetsuo Kiso, Masashi Kawasaki, Fuyuhiko Inagaki, Mitsuhiro Sekiguchi, Keitaro Mori, Masashi Yokochi, Toshio Teramura, and Ken Ichi Suzumura
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Magnetic Resonance Spectroscopy ,Recombinant Fusion Proteins ,Bioengineering ,Tacrolimus Binding Protein 1A ,Biology ,Ligands ,Biochemistry ,DSC ,Protein structure ,fusion protein ,Humans ,Enzyme Inhibitors ,Protein kinase A ,Molecular Biology ,PI3K/AKT/mTOR pathway ,Calorimetry, Differential Scanning ,TOR Serine-Threonine Kinases ,Original Articles ,Nuclear magnetic resonance spectroscopy ,Fusion protein ,NMR ,Protein Structure, Tertiary ,FKBP ,FRB domain ,Protein Binding ,Biotechnology - Abstract
Mammalian target of rapamycin (mTOR), a large multi-domain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein-drug interactions at the domain level, while NMR gave insights into the protein-drug interactions at the residue level. The use of the FKBP12-FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.
- Published
- 2011
66. Intramolecular Cyclization Reaction of Multiple Bonds and Its Application
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Fuyuhiko Inagaki
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Pharmacology ,chemistry.chemical_classification ,Bicyclic molecule ,Alkene ,Stereochemistry ,Allene ,Pharmaceutical Science ,Alkyne ,Total synthesis ,Stereoisomerism ,Alkenes ,Ring (chemistry) ,Cycloaddition ,Organic Chemistry Phenomena ,Alkadienes ,chemistry.chemical_compound ,Alkaloids ,Cycloisomerization ,chemistry ,Cyclization ,Alkynes ,Oximes ,Heterocyclic Compounds, 3-Ring ,Carbolines - Abstract
The cycloaddition and cycloisomerization of the allene with an alkyne, alkene, or an additional allene for construction of various monocyclic and bicyclic ring systems has been developed. The characteristic features of these methods using allene functionality instead of a simple alkene or alkyne include the reaction mode that originated from the double function as well as the high efficiency for the constructions of medium-sized rings. Furthermore, asymmetric formal synthesis of (+)-nakadomarin A and total synthesis of (+)-fawcettimine and (+)-lycoposerramine-B based on highly stereoselective Pauson-Khand reaction of alkene-alkynes were completed.
- Published
- 2011
67. A new procedure for the preparation of 2-vinylindoles and their [4+2] cycloaddition reaction
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Chisato Mukai, Fuyuhiko Inagaki, Ryohei Takahashi, and Y. A. Mohamed
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chemistry.chemical_compound ,Chemistry ,Carbazole ,Organic Chemistry ,Drug Discovery ,Organic chemistry ,SN2 reaction ,Biochemistry ,Chemical synthesis ,Cycloaddition ,Stille reaction ,Diels–Alder reaction - Abstract
A new approach for the synthesis of 2-vinylindole derivatives by 5-exo mode cyclization of 2-(3-silyloxymethylallenyl)anilines was developed. The starting allenylanilines were easily prepared by the Stille coupling of o-iodoaniline and allenylstannanes. The formed 2-vinylindole derivatives were transformed into several carbazole derivatives via the [4+2] cycloaddition reaction with suitable dienophiles.
- Published
- 2011
68. Rh(I)-catalyzed intramolecular [2 + 2 + 1] cycloaddition of allenenes: Construction of bicyclo[4.3.0]nonenones with an angular methyl group and tricyclo[6.4.0.01,5]dodecenone
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Yujiro Hayashi, Fuyuhiko Inagaki, Yumi Matsui, Naoya Itoh, and Chisato Mukai
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Bicyclic molecule ,Stereochemistry ,allene ,Allene ,Organic Chemistry ,chemistry.chemical_element ,Ring (chemistry) ,Full Research Paper ,carbonylative [2 + 2 + 1] cycloaddition ,Cycloaddition ,Rhodium ,lcsh:QD241-441 ,Chemistry ,chemistry.chemical_compound ,lcsh:Organic chemistry ,chemistry ,Intramolecular force ,Yield (chemistry) ,rhodium ,bicyclo[4.3.0] derivatives ,lcsh:Q ,lcsh:Science ,quaternary center construction ,Methyl group - Abstract
The [RhCl(CO)dppp]2-catalyzed intramolecular carbonylative [2 + 2 + 1] cycloaddition of allenenes was developed to prepare bicyclo[4.3.0]nonenones possessing a methyl group at the ring junction, which is difficult to achieve by the Pauson–Khand reaction of the corresponding enynes. This method also provided a new procedure for the construction of the tricyclo[6.4.0.01,5]dodecenone framework in a satisfactory yield.
- Published
- 2011
69. Phosphoinositide-incorporated lipid–protein nanodiscs: A tool for studying protein–lipid interactions
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Yoshihiro Kobashigawa, Kohsuke Harada, Kenji Ogura, Fuyuhiko Inagaki, and Naoki Yoshida
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Male ,Models, Molecular ,Magnetic Resonance Spectroscopy ,Immunoprecipitation ,Lipid Bilayers ,Biophysics ,Fluorescence Polarization ,Phosphatidylinositols ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Humans ,Nanotechnology ,Inositol ,Phosphatidylinositol ,Molecular Biology ,Nanodisc ,Apolipoprotein A-I ,Ligand binding assay ,Cell Membrane ,Membrane Proteins ,Water ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Cytosol ,chemistry ,Second messenger system ,Hydrophobic and Hydrophilic Interactions ,Fluorescence anisotropy ,Protein Binding - Abstract
Phosphatidylinositol (PtdIns) is phosphorylated at D-3, D-4, and/or D-5 of the inositol ring to produce seven distinct lipid second messengers known as phosphoinositides (PIs). The PI level is temporally and spatially controlled at the cytosolic face of the cellular membrane. Effectors containing PI-binding domains (e.g., PH, PX, FYVE, ENTH, FERM) associate with specific PIs. This process is crucial for the localization of a variety of cell-signaling proteins, thereby regulating intracellular membrane trafficking, cell growth and survival, cytoskeletal organization, and so on. However, quantitative assessments of protein–PI interactions are generally difficult due to insolubility of PIs in aqueous solution. Here we incorporated PIs into a lipid–protein nanoscale bilayer (nanodisc), which is applied for studying the protein–PI interactions using pull-down binding assay, fluorescence polarization, and nuclear magnetic resonance studies, each facilitating fast, quantitative, and residue-specific evaluation of the protein–PI interactions. Therefore, the PI-incorporated nanodisc could be used as a versatile tool for studying the protein–lipid interactions by various biochemical and biophysical techniques.
