Researcher Profile and Settings

Master

Affiliation (Master)

• Institute for Genetic Medicine　Disease Control

Affiliation (Master)

• Institute for Genetic Medicine　Disease Control

researchmap

Profile and Settings

Affiliation

• Hokkaido University, Institute for Genetic Medicine, Assistant professor

Profile and Settings

• Name (Japanese)

  Noshiro

• Name (Kana)

  Daisuke

• Name

  201801014910663266

Affiliation

• Hokkaido University, Institute for Genetic Medicine, Assistant professor

Achievement

Research Interests

• リポソーム   intrinsically disordered protein   liquid-liquid phase separation (LLPS)   protein science   autophagy   electrophysiology   high-speed atomic force microscopy   

Research Areas

• Life sciences / Pharmaceuticals - analytical and physicochemistry / imaging

Research Experience

• 2023/04 - Today Hokkaido University Institute for Genetic Medicine Assistant Professor
• 2022/04 - 2023/03 Hokkaido University Institute for Genetic Medicine
• 2018/04 - 2022/03 Microbial Chemistry Research Foundation
• 2017/10 - 2018/03 金沢大学ナノ生命科学研究所 博士研究員
• 2013/10 - 2017/09 金沢大学バイオAFM先端研究センター 博士研究員
• 2012/10 - 2013/09 University of Oxford
• 2012/04 - 2012/09 Kyoto University Institute for Chemical Research

Education

• 2009/04 - 2012/03  京都大学大学院
• 2007/04 - 2009/03  京都大学大学院
• 2003/04 - 2007/03  Kyoto University  Faculty of Pharmaceutical Sciences

Published Papers

• Complete set of the Atg8-E1-E2-E3 conjugation machinery forms an interaction web that mediates membrane shaping.

  Jahangir Md Alam, Tatsuro Maruyama, Daisuke Noshiro, Chika Kakuta, Tetsuya Kotani, Hitoshi Nakatogawa, Nobuo N Noda

  Nature structural & molecular biology 31 (1) 170 - 178 2024/01 [Refereed][Not invited]
   

  Atg8, a ubiquitin-like protein, is conjugated with phosphatidylethanolamine (PE) via Atg7 (E1), Atg3 (E2) and Atg12-Atg5-Atg16 (E3) enzymatic cascade and mediates autophagy. However, its molecular roles in autophagosome formation are still unclear. Here we show that Saccharomyces cerevisiae Atg8-PE and E1-E2-E3 enzymes together construct a stable, mobile membrane scaffold. The complete scaffold formation induces an in-bud in prolate-shaped giant liposomes, transforming their morphology into one reminiscent of isolation membranes before sealing. In addition to their enzymatic roles in Atg8 lipidation, all three proteins contribute nonenzymatically to membrane scaffolding and shaping. Nuclear magnetic resonance analyses revealed that Atg8, E1, E2 and E3 together form an interaction web through multivalent weak interactions, where the intrinsically disordered regions in Atg3 play a central role. These data suggest that all six Atg proteins in the Atg8 conjugation machinery control membrane shaping during autophagosome formation.
• Immobilization of lipid nanorods onto two-dimensional crystals of protein tamavidin 2 for high-speed atomic force microscopy.

  Daisuke Noshiro, Nobuo N Noda

  STAR protocols 4 (4) 102633 - 102633 2023/12/02 [Refereed][Not invited]
   

  High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action. For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1.
• Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

  Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda, Masaaki Komatsu

  The EMBO journal 42 (14) e113349  2023/07/17 [Refereed]
   

  NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.
• Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex

  Reo Kurusu, Yuki Fujimoto, Hideaki Morishita, Daisuke Noshiro, Shuhei Takada, Koji Yamano, Hideaki Tanaka, Ritsuko Arai, Shun Kageyama, Tomoko Funakoshi, Satoko Komatsu-Hirota, Hikari Taka, Saiko Kazuno, Yoshiki Miura, Masato Koike, Toshifumi Wakai, Satoshi Waguri, Nobuo N. Noda, Masaaki Komatsu

  Developmental Cell 58 (13) 1189 - 1205.e11 1534-5807 2023/07 [Refereed]
  
• The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy.

  Tomoyuki Fukuda, Kentaro Furukawa, Tatsuro Maruyama, Shun-Ichi Yamashita, Daisuke Noshiro, Chihong Song, Yuta Ogasawara, Kentaro Okuyama, Jahangir Md Alam, Manabu Hayatsu, Tetsu Saigusa, Keiichi Inoue, Kazuho Ikeda, Akira Takai, Lin Chen, Vikramjit Lahiri, Yasushi Okada, Shinsuke Shibata, Kazuyoshi Murata, Daniel J Klionsky, Nobuo N Noda, Tomotake Kanki

  Molecular cell 83 (12) 2045 - 2058 2023/06/15 [Refereed]
   

  Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.
• Development of hidden Markov modeling method for molecular orientations and structure estimation from high-speed atomic force microscopy time-series images.

