Phase separation promotes Atg8 lipidation for autophagy progression
Yuko Fujioka, Takuma Tsuji, Tetsuya Kotani, Hiroyuki Kumeta, Chika Kakuta, Toyoshi Fujimoto, Hitoshi Nakatogawa, Nobuo N Noda
Cold Spring Harbor Laboratory, 2024年08月30日
Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the pre-autophagosomal structure (PAS) in yeast, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS as a liquid droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets via specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution experiments reveal that E3-enriched early PAS droplets promote Atg8 lipidation and incorporate Atg8-coated vesicles to the interior, thereby protecting them from Atg4-mediated delipidation. These data suggest that the PAS utilizes its liquid-like property to function as an efficient production site for lipidated Atg8 and pool membrane seeds to drive autophagosome formation.

The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy
Yutaro Hama, Yuko Fujioka, Hayashi Yamamoto, Noboru Mizushima, Nobuo N. Noda
Cold Spring Harbor Laboratory, 2024年08月02日
Abstract

In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism of the ULK complex formation remain poorly understood. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 binds to the base of the two UBL domains in the FIP200 dimer using two phenylalanines and to the tandem MIT domain of ULK1, allowing for the 1:1:2 stoichiometry of the ULK1–ATG13–FIP200 complex. We confirmed the predicted interactions by point mutations and revealed the existence of direct triad interactions among ULK1, ATG13, and FIP200 in vitro and in cells, in which each interaction was additively important for autophagic flux. These results indicate that the ULK1–ATG13–FIP200 triadic interaction is crucial for autophagosome formation and provide a structural basis and insights into the regulation mechanism of autophagy initiation in mammals.

The UFM1 system: Working principles, cellular functions, and pathophysiology.
Masaaki Komatsu, Toshifumi Inada, Nobuo N Noda
Molecular cell, 84, 1, 156, 169, 2024年01月04日, ［国際誌］
英語, 研究論文（学術雑誌）, Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through UFMylation, a process similar to ubiquitylation. Growing lines of evidence regarding not only the structural basis of the components essential for UFMylation but also their biological properties shed light on crucial roles of the UFM1 system in the endoplasmic reticulum (ER), such as ER-phagy and ribosome-associated quality control at the ER, although there are some functions unrelated to the ER. Mouse genetics studies also revealed the indispensable roles of this system in hematopoiesis, liver development, neurogenesis, and chondrogenesis. Of critical importance, mutations of genes encoding core components of the UFM1 system in humans cause hereditary developmental epileptic encephalopathy and Schohat-type osteochondrodysplasia of the epiphysis. Here, we provide a multidisciplinary review of our current understanding of the mechanisms and cellular functions of the UFM1 system as well as its pathophysiological roles, and discuss issues that require resolution.

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, 170, 178, 2024年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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年12月02日, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Mechanisms of mitochondrial reorganization
Tatsuro Maruyama, Yutaro Hama, Nobuo N Noda
The Journal of Biochemistry, Oxford University Press (OUP), 2023年11月28日
研究論文（学術雑誌）, Abstract

The cytoplasm of eukaryotes is dynamically zoned by membrane-bound and membraneless organelles. Cytoplasmic zoning allows various biochemical reactions to take place at the right time and place. Mitochondrion is a membrane-bound organelle that provides a zone for intracellular energy production and metabolism of lipids and iron. A key feature of mitochondria is their high dynamics: mitochondria constantly undergo fusion and fission, and excess or damaged mitochondria are selectively eliminated by mitophagy. Therefore, mitochondria are appropriate model systems to understand dynamic cytoplasmic zoning by membrane organelles. In this review, we summarize the molecular mechanisms of mitochondrial fusion and fission as well as mitophagy unveiled through studies using yeast and mammalian models.

Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy.
Tomoyuki Fukuda, Kentaro Furukawa, Tatsuro Maruyama, Nobuo N Noda, Tomotake Kanki
Autophagy, 19, 11, 3019, 3021, 2023年11月, ［国際誌］
英語, 研究論文（学術雑誌）, Atg: autophagy related; IMM: inner mitochondrial membrane; IMS: intermembrane space; PAS: phagophore assembly site; SAR: selective autophagy receptor.

Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control
Ryosuke Ishimura, Sota Ito, Gaoxin Mao, Satoko Komatsu-Hirota, Toshifumi Inada, Nobuo N. Noda, Masaaki Komatsu
Science Advances, 9, 33, American Association for the Advancement of Science (AAAS), 2023年08月18日
研究論文（学術雑誌）, Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)–associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60 S subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.

The Atg1 complex, Atg9, and Vac8 recruit PI3K complex I to the pre-autophagosomal structure.
Kanae Hitomi, Tetsuya Kotani, Nobuo N Noda, Yayoi Kimura, Hitoshi Nakatogawa
The Journal of cell biology, 222, 8, 2023年08月07日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14-Vac8 interaction is constitutive, the Atg38-Atg1 complex interaction and the Vps30-Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis.

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, e113349, 2023年06月12日, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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, 2023年05月09日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis.

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.e9, Elsevier BV, 2023年05月, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

Autophagy and cancer: Basic mechanisms and inhibitor development
Yutaro Hama, Yuta Ogasawara, Nobuo N. Noda
Cancer Science, Wiley, 2023年04月20日, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）

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年12月21日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Qualitative differences in disease-associated MEK mutants reveal molecular signatures and aberrant signaling-crosstalk in cancer
Yuji Kubota, Yuko Fujioka, Ashwini Patil, Yusuke Takagi, Daisuke Matsubara, Masatomi Iijima, Isao Momose, Ryosuke Naka, Kenta Nakai, Nobuo N. Noda, Mutsuhiro Takekawa
Nature Communications, 13, 1, Springer Science and Business Media LLC, 2022年12月, ［査読有り］
研究論文（学術雑誌）, Abstract

Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes. By making use of such distinct characteristics of the MEK1 mutants, we identified cancer- and RASopathy-signature genes that may serve as diagnostic markers or therapeutic targets for these diseases. In particular, two AKT-inhibitor molecules, PHLDA1 and 2, are simultaneously upregulated by oncogenic ERK signaling, and mediate cancer-specific ERK-AKT crosstalk. The combined expression of PHLDA1/2 is critical to confer resistance to ERK pathway-targeted therapeutics on cancer cells. Finally, we propose a therapeutic strategy to overcome this drug resistance. Our data provide vital insights into the etiology, diagnosis, and therapeutic strategy of cancers and RASopathies.

Development of new tools to study membrane-anchored mammalian Atg8 proteins
Sang-Won Park, Pureum Jeon, Akinori Yamasaki, Hye Eun Lee, Haneul Choi, Ji Young Mun, Yong-Woo Jun, Ju-Hui Park, Seung-Hwan Lee, Soo-Kyeong Lee, You-Kyung Lee, Hyun Kyu Song, Michael Lazarou, Dong-Hyong Cho, Masaaki Komatsu, Nobuo N. Noda, Deok-Jin Jang, Jin-A Lee
Autophagy, 1, 20, Informa UK Limited, 2022年10月17日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

Targeting the ATG5-ATG16L1 Protein–Protein Interaction with a Hydrocarbon-Stapled Peptide Derived from ATG16L1 for Autophagy Inhibition
Jin Cui, Yuta Ogasawara, Ikuko Kurata, Kazuaki Matoba, Yuko Fujioka, Nobuo N. Noda, Masakatsu Shibasaki, Takumi Watanabe
Journal of the American Chemical Society, 144, 38, 17671, 17679, American Chemical Society (ACS), 2022年09月15日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

Lipid Transport from Endoplasmic Reticulum to Autophagic Membranes
Takuo Osawa, Kazuaki Matoba, Nobuo N. Noda
Cold Spring Harbor Perspectives in Biology, a041254, a041254, Cold Spring Harbor Laboratory, 2022年08月08日, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）