- Published
- 2011
70. Construction of Diverse Ring Systems Based on Allene-Multiple Bond Cycloaddition
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Shinji Kitagaki, Fuyuhiko Inagaki, and Chisato Mukai
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chemistry.chemical_classification ,Cyclic compound ,Alkene ,Allene ,Pauson–Khand reaction ,Organic Chemistry ,Alkyne ,Cycloaddition ,chemistry.chemical_compound ,Cycloisomerization ,chemistry ,Computational chemistry ,Physics::Chemical Physics ,Isomerization - Abstract
Cycloaddition and cycloisomerization based on the interaction between an allene and another multiple bond, such as an alkyne, alkene, or additional allene, enabled us to build a variety of useful cyclic structures. This account describes our research on allene cycloaddition and cycloisomerization, categorizing the reactions by the proper reaction mode and the cyclic framework of the product.
- Published
- 2011
71. Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway
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Yoshinori Ohsumi, Fuyuhiko Inagaki, Hiroyuki Kumeta, Masaya Yamaguchi, Nobuo N. Noda, and Hitoshi Nakatogawa
- Subjects
Autophagy-Related Protein 8 Family ,Cytoplasm ,Saccharomyces cerevisiae Proteins ,ATG8 ,Amino Acid Motifs ,Autophagy-Related Proteins ,Vacuole ,Ubiquitin-conjugating enzyme ,CVT pathway ,Biology ,Biochemistry ,Protein Structure, Secondary ,Fungal Proteins ,Structural motif ,Molecular Biology ,Phosphatidylethanolamines ,Autophagy ,Cell Biology ,Transport protein ,Cell biology ,Protein Structure, Tertiary ,Protein Transport ,Ubiquitin-Conjugating Enzymes ,Vacuoles ,Protein Structure and Folding ,Microtubule-Associated Proteins ,Signal Transduction - Abstract
The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.
- Published
- 2010
72. Selective Transport of α-Mannosidase by Autophagic Pathways
- Author
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Yasunori Watanabe, Yoshinori Ohsumi, Kuninori Suzuki, Fuyuhiko Inagaki, Hiroyuki Kumeta, and Nobuo N. Noda
- Subjects
ATG8 ,fungi ,Saccharomyces cerevisiae ,Cell Biology ,Vacuole ,Immunoglobulin domain ,Biology ,Protein degradation ,biology.organism_classification ,alpha-Mannosidase ,Biochemistry ,Transport protein ,Cell biology ,Protein structure ,Molecular Biology - Abstract
In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and α-mannosidase (Ams1) are selectively transported to the vacuole through the cytoplasm-to-vacuole targeting pathway under vegetative conditions and through autophagy under starvation conditions. Atg19 plays a central role in these processes by linking Ams1 and prApe1 to Atg8 and Atg11. However, little is known about the molecular mechanisms of cargo recognition by Atg19. Here, we report structural and functional analyses of Atg19 and its paralog, Atg34. A protease-resistant domain was identified in the C-terminal region of Atg19, which was also conserved in Atg34. In vitro pulldown assays showed that the C-terminal domains of both Atg19 and Atg34 are responsible for Ams1 binding; these domains are hereafter referred to as Ams1-binding domains (ABDs). The transport of Ams1, but not prApe1, was blocked in atg19Δatg34Δ cells expressing Atg19ΔABD, indicating that ABD is specifically required for Ams1 transport. We then determined the solution structures of the ABDs of Atg19 and Atg34 using NMR spectroscopy. Both ABD structures have a canonical immunoglobulin fold consisting of eight β-strands with highly conserved loops clustered at one side of the fold. These facts, together with the results of a mutational analysis, suggest that ABD recognizes Ams1 using these conserved loops.
- Published
- 2010
73. Structure determination of proteins in 2H2O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment
- Author
-
Kenji Ogura, Fuyuhiko Inagaki, and Hiroyuki Kumeta
- Subjects
Carbon Isotopes ,Protein Conformation ,Chemistry ,Stereochemistry ,Chemical shift ,Carbon-13 ,Proteins ,Pulse sequence ,Hydrogen-Ion Concentration ,Resonance (chemistry) ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Deuterium ,Amide ,Peptide bond ,Deuterium Oxide ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone (1)Halpha and (13)Calpha to those of (13)C' in the preceding residue. By applying (2)H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone (13)C' and amide (15)N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for (13)C/(15)N-labeled GB1 dissolved in (2)H(2)O. The quality of the GB1 structure determined in (2)H(2)O was similar to that determined in H(2)O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in (2)H(2)O or H(2)O at high pH values.