  Tomonori Ogane, Daisuke Noshiro, Toshio Ando, Atsuko Yamashita, Yuji Sugita, Yasuhiro Matsunaga

  PLoS computational biology 18 (12) e1010384  2022/12/29 [Refereed]
   

  High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between H frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is applicable in integrative modeling of conformational dynamics using HS-AFM data.
• The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

  Ryosuke Ishimura, Afnan H El-Gowily, Daisuke Noshiro, Satoko Komatsu-Hirota, Yasuko Ono, Mayumi Shindo, Tomohisa Hatta, Manabu Abe, Takefumi Uemura, Hyeon-Cheol Lee-Okada, Tarek M Mohamed, Takehiko Yokomizo, Takashi Ueno, Kenji Sakimura, Tohru Natsume, Hiroyuki Sorimachi, Toshifumi Inada, Satoshi Waguri, Nobuo N Noda, Masaaki Komatsu

  Nature communications 13 (1) 7857 - 7857 2022/12/21 [Refereed]
   

  Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.
• A glutamine sensor that directly activates TORC1

  Mirai Tanigawa, Katsuyoshi Yamamoto, Satoru Nagatoishi, Koji Nagata, Daisuke Noshiro, Nobuo N. Noda, Kouhei Tsumoto, Tatsuya Maeda

  Communications Biology 4 (1) 2021/09 [Refereed]
   

  TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells.
• Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy

  Noriyuki Kodera, Daisuke Noshiro, Sujit K. Dora, Tetsuya Mori, Johnny Habchi, David Blocquel, Antoine Gruet, Marion Dosnon, Edoardo Salladini, Christophe Bignon, Yuko Fujioka, Takashi Oda, Nobuo N. Noda, Mamoru Sato, Marina Lotti, Mineyuki Mizuguchi, Sonia Longhi, Toshio Ando

  Nature Nanotechnology 16 (2) 181 - 189 1748-3387 2021/02 [Refereed]
  
• p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

  Shun Kageyama, Sigurdur Runar Gudmundsson, Yu-Shin Sou, Yoshinobu Ichimura, Naoki Tamura, Saiko Kazuno, Takashi Ueno, Yoshiki Miura, Daisuke Noshiro, Manabu Abe, Tsunehiro Mizushima, Nobuaki Miura, Shujiro Okuda, Hozumi Motohashi, Jin-A Lee, Kenji Sakimura, Tomoyuki Ohe, Nobuo N. Noda, Satoshi Waguri, Eeva-Liisa Eskelinen, Masaaki Komatsu

  Nature Communications 12 (1) 2021/01 [Refereed]
   

  Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.
• Author Correction: Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.

  Kazuaki Matoba, Tetsuya Kotani, Akihisa Tsutsumi, Takuma Tsuji, Takaharu Mori, Daisuke Noshiro, Yuji Sugita, Norimichi Nomura, So Iwata, Yoshinori Ohsumi, Toyoshi Fujimoto, Hitoshi Nakatogawa, Masahide Kikkawa, Nobuo N Noda

  Nature structural & molecular biology 27 (12) 1209 - 1209 2020/12 

  An amendment to this paper has been published and can be accessed via a link at the top of the paper.
• Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.

  Kazuaki Matoba, Tetsuya Kotani, Akihisa Tsutsumi, Takuma Tsuji, Takaharu Mori, Daisuke Noshiro, Yuji Sugita, Norimichi Nomura, So Iwata, Yoshinori Ohsumi, Toyoshi Fujimoto, Hitoshi Nakatogawa, Masahide Kikkawa, Nobuo N Noda

  Nature structural & molecular biology 27 (12) 1185 - 1193 2020/12 [Refereed]
   

  The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
• Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM

  Yuki Kawasaki, Hirotaka Ariyama, Hajime Motomura, Daisuke Fujinami, Daisuke Noshiro, Toshio Ando, Daisuke Kohda

  Journal of Molecular Biology 432 (22) 5951 - 5965 0022-2836 2020/11 [Refereed]
   

  Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin-avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle.
• Liquidity Is a Critical Determinant for Selective Autophagy of Protein Condensates.

  Akinori Yamasaki, Jahangir Md Alam, Daisuke Noshiro, Eri Hirata, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N Noda

  Molecular cell 77 (6) 1163 - 1175 2020/03/19 [Refereed][Not invited]
   

  Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.
• Phase separation organizes the site of autophagosome formation.

  Yuko Fujioka, Jahangir Md Alam, Daisuke Noshiro, Kazunari Mouri, Toshio Ando, Yasushi Okada, Alexander I May, Roland L Knorr, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N Noda

  Nature 578 (7794) 301 - 305 2020/02 [Refereed][Not invited]
   

  Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.
• The induction of RANKL molecule clustering could stimulate early osteoblast differentiation.