- Published
- 2010
74. Solution Structure of a Novel Cdc42 Binding Module of Bem1 and Its Interaction with Ste20 and Cdc42
- Author
-
Fuyuhiko Inagaki, Naoki Yoshida, Tomoyuki Takaku, Kenji Ogura, and Hiroyuki Kumeta
- Subjects
Scaffold protein ,Magnetic Resonance Spectroscopy ,Saccharomyces cerevisiae Proteins ,Proline ,Molecular Sequence Data ,Protein domain ,Saccharomyces cerevisiae ,macromolecular substances ,Plasma protein binding ,Protein Serine-Threonine Kinases ,Biology ,Crystallography, X-Ray ,Biochemistry ,SH3 domain ,Protein–protein interaction ,Protein structure ,Cell polarity ,Escherichia coli ,Amino Acid Sequence ,Kinase activity ,cdc42 GTP-Binding Protein ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Sequence Homology, Amino Acid ,Cell Membrane ,fungi ,Intracellular Signaling Peptides and Proteins ,Cell Polarity ,Cell Biology ,MAP Kinase Kinase Kinases ,Protein Structure and Folding ,Biophysics ,biological phenomena, cell phenomena, and immunity ,Protein Binding - Abstract
Bem1 is a scaffold protein essential for the establishment of cell polarity in Saccharomyces cerevisiae. This work reports the solution structure of a Cdc42 binding module of Bem1 comprising the second SH3 domain (SH3b) and its C-terminal flanking region termed Cdc42 interacting (CI). First, the structure of Bem1 SH3b-CI was determined by NMR spectroscopy, which shows that Bem1 SH3b-CI is a structurally and functionally related domain that binds Cdc42. Next, the solution structure of Bem1 SH3b-CI in complex with the proline-rich region of p21-activated kinase Ste20 (Ste20 PRR) was determined. Finally, the interaction surface of Bem1 SH3b-CI with Cdc42 was identified based on chemical shift perturbation studies which reveals that Bem1 SH3b-CI interacts simultaneously with both Ste20 PRR and Cdc42 using the opposite surfaces. Thus, Bem1 can tether Cdc42 and Ste20 in close proximity so that Cdc42 can efficiently interact with Ste20 Cdc42 and Rac interactive binding (CRIB). Based on the present results together with the previous biochemical studies (Lamson, R. E., Winters, M. J., and Pryciak, P. M. (2002) Mol. Cell. Biol. 22, 2939–2951 and Winters, M. J., and Pryciak, P. M. (2005) Mol. Cell. Biol. 25, 2177–2190), a model was suggested that the autoinhibition of Ste20 kinase activity by CRIB is released through the Cdc42-CRIB interaction, which is mediated by Bem1, and Ste20 is subsequently activated, an initial step for the establishment of the cell polarity.
- Published
- 2010
75. Corrigendum to 'Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein Atg8 through Multi-step Recognition' [J. Mol. Biol. 430 (3) (Feb 2, 2018) 249–257]
- Author
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Masaya Yamaguchi, Yoshinori Ohsumi, Yuko Fujioka, Hironori Suzuki, Nobuo N. Noda, Kenji Satoo, and Fuyuhiko Inagaki
- Subjects
Ubiquitin ,biology ,Structural Biology ,Chemistry ,ATG8 ,Autophagy ,biology.protein ,Molecular Biology ,Cell biology - Published
- 2018
76. The NMR structure of the autophagy-related protein Atg8
- Author
-
Yuko Fujioka, Masahiro Watanabe, Masaya Yamaguchi, Wakana Adachi, Hiroyuki Kumeta, Kenji Ogura, Yoshinori Ohsumi, Hitoshi Nakatogawa, Fuyuhiko Inagaki, and Nobuo N. Noda
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Sequence Homology, Amino Acid ,Chemistry ,ATG8 ,Molecular Sequence Data ,Autophagy ,Proteins ,Nuclear magnetic resonance spectroscopy of nucleic acids ,Autophagy-Related Protein 8 Family ,Nuclear magnetic resonance crystallography ,Fluorine-19 NMR ,Nuclear magnetic resonance spectroscopy ,Biochemistry ,Transport protein ,Nuclear magnetic resonance ,Mutation ,Mutagenesis, Site-Directed ,Transverse relaxation-optimized spectroscopy ,Amino Acid Sequence ,Microtubule-Associated Proteins ,Nuclear Magnetic Resonance, Biomolecular ,Sequence Alignment ,Spectroscopy - Published
- 2010
77. Systematic characterization by mass spectrometric analysis of phosphorylation sites in IRF-3 regulatory domain activated by IKK-i
- Author
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Kiyonaga Fujii, Kiyohiro Takahashi, Shingo Nakamura, and Fuyuhiko Inagaki
- Subjects
Phosphopeptides ,Proteomics ,Proteome ,Biophysics ,Models, Biological ,environment and public health ,Biochemistry ,Mass Spectrometry ,Cell Line ,Humans ,Protein phosphorylation ,Phosphorylation ,Transcription factor ,Binding Sites ,Molecular mass ,Chemistry ,Kinase ,Molecular biology ,Recombinant Proteins ,I-kappa B Kinase ,Protein Structure, Tertiary ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Interferon Regulatory Factor-3 ,Peptides ,Interferon regulatory factors - Abstract
Interferon regulatory factor 3 (IRF-3) is a critical transcription factor that regulates innate immune responses against viral infection. Upon infection, IRF-3 is activated through phosphorylation of Ser/Thr residues in its C-terminal domain by the kinases, IKK-i and/or TBK-1. This phosphorylation triggers IRF-3 to interact with the co-activators to form a complex that activates target genes in the nucleus. However, the phosphorylation sites that determine the active/inactive status of IRF-3, estimated using biochemical methods such as mutagenesis and kinase assays, remain controversial. In the present study, phosphorylated IRF-3 189-427 (IRF-3 189C) was prepared by co-expression with IKK-i and was specifically fractionated into 3 major phosphorylation forms using anion-exchange chromatography. Identification of the phosphorylation sites was performed using systematic mass spectrometry approaches as follows: intact molecular mass analysis by nanoESI-MS, MS survey of phosphopeptides, and targeted MS/MS analysis of LC-MS/MS-based proteomics using a high-resolution Orbitrap mass spectrometer. Phosphorylated IRF-3 189C was clearly identified to exist as a mono-phosphoprotein (at Ser-402), and in two di-phosphoprotein forms (at Ser-386, -402 and Ser-396, -402). Thus, we demonstrated that Ser-386, -396 and -402 are directly phosphorylated by IKK-i in the co-expression system. These results will help provide new insights into the IRF-3 activation mechanism.
- Published
- 2010
78. Rhodium(I)-Catalyzed Intramolecular Carbonylative [2+2+1] Cycloadditions and Cycloisomerizations of Bis(sulfonylallene)s
- Author
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Takamasa Kawamura, Fuyuhiko Inagaki, Syu Narita, Chisato Mukai, Shuichi Hirata, Shinji Kitagaki, and Yasuhito Takahashi
- Subjects
chemistry.chemical_classification ,Molecular Structure ,Double bond ,Bicyclic molecule ,Stereochemistry ,Organic Chemistry ,Stereoisomerism ,General Chemistry ,Catalysis ,Cycloaddition ,Alkadienes ,chemistry.chemical_compound ,Cyclononene ,Cycloisomerization ,chemistry ,Cyclization ,Cyclooctene ,Intramolecular force ,Combinatorial Chemistry Techniques ,Cycloheptene ,Rhodium ,Sulfones - Abstract
Novel [{RhCl(CO)dppp}(2)]-catalyzed intramolecular carbonylative [2+2+1] cycloadditions of bis(phenylsulfonylallene) derivatives under CO, leading to the facile formation of bis(phenylsulfonyl)bicyclo[n.3.0] frameworks (n=4-6), have been developed. The terminal double bonds of both allenyl moieties served exclusively as the two pi-components. In particular, this newly developed method was shown to be a powerful tool for the construction of bicyclo[6.3.0]undecadienones, which have hardly been prepared by the known Pauson-Khand (-type) reactions. The bicyclo[7.3.0]dodecadienone homologue (one extra carbon) could be formed in rather low yields. Alternatively, novel cycloisomerizations of bis(phenylsulfonylallene) derivatives with catalysis by the same Rh(I) complex under N(2) readily produced the 3,4-dimethylene-2,5-bis(phenylsulfonyl)cyclononene and the corresponding cyclooctene and cycloheptene frameworks.
- Published
- 2010
79. Rhodium(I)-Catalyzed Intramolecular [5+2] Cycloaddition Reactions of Alkynes and Allenylcyclopropanes: Construction of Bicyclo[5.4.0]undecatrienes and Bicyclo[5.5.0]dodecatrienes
- Author
-
Chisato Mukai, Fuyuhiko Inagaki, Katsuya Sugikubo, and Yusuke Miyashita
- Subjects
Cyclopropanes ,Bicyclic molecule ,Stereochemistry ,Allene ,chemistry.chemical_element ,General Medicine ,General Chemistry ,Catalysis ,Cycloaddition ,Cyclopropane ,Rhodium ,Alkadienes ,Bridged Bicyclo Compounds ,chemistry.chemical_compound ,chemistry ,Alkynes ,Intramolecular force - Published
- 2010
80. Characterization of the Atg17–Atg29–Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae
- Author
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Fuyuhiko Inagaki, Yuko Fujioka, Nobuo N. Noda, Yoshinori Ohsumi, Kuninori Suzuki, and Yukiko Kabeya
- Subjects
Autophagosome ,Saccharomyces cerevisiae Proteins ,biology ,Molecular mass ,Autophagy ,Saccharomyces cerevisiae ,Biophysics ,Autophagy-Related Proteins ,Cell Biology ,Vacuole ,Phosphoproteins ,biology.organism_classification ,Biochemistry ,Cell biology ,Cytosol ,Phagosomes ,Ultracentrifuge ,Carrier Proteins ,Ultracentrifugation ,Molecular Biology ,Ternary complex ,Phagosome - Abstract
Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.
- Published
- 2009
81. Solution structure of the silkworm βGRP/GNBP3 N-terminal domain reveals the mechanism for β-1,3-glucan-specific recognition
- Author
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Masataka Horiuchi, Hiroyuki Kumeta, Fuyuhiko Inagaki, Masanori Ochiai, Kenji Ogura, Kiyohiro Takahasi, and Masaaki Ashida
- Subjects
Models, Molecular ,Protein Folding ,Magnetic Resonance Spectroscopy ,beta-Glucans ,Protein Conformation ,DNA Mutational Analysis ,Molecular Sequence Data ,Sequence alignment ,Plasma protein binding ,Protein structure ,Animals ,Amino Acid Sequence ,Peptide sequence ,Bombyx ,Multidisciplinary ,Innate immune system ,Base Sequence ,biology ,Prophenoloxidase ,Biological Sciences ,biology.organism_classification ,Immunity, Innate ,Biochemistry ,Insect Proteins ,Protein folding ,Carrier Proteins ,Sequence Alignment ,Protein Binding - Abstract
The beta-1,3-glucan recognition protein (betaGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The betaGRP consists of an N-terminal beta-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal beta-1,3-glucan recognition domain of silkworm betaGRP. Although the N-terminal domain of betaGRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that betaGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that betaGRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.