  Sone E, Noshiro D, Ikebuchi Y, Nakagawa M, Khan M, Tamura Y, Ikeda M, Oki M, Murali R, Fujimori T, Yoda T, Honma M, Suzuki H, Ando T, Aoki K

  Biochemical and biophysical research communications 509 (2) 435 - 440 0006-291X 2019/02 [Refereed][Not invited]
   

  We recently found that the membrane-bound receptor activator of NF-kappa B ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. lmmunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation. (C) 2018 Elsevier Inc. All rights reserved.
• Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

  Daisuke Noshiro, Toshio Ando

  Philosophical Transactions of the Royal Society B: Biological Sciences 373 (1749) 1471-2970 2018/06/19 [Refereed][Not invited]
   

  A double-ring-shaped tetradecameric GroEL complex assists proper protein folding in cooperation with the cochaperonin GroES. The dynamic GroEL– GroES interaction reflects the allosteric intra- and inter-ring communications and the chaperonin reaction. Therefore, revealing this dynamic interaction is essential to understanding the allosteric communications and the operation mechanism of GroEL. Nevertheless, how this interaction proceeds in the chaperonin cycle has long been controversial. Here, we directly image the dynamic GroEL–GroES interaction under conditions with and without foldable substrate protein using high-speed atomic force microscopy. Then, the imaging results obtained under these conditions and our previous results in the presence of unfoldable substrate are compared. The molecular movies reveal that the entire reaction pathway is highly complicated but basically identical irrespective of the substrate condition. A prominent (but moderate) difference is in the population distribution of intermediate species: symmetric GroEL: GroES2 and asymmetric GroEL: GroES1 complexes, and GroES– unbound GroEL. This difference is mainly attributed to the longer lifetime of GroEL: GroES1 complexes in the presence of foldable substrate. Moreover, the inter-ring communication, which is the basis for the alternating action of the two rings, occurs at two distinct (GroES association and dissociation) steps in the main reaction pathway, irrespective of the substrate condition. This article is part of a discussion meeting issue ‘Allostery and molecular machines’.
• Calmodulin EF-hand peptides as Ca2+-switchable recognition tags

  Akihiko Oku, Miki Imanishi, Daisuke Noshiro, Tomo Murayama, Toshihide Takeuchi, Ikuhiko Nakase, Shiroh Futaki

  BIOPOLYMERS 108 (1) 0006-3525 2017/01 [Refereed][Not invited]
   

  Calmodulin is a representative calcium-binding protein comprised of four Ca2+-binding motifs with a helix-loop-helix structure (EF-hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF-hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N- or C-terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild-type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+-switchable recognition tags. An example of an "EF-tag" system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented.
• The Intrinsically Disordered Protein Atg13 Mediates Supramolecular Assembly of Autophagy Initiation Complexes

  Hayashi Yamamoto, Yuko Fujioka, Sho W. Suzuki, Daisuke Noshiro, Hironori Suzuki, Chika Kondo-Kakuta, Yayoi Kimura, Hisashi Hirano, Toshio Ando, Nobuo N. Noda, Yoshinori Ohsumi

  DEVELOPMENTAL CELL 38 (1) 86 - 99 1534-5807 2016/07 [Refereed][Not invited]
   

  Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
• Extramembrane control of ion channel peptide assemblies, using alamethicin as an example

  Shiroh Futaki, Daisuke Noshiro, Tatsuto Kiwada, Koji Asami

  Accounts of Chemical Research 46 (12) 2924 - 2933 0001-4842 2013/12/17 [Refereed][Not invited]
   

  Ion channels allow the influx and efflux of specific ions through a plasmamembrane. Many ion channels can sense, for example, the membrane potential (the voltage gaps between the inside and the outside of the membrane), specific ligands such as neurotransmitters, and mechanical tension within the membrane. They modulate cell function in response to these stimuli. Researchers have focused on developing peptide- and non-peptide-based model systems to elucidate ion-channel protein functions and to create artificial sensing systems.In this Account, we employed a typical peptide that forms ion channels,alamethicin, as a model to evaluate our methodologies for controlling the assembly states of channel-forming molecules in membranes. As alamethicin self-assembles in membranes, it prompts channel formation, but number of peptide molecules in these channels is not constant. Using planar-lipid bilayer methods, we monitored the association states of alamethicin in real time.Many ligand-gated, natural-ion channel proteins have large extramembranedomains. As these proteins interact with specific ligands, those conformational alterations in the extramembrane domains are transmitted to the transmembrane, pore-forming domains to open and close the channels. We hypothesized that if we conjugated suitable extramembrane segments to alamethicin, ligand binding to the extramembrane segments could alter the structure of the extramembrane domains and influence the association states or association numbers of alamethicin in the membranes. We could then assess those changes by using single-channel current recording. We found that we could modulate channel assembly and eventual ion flux with attached leucine-zipper extramembrane peptide segments. Using conformationally switchable leucine-zipper extramembrane segments that respond to Fe 3+, we fabricated an artificial Fe3+-sensitive ion channel a decrease in the helical content of the extramembrane segment led to an increase in the channel current.When we added a calmodulin C-terminus segment, we formed a channel that was sensitive to Ca2+. This result demonstrated that we could prepare artificial channels that were sensitive to specific ligands by adding appropriate extramembrane segments from natural protein motifs that respond to external stimuli.In conclusion, our research points to the possibility of creating tailored sensor or signal transduction systems through the conjugation of a conformationally switchable extramembrane peptide/protein segment to a suitable transmembrane peptide segment. © 2013 American Chemical Society.
• Construction of a Ca2+-gated artificial channel by fusing alamethicin with a calmodulin-derived extramembrane segment

  Daisuke Noshiro, Kazuhiro Sonomura, Hao-Hsin Yu, Miki Imanishi, Koji Asami, Shiroh Futaki

  Bioconjugate Chemistry 24 (2) 188 - 195 1043-1802 2013/02/20 [Refereed][Not invited]
   

  Using native chemical ligation, we constructed a Ca2+-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca2+, this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca2+ significantly lengthened the specific channel open state and increased the mean channel current, while Mg2+ produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca 2+-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein. © 2012 American Chemical Society.
• Control of leakage activities of alamethicin analogs by metals: Side chain-dependent adverse gating response to Zn2+

  Daisuke Noshiro, Koji Asami, Shiroh Futaki

  BIOORGANIC & MEDICINAL CHEMISTRY 20 (23) 6870 - 6876 0968-0896 2012/12 [Refereed][Not invited]
   

  Alamethicin (Alm), an antimicrobial peptide rich in alpha-aminoisobutyric acid (Aib), is known to self-assemble to form channels in the membranes. Previously, we reported that HG-Alm, an Alm analog with a single His residue at the N-terminus, forms channel assemblies with extremely long lifetimes in the presence of Zn2+. In this study, HG-Alm analogs, in the sequences of which all Aib residues were substituted by Leu, norvaline (Nva), or norleucine (Nle), were synthesized and their leakage activities were measured using fluorescent dye-loaded liposomes. We found that these peptides could be categorized into two classes with different gating responses to Zn2+. (C) 2012 Elsevier Ltd. All rights reserved.
• Metal-Stimulated Regulation of Transcription by an Artificial Zinc-Finger Protein

  Miki Imanishi, Tomohiro Nakaya, Tatsuya Morisaki, Daisuke Noshiro, Shiroh Futaki, Yukio Sugiura

  CHEMBIOCHEM 11 (12) 1653 - 1655 1439-4227 2010/08 [Refereed][Not invited]
  
• Metal-Assisted Channel Stabilization: Disposition of a Single Histidine on the N-terminus of Alamethicin Yields Channels with Extraordinarily Long Lifetimes

  Daisuke Noshiro, Koji Asami, Shiroh Futaki

  BIOPHYSICAL JOURNAL 98 (9) 1801 - 1808 0006-3495 2010/05 [Refereed][Not invited]
   

  Alamethicin, a member of the peptaibol family of antibiotics, is a typical channel-forming peptide with a helical structure. The self-assembly of the peptide in the membranes yields voltage-dependent channels. In this study, three alamethicin analogs possessing a charged residue (His, Lys, or Glu) on their N-termini were designed with the expectation of stabilizing the transmembrane structure. A slight elongation of channel lifetime was observed for the Lys and Glu analogs. On the other hand, extensive stabilization of certain channel open states was observed for the His analog. This stabilization was predominantly observed in the presence of metal ions such as Zn(2+), suggesting that metal coordination with His facilitates the formation of a supramolecular assembly in the membranes. Channel stability was greatly diminished by acetylation of the N-terminal amino group, indicating that the N-terminal amino group also plays an important role in metal coordination.
• Creation of metal sensitive channel-forming peptides: Metal-assisted peptide assembly and its application

  Daisuke Noshiro, Koji Asami, Shiroh Futaki

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 128 41 - 41 0031-6903 2008 [Not refereed][Not invited]
  

MISC

• オートファジー研究から拡大する細胞質ゾーニングの世界 液-液相分離で形成される"p62 body"の新規構成成分の同定とその選択的オートファジーによる分解意義の解明

  森下 英晃, 来栖 玲央, 藤本 侑生, 能代 大輔, 高田 周平, 山野 晃史, 田中 秀明, 荒井 律子, 蔭山 俊, 船越 智子, 小松 聡子[廣田], 高 ひかり, 數野 彩子, 三浦 芳樹, 小池 正人, 若井 俊文, 和栗 聡, 野田 展生, 小松 雅明  日本生化学会大会プログラム・講演要旨集  96回-  [3S01m  -03]  2023/10
• ULK1キナーゼによるp62の液-液相分離制御

  池田良, 一村義信, 森下英晃, 能代大輔, 船越智子, 小松聡子, 蔭山俊, 野田展生, 小松雅明  日本Cell Death学会学術集会プログラム抄録集  30th-  2022
• UFM1システム:Alphafold2構造予測からわかったUFM1システムの作動原理

  石村亮輔, 能代大輔, 植村武文, 和栗聡, 野田展生, 小松雅明  日本Cell Death学会学術集会プログラム抄録集  30th-  2022
• 【どうして自分だけ狙われる?選択的オートファジー 既成概念を覆す分子機構と生理作用】液-液相分離と選択的オートファジー

  能代 大輔, 野田 展生  実験医学  39-  (13)  2046  -2051  2021/08  

  液-液相分離はその生物学的な重要性が認識されはじめて久しいが、選択的オートファジーにおいても重大な役割を担っている。選択的オートファジーは、液-液相分離により液滴状態となったタンパク質を分解対象のひとつとしており、その際は液滴の適度な液体度と、レセプターを介した液滴-隔離膜間相互作用が重要な役割を担う。本稿では、p62液滴、ストレス顆粒、そしてエンドサイトーシス異常の際に形成されるEND液滴の例を紹介するとともに、液滴選択的オートファジーのメカニズムを議論する。(著者抄録)
• Liquidity is a critical determinant for selective autophagy of protein condensates.