- Published
- 2009
82. Synthesis of a Core Carbon Framework of Cyanosporasides A and B
- Author
-
Fuyuhiko Inagaki, Chisato Mukai, Daisuke Aburano, and Shoichirou Tomonaga
- Subjects
Molecular Structure ,Organic Chemistry ,Chemical modification ,Stereoisomerism ,Lipase ,Sigmatropic reaction ,Pseudomonas fluorescens ,Chemical synthesis ,Medicinal chemistry ,Carbon ,Catalysis ,chemistry.chemical_compound ,Indenes ,chemistry ,Cyclization ,Yield (chemistry) ,Organometallic Compounds ,Organic chemistry ,Rhodium ,Glycosides ,Indene ,Carbonylation ,Carbon monoxide - Abstract
Treatment of 3-(2-ethynylphenyl)prop-2-ynyl benzenesulfinate with 2.5 mol % of [RhCl(CO)(2)](2) at 40 degrees C under an atmosphere of CO effected the successive 2,3-sigmatropic rearrangement and carbonylative [2 + 2 + 1] ring-closing reaction to afford the 8-(phenylsulfonyl)-1H-cyclopent[a]-inden-2-one in a high yield. Chemical modification of the ring-closed product via lipase-mediated optical resolution produced the optically active 3-acetoxy-3a-cyclohexyloxy-3,3a-dihydrocyclopent-[a]indene skeleton, the core carbon framework of cyanosporasides A and B.
- Published
- 2009
83. Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains
- Author
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Natsuko Tsuduki, Ryo Narita, Hiroyuki Kumeta, Takashi Fujita, Fuyuhiko Inagaki, Reiko Hirai, Mitsutoshi Yoneyama, Taeko Shigemoto, Masataka Horiuchi, Kiyohiro Takahasi, and Kenji Ogura
- Subjects
RIG-I ,viruses ,fungi ,LGP2 ,RNA ,MDA5 ,Cell Biology ,Biology ,RIG-I-like receptor ,Biochemistry ,Cell biology ,RNA silencing ,CTD ,DEAD Box Protein 58 ,Molecular Biology - Abstract
The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.
- Published
- 2009
84. Two-point anchoring of a lanthanide-binding peptide to a target protein enhances the paramagnetic anisotropic effect
- Author
-
Fuyuhiko Inagaki, Masashi Yokochi, Yoshihiro Kobashigawa, Tomohide Saio, and Kenji Ogura
- Subjects
Lanthanide ,chemistry.chemical_classification ,Protein Conformation ,Chemistry ,Proteins ,Peptide ,Lanthanoid Series Elements ,Biochemistry ,Protein Structure, Secondary ,Ion ,Paramagnetism ,Crystallography ,Dipole ,Nuclear magnetic resonance ,Protein structure ,Residual dipolar coupling ,Target protein ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Abstract
Paramagnetic lanthanide ions fixed in a protein frame induce several paramagnetic effects such as pseudo-contact shifts and residual dipolar couplings. These effects provide long-range distance and angular information for proteins and, therefore, are valuable in protein structural analysis. However, until recently this approach had been restricted to metal-binding proteins, but now it has become applicable to non-metalloproteins through the use of a lanthanide-binding tag. Here we report a lanthanide-binding peptide tag anchored via two points to the target proteins. Compared to conventional single-point attached tags, the two-point linked tag provides two to threefold stronger anisotropic effects. Though there is slight residual mobility of the lanthanide-binding tag, the present tag provides a higher anisotropic paramagnetic effect.
- Published
- 2009
85. The NMR structure of the TC10- and Cdc42-interacting domain of CIP4
- Author
-
Yoshihiro Kobashigawa, Fuyuhiko Inagaki, Hiroyuki Kumeta, and Daisuke Kanoh
- Subjects
Models, Molecular ,rho GTP-Binding Proteins ,Coiled coil ,Binding Sites ,Chemistry ,Structure (category theory) ,Biochemistry ,Protein Structure, Tertiary ,Domain (software engineering) ,Crystallography ,Protein structure ,cdc42 GTP-Binding Protein ,Microtubule-Associated Proteins ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy - Published
- 2009
86. The Domain Organization of p67phox, a Protein Required for Activation of the Superoxide-Producing NADPH Oxidase in Phagocytes
- Author
-
Hideki Sumimoto, Fuyuhiko Inagaki, Kazuya Honbou, Satoru Yuzawa, and Kei Miyano
- Subjects
Innate immune system ,NADPH oxidase ,COS cells ,biology ,Phagocyte ,Superoxide ,Chinese hamster ovary cell ,Phagocytosis ,fungi ,Cell biology ,Enzyme activator ,chemistry.chemical_compound ,medicine.anatomical_structure ,Biochemistry ,chemistry ,biology.protein ,medicine ,Immunology and Allergy - Abstract
The phagocyte NADPH oxidase, crucial for innate immunity, is dormant in resting cells, but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. In activation of the oxidase, the multidomain protein p67phoxplays a central role: it translocates to the membrane as a ternary complex with p47phoxand p40phox, and interacts with the small GTPase Rac to assemble with the membrane-integrated catalytic protein gp91phox, leading to superoxide production. Here we show, using small-angle X-ray scattering (SAXS) analysis, that p67phoxadopts an elongated conformation when it exists not only as a monomer but also as the heterotrimer. Although p67phoxharbors an N-terminal TPR domain for binding to Rac and a p40phox-interacting PB1 domain, followed by an SH3 domain that associates with p47phox, the present model suggests that no or few apparent associations occur between the domains. The positions of the protein-interaction domains in p67phoxcontribute to activation of the phagocyte NADPH oxidase: the first SH3 domain that is located between the TPR and PB1 domains positively regulates oxidase activation only when it is present at the correct position; the PB1 domain placed at this SH3 domain position inhibits the oxidase by interacting with p40phox.