  山崎章徳, 山崎章徳, ALAM Jahangir MD., 能代大輔, 平田恵理, 藤岡優子, MAY Alexander I., 鈴木邦律, 鈴木邦律, 鈴木邦律, 大隅良典, 野田展生  日本細胞生物学会大会(Web)  73rd-  2021
• Pib2 is a glutamine sensor that directly activates TORC1

  谷川美頼, 山本勝良, 長門石曉, 津本浩平, 能代大輔, 野田展生, 永田宏次, 前田達哉, 前田達哉  日本農芸化学会大会講演要旨集(Web)  2021-  2021
• p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

  蔭山俊, GUDMUNDSSON Sigurdur, SOU Yu-Shin, 一村義信, 田村直輝, 數野彩子, 上野隆, 三浦芳樹, 能代大輔, 阿部学, 水島恒裕, 三浦信明, 奥田修二郎, 本橋ほづみ, LEE Jin-A, 崎村建司, 大江知之, 野田展生, 和栗聡, ESKELINEN Eeva-Liisa, 小松雅明  日本細胞生物学会大会(Web)  73rd-  2021
• マルチモードオートファジー カーゴの液体度が選択的オートファジーでの取り込みを左右する(Liquidty is a critical determinant for selective autophagy of protein condensates)

  山崎 章徳, Alam Jahangir, 能代 大輔, 平田 恵理, 藤岡 優子, 鈴木 邦律, 大隅 良典, 野田 展生  日本細胞生物学会大会講演要旨集  72回-  S9  -4  2020/06
• 【イメージング時代の構造生命科学 細胞の動態、膜のないオルガネラ、分子の構造変化をトランススケールに観る】(第2章)構造生命科学からトランススケール・イメージングによる細胞動態学へ トランススケールな解析が待たれる生命科学の未解決課題 柔らかい構造の可視化 LLPSと膜動態を例に

  能代 大輔, 野田 展生  実験医学  38-  (5)  750  -755  2020/03  

  タンパク質や核酸がLLPSすることで形成される液滴は、時間経過やストレス、変異等で固体化が進みゲルや凝集体となる。オートファジーは液滴を選択的に分解するが、効率的分解には液滴の適度な固体化が重要なようである。またLLPSはオートファジー関連因子の集積を通してオートファジーのマシーナリーの制御も担う。高速AFMは液滴の構造解析に適しており、同じタンパク質に関して液滴状態とゲル状態の構造的差異を見分けることが可能である。(著者抄録)
• Pib2はTORC1を直接活性化する細胞内グルタミンセンサーである

  谷川美頼, 山本勝良, 長門石曉, 永田宏次, 能代大輔, 野田展生, 津本浩平, 津本浩平, 前田達哉, 前田達哉  日本分子生物学会年会プログラム・要旨集(Web)  43rd-  2020
• 統合的なAFM,NMR,電顕単粒子解析により明らかにされたオリゴ糖転移酵素の動的構造

  川崎由貴, 有山弘高, 藤浪大輔, 本村肇, SRIVASTAVA Ashutosh, TAMA Florence, 能代大輔, 安藤敏夫, 真柳浩太, 神田大輔  日本糖質学会年会要旨集  38th-  2019
• 相分離したタンパク質の選択的オートファジーの試験管内再構成

  山崎章徳, ALAM Jahangir Md., 能代大輔, 野田展生  日本細胞生物学会大会(Web)  71st-  2019
• 細胞膜上RANKL分子のクラスター化誘導による骨芽細胞分化促進作用

  曽根 絵梨, 田村 幸彦, 菅森 泰隆, 依田 哲也, 青木 和広, 能代 大輔, 本間 雅, 池淵 祐樹  Journal of Oral Biosciences Supplement  2018-  340  -340  2018/09  [Not refereed][Not invited]
  
• オートファジーの足場タンパク質Atg17-Atg29-Atg31複合体の高速AFM観察

  能代 大輔, 藤岡 優子, 野田 展生, 安藤 敏夫  日本生化学会大会プログラム・講演要旨集  91回-  [2P  -003]  2018/09  [Not refereed][Not invited]
  
• RANKL結合ペプチドによる骨形成促進メカニズムの検討

  曽根絵梨, MASUD Khan, 能代大輔, 池淵祐樹, 本間雅, 田村幸彦, 菅森泰隆, 鈴木洋史, 宇田川信之, 青木和広  日本骨形態計測学会雑誌  28-  (2)  2018
• 実験・シミュレーション・データ科学の融合による遺伝情報分子システムの生物物理 高速AFMによる天然変性タンパク質の構造動態解析(Biophysics of genetic information molecules and systems: Integrated approach of experiments, simulations, and data science Structural dynamics analysis of intrinsically disordered proteins by high-speed AFM)

  Kodera Noriyuki, Noshiro Daisuke, Dora Sujit, Ando Toshio  生物物理  57-  (Suppl.1-2)  S191  -S191  2017/08  [Not refereed][Not invited]
  
• ビオチン化した生体分子の迅速簡便な固定化法のための、タマビジン2の二次元結晶(Two-dimensional crystals of tamavidin 2 for a quick and easy method of immobilization of biotinylated biomolecules)

  Noshiro Daisuke, Kodera Noriyuki, Ando Toshio  生物物理  57-  (Suppl.1-2)  S268  -S268  2017/08  [Not refereed][Not invited]
  
• 味覚受容体細胞外領域ヘテロ二量体の発現・精製および性状解析(Expression, purification, and characterization of the entire heterodimeric extracellular regions of fish taste receptor)

  Maruhashi Hiroki, Noshiro Daisuke, Yasui Norihisa, Ando Toshio, Yamashita Atsuko  生物物理  57-  (Suppl.1-2)  S313  -S313  2017/08  [Not refereed][Not invited]
  
• 高速AFMによる古細菌S.solfataricus由来ミニ染色体維持(ssoMCM)タンパク質複合体の観察(Observation of S. solfataricus archaeal minichromosome maintenance(ssoMCM) protein complex by high-speed AFM)

  Noshiro Daisuke, Kodera Noriyuki, Ando Toshio  生物物理  56-  (Suppl.1-2)  S276  -S276  2016/10  [Not refereed][Not invited]
  
• 高速AFMにより観察されたバクテリアマイクロコンパートメントの小平面

  能代 大輔  ファルマシア  52-  (9)  881  -881  2016  [Not refereed][Not invited]
   

  バクテリアマイクロコンパートメント（bacterial microcompart‐ment：BMC）は，正二十面体様のタンパク質の殻によって形成される，広く細菌類にみられるオルガネラである．内部に炭素固定に関連する重要な酵素類を含むカルボキシソームは，その代表例である．空間を区画化することで目的の反応に必要な酵素類の濃度を高め，反応を阻害する外部の要因から防御する役割を持つため，BMCは効率よく反応を行うナノリアクターとしての応用価値も見いだされている．BMCの殻の小平面は，主に単一の六量体形成シェルタンパク質BMC-Hにより形成されているが，シェルタンパク質がどのように自己集合して高次構造を形成するのかは未解明のままである．一方，高速AFM（high-speed atomic force microscopy：HS-AFM）は，生理的条件下に近い水溶液中でのタンパク質の動的挙動をリアルタイムで直接観察することが可能な装置である．これまでにアクチン上でのミオシンVの歩行運動や，回転軸を除いたF1-ATPaseの回転運動，バクテリオロドプシンの光励起による構造変化などの観察が行われた．最近Sutterらにより，高速AFMを用いて，BMC-H六量体の自己集合ダイナミクスが初めて観察されたので，本稿で紹介する．
  なお，本稿は下記の文献に基づいて，その研究成果を紹介するものである.
  1) Chowdhury C. et al., Microbiol. Mol. Biol. Rev., 78, 438-468 (2014).
  2) Frank S. et al., J. Biotechnol., 163, 273-279 (2013).
  3) Ando T. et al., Annu. Rev. Biophys., 42, 393-414 (2013).
  4) Sutter M. et al., Nano Lett., 16, 1590-1595 (2016).
• Construction of a Calcium-gated Artificial Channel by Fusing Alamethicin with a Calcium Sensor Protein

  NOSHIRO Daisuke, SONOMURA Kazuhiro, YU Hao-Hsin, IMANISHI Miki, ASAMI Koji, FUTAKI Shiroh  Peptide science : proceedings of the ... Japanese Peptide Symposium  2012-  183  -184  2013/03/01  [Not refereed][Not invited]
  
• Single-stranded DNA detection via chemically modified alamethicin nanopore at single molecule level

  R. Kawano, D. Noshiro, T. Osaki, T. Osaki, K. Kamiya, K. Asami, S. Futaki, S. Takeuchi, S. Takeuchi  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  1-  32  -34  2013/01/01  [Not refereed][Not invited]
   

  This paper describes the electrical detection of single-stranded DNA (ssDNA) at the single molecule level using an engineered alamethicin (Aim) channel embedded in bilayer lipid membranes (BLMs). Nanopore sequencing has the potential to become a direct, fast and inexpensive DNA sequencing methodology (Fig. 1). We propose here the chemically covalent dimmers of Aim (di-Alm) in which monomers were linked at their N-terminal ends and the di-Alm forms mainly the 6-mers (1.3 nm dia.). In addition, the conductance states were stabilized with lifetimes up to 200-fold longer than the same sates observed with monomers. Using the di-Alm nanopore, we can observe the slow ssDNA translocation and this finding highlight the importance of the di-Alm in the future ofj nanopore sequencing. Copyright © (2013) by the Chemical and Biological Microsystems Society All rights reserved.
• リポソーム内物質のリーケージを金属により制御可能なチャネルペプチドの創製