- Published
- 2009
87. Intramolecular Cycloaddition Between Allenyl .PI.-Bond and Multiple Bond
- Author
-
Fuyuhiko Inagaki, Shinji Kitagaki, and Chisato Mukai
- Subjects
chemistry.chemical_compound ,Cycloisomerization ,chemistry ,Bicyclic molecule ,Allene ,Intramolecular force ,Organic Chemistry ,Cycloheptene ,Organic synthesis ,Pi bond ,Medicinal chemistry ,Cycloaddition - Abstract
Allene functionality has the potential to create interesting and useful reactions in organic synthesis. We have developed the following several novel cycloaddition reactions between allenyl π-bond and carbon-carbon multiple bond: (i) Intramolecular Pauson-Khand-type reaction of allene-alkyne substrates (allenynes) leading to construction of bicyclo[5.3.0]decadienone framework has been realized with the aid of Rh(I) catalyst under CO atmosphere. (ii) The allenynes were found to produce cycloheptene derivatives and/or bicyclo[5.2.0]nonadiene compounds depending on the substitution pattern at the allenic terminus under N2. (iii) Thermal[2+2]cycloaddition of allenynes was shown to be applicable to the construction of bicyclo[6.2.0]skeleton. (iv) We also found that the Pauson-Khand-type reaction of allene-alkenes (allenenes) and bisallenes resulted in the easy preparation of bicyclo[4.3.0]nonenone skeleton having an alkyl appendage at the ring juncture and bicyclo[6.3.0]undecadienone derivatives, respectively.
- Published
- 2009
88. Structural basis of target recognition by Atg8/LC3 during selective autophagy
- Author
-
Wakana Adachi, Kenji Satoo, Nobuo N. Noda, Hiroyuki Kumeta, Yuko Fujioka, Fuyuhiko Inagaki, Junko Ishii, Yoshinori Ohsumi, and Hitoshi Nakatogawa
- Subjects
Autophagy-Related Protein 8 Family ,Membrane ,Cytoplasm ,ATG8 ,Vesicle ,Autophagy ,Genetics ,Cell Biology ,Biology ,Protein aggregation ,Transport protein ,Cell biology - Abstract
Autophagy is a non-selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double-membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin-like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease-related protein aggregates, respectively. Using X-ray crystallography and NMR, we herein report the structural basis for Atg8–Atg19 and LC3–p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side-chains of Trp and Leu in a four-amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL-containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.
- Published
- 2008
89. Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor
- Author
-
Takanori Shiga, Terrence R. Burke, Satoru Yuzawa, Fuyuhiko Inagaki, Kenji Ogura, and Masashi Yokochi
- Subjects
Models, Molecular ,Protein Conformation ,Recombinant Fusion Proteins ,Dimer ,Crystal structure ,Crystallography, X-Ray ,SH2 domain ,Peptides, Cyclic ,Biochemistry ,Article ,src Homology Domains ,Structure-Activity Relationship ,chemistry.chemical_compound ,Biomimetics ,Escherichia coli ,Spectroscopy ,Nuclear Magnetic Resonance, Biomolecular ,GRB2 Adaptor Protein ,Glutathione Transferase ,Binding Sites ,Molecular Structure ,biology ,Chemistry ,Intermolecular force ,Solution structure ,Protein Subunits ,Crystallography ,Models, Chemical ,Chromatography, Gel ,biology.protein ,GRB2 ,biological phenomena, cell phenomena, and immunity ,Hinge region ,Protein Binding - Abstract
The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.
- Published
- 2008
90. The Structure of the Aggregate Form of Bacteriochlorophyll c Showing the Qy Absorption above 740 nm as Determined by the Ring-current Effects on 1H and 13C Nuclei and by 1H-1H Intermolecular NOE Correlations
- Author
-
Fuyuhiko Inagaki, Shun Sakamoto, Yasushi Koyama, Kenji Ogura, and Tadashi Mizoguchi
- Subjects
Stereochemistry ,Chemical shift ,Intermolecular force ,General Medicine ,Nuclear Overhauser effect ,Biochemistry ,chemistry.chemical_compound ,Crystallography ,chemistry ,Heteronuclear molecule ,Molecule ,Physical and Theoretical Chemistry ,Methylene ,Absorption (chemistry) ,Spectroscopy - Abstract
13C-enriched bacteriochlorophyll c (S[I, E] BChl cF) was suspended in a 1:3 mixture of methylene chloride and carbon tetrachloride to form an aggregate showing the Qy absorption above 740 nm; changes in the 13C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and then, the changes were correlated to the ring-current effects due to the neighboring macrocycles in the aggregate. A pair of aggregate structures has been proposed based on the ring-current effects on both 1H and 13C nuclei; the monomeric units are stacked together to form an inclined column with different sliding directions, in which the y-axis of the molecule is parallel to the long axis of the column. In order to confirm this pair of models, the ring-current effects on the 1H and 13C nuclei were calculated based on both the magnetic-dipole and the loop-current approximations. Further, an application of three-dimensional F1 13C-edited F3 13C-filtered heteronuclear single-quantum correlation-nuclear Overhauser effect spectroscopy to the above aggregate consisting of a 1:1 mixture of 13C-labeled and unlabeled BChl c succeeded in detecting selectively the 1H–1H intermolecular nuclear Overhauser effect correlations, which established the coexistence of the above pair of stacked structures in the aggregate.