  能代大輔, 浅見耕司, 二木史朗  日本ケミカルバイオロジー学会 第7回年会 ,京都（京都大学百周年時計台記念館）  2012/06/08  [Not refereed][Not invited]
  
• Design and Creation of a Ca2+-sensitive Artificial Channel Protein

  NOSHIRO, Daisuke, SONOMURA, Kazuhiro, Yu H.H, IMANISHI, Miki, ASAMI, Kouji, FUTAKI, Shiroh  International Symposium on Innovative Nanobiodevices ISIN 2012 ,Nagoya, Japan  2012/03/21  [Not refereed][Not invited]
  
• Metal-Mediated Leakage Control by Histidine-Containing Alamethicin Analogs

  NOSHIRO Daisuke, ASAMI Koji, FUTAKI Shiroh  Peptide science : proceedings of the ... Japanese Peptide Symposium  2011-  189  -190  2012/03/01  [Not refereed][Not invited]
  
• ヒスチジン含有アラメチシン誘導体を用いた金属イオンによるリーケージ制御

  能代大輔, 浅見耕司, 二木史朗  第48回ペプチド討論会 ,札幌（札幌コンベンションセンター）  2011/09/27  [Not refereed][Not invited]
  
• 新規アラメチシン誘導体による金属を用いたリーケージ制御

  能代大輔, 浅見耕司, 二木史朗  第５回バイオ関連化学シンポジウム ,つくば（つくば国際会議場「エポカルつくば」）  2011/09/13  [Not refereed][Not invited]
  
• Alamethicinをベースにした金属イオン感受性人工イオンチャネルの創製

  能代大輔, 浅見耕司, 二木史朗  日本ケミカルバイオロジー学会第6回年会 ,東京（東京工業大学）  2011/05/24  [Not refereed][Not invited]
  
• Design and Creation of Metal-Responsive DNA-Binding Proteins

  FUTAKI Shiroh, IMANISHI Miki, NAKAYA Tomohiro, MORISAKI Tatsuya, NOSHIRO Daisuke, SUGIURA Yukio  Peptide science : proceedings of the ... Japanese Peptide Symposium  2010-  16  -16  2011/03/01  [Not refereed][Not invited]
  
• Metal-stimulated transcriptional control system using CDH_2-type zinc finger peptides

  NAKAYA Tomohiro, IMANISHI Miki, MORISAKI Tatsuya, NOSHIRO Daisuke, SUGIURA Yukio, FUTAKI Shiroh  Peptide science : proceedings of the ... Japanese Peptide Symposium  2010-  231  -231  2011/03/01  [Not refereed][Not invited]
  
• Metal-Assisted Stabilization of the Channel Assembly of Alamethicin : Metal Coordination with an Additional Histidine Yields Extremely Stable Channels

  NOSHIRO Daisuke, ASAMI Koji, FUTAKI Shiroh  Peptide science : proceedings of the ... Japanese Peptide Symposium  2010-  177  -177  2011/03/01  [Not refereed][Not invited]
  
• Creation of Metal Sensitive Artificial Ion Channels

  D. Noshiro, K. Asami, S. Futaki  BIOPOLYMERS  96-  (4)  500  -500  2011  [Not refereed][Not invited]
  
• Creation of a zinc finger type transcription factor responsive to Zn(II) concentration

  中屋智博, 今西未来, 森崎 達也, 能代 大輔, 二木史朗, 杉浦 幸雄  金属の関与する生体関連反応シンポジウム ,徳島  2010/06/25  [Not refereed][Not invited]
  
• 亜鉛濃度に依存した新規転写調節系の開発

  中屋智博, 森崎 達也, 能代 大輔, 今西未来, 二木史朗, 杉浦 幸雄  日本ケミカルバイオロジー学会 第５回 年会 ,横浜  2010/05/18  [Not refereed][Not invited]
  
• アラメチシンのアミノ酸置換とチャネル活性

  二木史朗, 野口晴加, 能代大輔, 浅見耕司  第46回ペプチド討論会 ,北九州  2009/11/05  [Not refereed][Not invited]
  
• 金属イオンによる会合制御が可能なチャネル形成ペプチドの創製とその応用

  能代大輔, 浅見耕司, 二木史朗  第18回金属の関与する生体関連反応シンポジウム（SRM2008） ,名古屋  2008/06/05  [Not refereed][Not invited]
  
• キレート形成原子団導入によるアラメチシンの会合安定化

  能代大輔, 浅見孝司, 二木史朗  第57回日本薬学会近畿支部大会 ,高槻  2007/10/27  [Not refereed][Not invited]
  
• 金属によるチャンネル形成ペクチドの会合安定

  能代大輔, 浅見耕司, 二木史朗  第２２回生体機能関連化学シンポジウム ,仙台  2007/09/28  [Not refereed][Not invited]
  
• 金属イオンによるチャネル形成ペプチドの会合安定化 Metal-mediated stabilization of channel peptide assemblies