- Published
- 2008
91. The Atg12-Atg5 Conjugate Has a Novel E3-like Activity for Protein Lipidation in Autophagy
- Author
-
Nobuo N. Noda, Yuko Fujioka, Yoshinobu Ichimura, Yoshinori Ohsumi, Yoshinori Satomi, Takao Hanada, Toshifumi Takao, and Fuyuhiko Inagaki
- Subjects
Autophagy-Related Protein 8 Family ,Saccharomyces cerevisiae Proteins ,Time Factors ,Ubiquitin-Protein Ligases ,ATG8 ,ATG5 ,Saccharomyces cerevisiae ,Biology ,Protein lipidation ,Models, Biological ,Biochemistry ,Autophagy-Related Protein 5 ,ATG12 ,Gene Expression Regulation, Fungal ,Autophagy ,Escherichia coli ,Molecular Biology ,Ubiquitin ,Cell Biology ,Lipids ,Cell biology ,Liposomes ,Autophagosome membrane ,Protein Processing, Post-Translational ,Autophagy-Related Protein 12 ,Conjugate - Abstract
Autophagy is a bulk degradation process in eukaryotic cells; autophagosomes enclose cytoplasmic components for degradation in the lysosome/vacuole. Autophagosome formation requires two ubiquitin-like conjugation systems, the Atg12 and Atg8 systems, which are tightly associated with expansion of autophagosomal membrane. Previous studies have suggested that there is a hierarchy between these systems; the Atg12 system is located upstream of the Atg8 system in the context of Atg protein organization. However, the concrete molecular relationship is unclear. Here, we show using an in vitro Atg8 conjugation system that the Atg12-Atg5 conjugate, but not unconjugated Atg12 or Atg5, strongly enhances the formation of the other conjugate, Atg8-PE. The Atg12-Atg5 conjugate promotes the transfer of Atg8 from Atg3 to the substrate, phosphatidylethanolamine (PE), by stimulating the activity of Atg3. We also show that the Atg12-Atg5 conjugate interacts with both Atg3 and PE-containing liposomes. These results indicate that the Atg12-Atg5 conjugate is a ubiquitin-protein ligase (E3)-like enzyme for Atg8-PE conjugation reaction, distinctively promoting protein-lipid conjugation.
- Published
- 2007
92. An efficient method for protein phosphorylation using the artificially introduced of cognate-binding modules into kinases and substrates
- Author
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Yoshihiro Kobashigawa, Masato Naito, and Fuyuhiko Inagaki
- Subjects
inorganic chemicals ,Genetic Vectors ,Molecular Sequence Data ,Bioengineering ,macromolecular substances ,Biology ,environment and public health ,Applied Microbiology and Biotechnology ,SH3 domain ,Substrate Specificity ,Phosphorylation cascade ,Mice ,Animals ,Humans ,Protein phosphorylation ,Amino Acid Sequence ,Phosphorylation ,Proto-Oncogene Proteins c-abl ,Proto-Oncogene Proteins c-vav ,GRB2 Adaptor Protein ,MAPK14 ,chemistry.chemical_classification ,Base Sequence ,Kinase ,Phosphotransferases ,General Medicine ,Proto-Oncogene Proteins c-crk ,enzymes and coenzymes (carbohydrates) ,Enzyme ,chemistry ,Biochemistry ,bacteria ,Signal transduction ,Biotechnology - Abstract
Protein phosphorylation is a major post-translational modification that regulates cellular signal transduction. The phosphorylation of substrate proteins by kinases requires cognate pairs of substrates and kinases. In addition, phosphorylation is mediated through both indirect and direct interaction between these kinases and substrates, which makes it difficult to effectively prepare large quantities of recombinant phosphorylated proteins. Here, we report a novel protein phosphorylation method involving the artificial introduction of cognate-binding modules into substrates and enzymes. This enhances the local concentration of substrates around enzymes so that the enzymatic reaction proceeds more efficiently. We prepared substrate proteins containing an SH3 domain at their N-terminus, and a kinase containing an SH3-binding motif at its C-terminus. This method was successfully applied to the phosphorylation of CrkII and the Vav DH domain, and we prepared (15)N-labelled phosphorylated CrkII for NMR analysis.
- Published
- 2007
93. Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK
- Author
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Kenji Ogura, Yoshinori Makino, Yoshihiro Kobashigawa, Fuyuhiko Inagaki, Hiroyuki Kumeta, Mieko Sakai, Masato Naito, Shinya Tanaka, and Masashi Yokochi
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,animal structures ,DNA Mutational Analysis ,Molecular Sequence Data ,Small G Protein ,Plasma protein binding ,Biology ,Adapter molecule crk ,Structural Biology ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Signal transducing adaptor protein ,Proto-Oncogene Proteins c-crk ,Protein Structure, Tertiary ,Cell biology ,Cell Transformation, Neoplastic ,Bromodeoxyuridine ,Biochemistry ,Mutation ,Phosphorylation ,Signal transduction ,Tyrosine kinase ,Protein Binding ,Signal Transduction - Abstract
CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.
- Published
- 2007
94. The NMR structure of the domain II of a chloroplastic NifU-like protein OsNifU1A
- Author
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Kenji Ogura, Munehiko Asayama, Hiroyuki Kumeta, Fuyuhiko Inagaki, Keizo Teshima, Shizue Katoh, and Etsuko Katoh
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Chloroplasts ,Tandem ,Stereochemistry ,Static Electricity ,Protein domain ,Oryza ,Biochemistry ,Protein Structure, Secondary ,Protein Structure, Tertiary ,Amino acid ,Structural genomics ,Crystallography ,Protein structure ,chemistry ,Static electricity ,Cluster (physics) ,Nuclear Magnetic Resonance, Biomolecular ,Spectroscopy ,Ferredoxin ,Plant Proteins - Abstract
NifU-like proteins are a highly conserved protein that serves as the scaffold for assembly of Fe-S clusters. Chloroplastic NifU-like proteins have tandem NifU like domains, named domain I and domain II. Although the amino acid sequences of these domains are very similar to each other, the predicted functional region for the Fe-S cluster assembly, the CXXC motif, exists only in domain I. The structure of the domain II of chloroplastic NifU-like protein OsNifU1A has an alpha-beta sandwich structure containing two alpha helices located on one side of the beta-sheet. The electrostatic surface potential of OsNifU1A domain II is predominantly positively charged. Chloroplastic NifU-like proteins are targeted to ferredoxin for transferring the Fe-S cluster. The ferredoxin presents an overall negatively charged surface, which may evoke an electrostatic association with OsNifU1A domain II.