  能代大輔, 浅見孝司, 二木史朗  第22回生体機能関連化学シンポジウム ,仙台  2007/09/28  [Not refereed][Not invited]
  
• 金属を認識する人工受容体型チャネルタンパク質の設計 Designing Artificial Metal-gated Ion Channels

  能代大輔, 園田和弘, 東野俊介, 浅見孝司, 二木史朗  日本ケミカルバイオロジー研究会 第2回年会 ,京都  2007/05/10  [Not refereed][Not invited]
  

Association Memberships

• THE JAPANESE BIOCHEMICAL SOCIETY   THE BIOPHYSICAL SOCIETY OF JAPAN   THE PHARMACEUTICAL SOCIETY OF JAPAN   

Research Projects

• 相分離液滴のオートファジー分解効率を規定する構造基盤の解明

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2023/04 -2027/03 

  Author : 能代 大輔
• 液－液相分離によって生じるオートファゴソーム形成場の高速AFMによる観察

  日本学術振興会：若手研究

  Date (from‐to) : 2019/04 -2023/03 

  Author : 能代 大輔

   

  オートファジーは、酵母からヒトに至る真核生物に保存された機構であり、タンパク質やオルガネラをリソソーム（液胞）へ輸送し分解する。オートファジーが始動すると隔離膜が分解対象物を取り囲み、オートファゴソームが形成されリソソーム（液胞）と融合する。酵母において、隔離膜形成には前オートファゴソーム構造体(PAS)が重要な役割を持ち、その足場はAtg1-Atg13-Atg17-Atg29-Atg31タンパク質複合体によって形成されるが、この複合体は液―液相分離して「液滴」として存在することが見出された。本研究は、オートファゴソーム形成メカニズムの解明のため、高速原子間力顕微鏡(AFM)を用いて液滴の構造的な理解を深めることを目指す。 本年度は前年度に続き、哺乳動物への研究の展開として、ヒトの選択的オートファジー受容体タンパク質p62/SQSTM1（以降p62）液滴について実験を進めた。p62はポリユビキチン鎖との結合により液滴を形成する。p62のユビキチン結合領域UBAのS403, S407のリン酸化はユビキチンとの結合を増強させるが、そのリン酸化ミミック体p62S403E_S407Eはポリユビキチン鎖と結合すると、野生型の場合よりサイズが大きく、より球形に近い液滴を形成することを明らかにした。また、p62はストレスセンサータンパク質Keap1の結合領域KIRを持つが、KIRのS349のリン酸化はKeap1との結合を増強させる。そのリン酸化ミミック体p62S349EとKeap1を結合させた後ポリユビキチン鎖を加えた場合、液滴状の構造物ではなく、細い繊維状の凝集体が形成された。野生型やp62S403E_S407Eを用いた場合はKeap1の有無に関わらず液滴を形成したため、S349のリン酸化及びそれに続くKeap1の結合は、液滴の形成を妨げることが示唆された。
• 高速AFMによる古細菌由来MCMタンパク質複合体の観察

  日本学術振興会：若手研究（B）

  Date (from‐to) : 2017/04 -2019/03 

  Author : 能代大輔
• センサー機能を有する人工イオンチャネルタンパク質の創製と展開

  日本学術振興会：特別研究員（DC1）

  Date (from‐to) : 2009/04 -2012/03 

  Author : 能代大輔

   

  アラメチシン(Alm)は,非タンパク質構成アミノ酸であるα-aminoisobutyric acid(Aib)を含む20アミノ酸残基から成る抗菌ペプチドである。両親媒性helix構造をとり,脂質二分子膜に電圧をかけると脂質中に入り込み,3分子～12分子が会合してイオンチャネルを形成する。本研究では,配列中にAibを含まずに安定なチャネルポアを形成するペプチドの創製,および外部刺激に応じてチャネル電流の変化する人工イオンチャネルへの応用を目指した。これまでに,AlmのN末端にhistidine残基を導入したペプチドHG-Almが,Zn(II)イオン等の金属イオンの存在下において,膜中に安定なチャネルポアを形成することを報告したが,これをAlmに含まれるすべてのAibをleucineに置換したAlm誘導体[Leu]Almに応用し,HG[Leu]Almを作製,チャネル電流測定により,安定なチャネルポアを形成するか評価を行った。その結果,HG-[Leu]Almは,HG-Almと異なり,Zn(II)イオンの存在下においても安定なボアを形成しないことがわかった。本年度は,この原因を探るため,Aib残基をnorvaline(Nva), norleucine(Nle)にそれぞれ置換したHG-Alm誘導体を合成し,蛍光色素封入リポソームを用いたリーケージアッセイにより,これらのペプチドの膜への作用様式を調べた。その結果,HG-AlkおよびHG-[Nva]Almについては,Zn(II)イオンによるリーケージ活性の増大が観察されたが,HG-[Leu]AlmおよびHG-[Nle]Almについては,逆にZn(II)イオンによるリーケージ活性の著しい阻害が観察された。また,これらのHG-Alm誘導体を用いて,Zn(II)イオンの添加と除去による溶出制御が可能であることが示された。