- Published
- 2007
95. The crystal structure of ATG3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation
- Author
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Yuya Yamada, Hiroyuki Kumeta, Yoshinobu Ichimura, Yuko Fujioka, Fuyuhiko Inagaki, Yoshinori Ohsumi, Takao Hanada, and Nobuo Suzuki
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Protein Conformation ,ATG8 ,Saccharomyces cerevisiae ,Molecular Sequence Data ,Autophagy-Related Proteins ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,Autophagy-Related Protein 7 ,Protein Structure, Secondary ,Protein structure ,Ubiquitin ,Catalytic Domain ,Autophagy ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Glutathione Transferase ,chemistry.chemical_classification ,DNA ligase ,biology ,Sequence Homology, Amino Acid ,Chemistry ,Cell Biology ,Autophagy-Related Protein 8 Family ,biology.organism_classification ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Biophysics ,Microtubule-Associated Proteins ,Protein Binding - Abstract
Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-A resolution. Atg3 has an alpha/beta-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of approximately 80 residues and has a random coil structure in solution and another with a long alpha-helical structure that protrudes from the core region as far as 30 A. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.
- Published
- 2007
96. Structure of Atg5·Atg16, a Complex Essential for Autophagy
- Author
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Keisuke Obara, Yuko Fujioka, Fuyuhiko Inagaki, Nobuo Suzuki, Minako Matsushita, and Yoshinori Ohsumi
- Subjects
Saccharomyces cerevisiae Proteins ,Multiprotein complex ,Protein Conformation ,ATG5 ,Mutant ,Autophagy-Related Proteins ,Biology ,Crystallography, X-Ray ,medicine.disease_cause ,BAG3 ,Biochemistry ,Autophagy-Related Protein 5 ,ATG12 ,Yeasts ,Autophagy ,medicine ,Animals ,Humans ,Molecular Biology ,Mutation ,Ubiquitin ,Phosphatidylethanolamines ,Cell Biology ,Cell biology ,Multiprotein Complexes ,Carrier Proteins ,Microtubule-Associated Proteins ,Protein Processing, Post-Translational ,Protein Binding ,Conjugate - Abstract
Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5·Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97Å resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.
- Published
- 2007
97. Structural Biology of Autophagy
- Author
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Fuyuhiko Inagaki and Nobuo Suzuki
- Subjects
Structural biology ,Chemistry ,Autophagy ,Cell biology - Published
- 2007
98. ChemInform Abstract: Concise Construction of Bicyclo[6.4.0] and -[7.4.0] Frameworks by [4 + 2] Cycloaddition of 3,4-Dimethylene-2,5-bis(phenylsulfonyl)cycloalk-1-enes
- Author
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Yasuhito Takahashi, Masanobu Ueda, Chisato Mukai, and Fuyuhiko Inagaki
- Subjects
chemistry.chemical_compound ,Cyclononene ,chemistry ,Bicyclic molecule ,Cyclooctene ,Intermolecular force ,chemistry.chemical_element ,General Medicine ,Medicinal chemistry ,Carbon ,Vicinal ,Cycloaddition - Abstract
The thermal intermolecular [4+2] cycloadditions of vicinal bis(exo-methylene)cyclooctene with carbon dienophiles efficiently produce the bicyclo[6.4.0] framework. Similar cycloadditions with the one-carbon-homologated cyclononene derivatives result in the formation of the bicyclo[7.4.0] skeleton. Both aza- and oxadieonphiles are suitable for this [4+2] reaction and provide the corresponding hetero-Diels–Alder-type products.
- Published
- 2015
99. The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy*
- Author
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Takayuki Shima, Rinji Akada, Hayashi Yamamoto, Chika Kondo-Kakuta, Yuh Mochizuki, Yoshinori Ohsumi, Takehiko Itoh, Hisashi Hoshida, Masaya Yamaguchi, Nobuo N. Noda, Soichiro Kakuta, and Fuyuhiko Inagaki
- Subjects
endocrine system ,Protein Denaturation ,Protein Folding ,Magnetic Resonance Spectroscopy ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,education ,Green Fluorescent Proteins ,Biochemistry ,Kluyveromyces ,Open Reading Frames ,Kluyveromyces marxianus ,Autophagy ,Fluorometry ,Molecular Biology ,biology ,Computational Biology ,Cell Biology ,biology.organism_classification ,In vitro ,Yeast ,Recombinant Proteins ,Open reading frame ,Microscopy, Electron ,Microscopy, Fluorescence ,Solubility ,Function (biology) - Abstract
Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.
- Published
- 2015
100. ChemInform Abstract: Rhodium(I)-Catalyzed Cycloisomerization of Allene-Allenylcyclopropanes
- Author
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Yasuaki Kawaguchi, Fuyuhiko Inagaki, Katsuya Sugikubo, Chisato Mukai, and Takamasa Kawamura
- Subjects
chemistry.chemical_compound ,Ethylene ,Cycloisomerization ,chemistry ,Bicyclic molecule ,Allene ,Moiety ,chemistry.chemical_element ,General Medicine ,Medicinal chemistry ,Cyclopropane ,Catalysis ,Rhodium - Abstract
Under conditions A), substrates bearing electron-deficient substituents at both allenyl sites produce a bicyclo[5.3.0]deca-1,6,8-triene skeleton with incorporation of the cyclopropane moiety as C1-building block and concomitant elimination of ethylene.
- Published
- 2015
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