Cytoskeleton grows p62 condensates for autophagy
Nobuo N. Noda
Cell Research, 32, 7, 607, 608, Springer Science and Business Media LLC, 2022年05月19日, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
研究論文（学術雑誌）

Phosphorylation by casein kinase 2 enhances the interaction between ER‐phagy receptor TEX264 and ATG8 proteins
Haruka Chino, Akinori Yamasaki, Koji L Ode, Hiroki R Ueda, Nobuo N Noda, Noboru Mizushima
EMBO reports, 23, 6, EMBO, 2022年04月13日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

Phosphorylation by casein kinase 2 ensures ER-phagy receptor TEX264 binding to ATG8 proteins
Haruka Chino, Akinori Yamasaki, Koji L Ode, Hiroki R Ueda, Nobuo N Noda, Noboru Mizushima
Cold Spring Harbor Laboratory, 2022年02月11日
研究論文（学術雑誌）

Update and nomenclature proposal for mammalian lysophospholipid acyltransferases, which create membrane phospholipid diversity.
William J Valentine, Keisuke Yanagida, Hiroki Kawana, Nozomu Kono, Nobuo N Noda, Junken Aoki, Hideo Shindou
The Journal of biological chemistry, 298, 1, 101470, 101470, 2022年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.

Phase-separated protein droplets of amyotrophic lateral sclerosis-associated p62/SQSTM1 mutants show reduced inner fluidity
Mohammad Omar Faruk, Yoshinobu Ichimura, Shun Kageyama, Satoko Komatsu-Hirota, Afnan H. El-Gowily, Yu-shin Sou, Masato Koike, Nobuo N. Noda, Masaaki Komatsu
Journal of Biological Chemistry, 297, 6, 101405, 101405, Elsevier {BV}, 2021年12月, ［国際誌］
英語, 研究論文（学術雑誌）, Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget's disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the antioxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system, and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.

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, 1093, 1093, Springer Science and Business Media LLC, 2021年12月, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Atg12-Interacting Motif Is Crucial for E2–E3 Interaction in Plant Atg8 System
Kazuaki Matoba, Nobuo N. Noda
Biological and Pharmaceutical Bulletin, 44, 9, 1337, 1343, Pharmaceutical Society of Japan, 2021年09月01日, ［査読有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）

Delineating the lipidated Atg8 structure for unveiling its hidden activity in autophagy
Tatsuro Maruyama, Nobuo N. Noda
Autophagy, 1, 2, Informa UK Limited, 2021年08月12日, ［最終著者, 責任著者］
研究論文（学術雑誌）

Atg2 and Atg9: Intermembrane and interleaflet lipid transporters driving autophagy
Nobuo N. Noda
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1866, 8, 158956, 158956, Elsevier BV, 2021年08月, ［査読有り］, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
研究論文（学術雑誌）

Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.
Maruyama, T, Alam, J. M, Fukuda, T, Kageyama, S, Kirisako, H, Ishii, Y, Shimada, I, Ohsumi, Y, Komatsu, M, Kanki, T, Nakatogawa, H, Noda, N. N
Nature Structural & Molecular Biology, 28, 7, 583, 593, 2021年07月08日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.

Structural catalog of core Atg proteins opens new era of autophagy research
Kazuaki Matoba, Nobuo N Noda
The Journal of Biochemistry, 169, 5, 517, 525, Oxford University Press (OUP), 2021年07月03日, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）,
Autophagy, which is an evolutionarily conserved intracellular degradation system, involves de novo generation of autophagosomes that sequester and deliver diverse cytoplasmic materials to the lysosome for degradation. Autophagosome formation is mediated by approximately 20 core autophagy-related (Atg) proteins, which collaborate to mediate complicated membrane dynamics during autophagy. To elucidate the molecular functions of these Atg proteins in autophagosome formation, many researchers have tried to determine the structures of Atg proteins by using various structural biological methods. Although not sufficient, the basic structural catalog of all core Atg proteins was established. In this review article, we summarize structural biological studies of core Atg proteins, with an emphasis on recently unveiled structures, and describe the mechanistic breakthroughs in autophagy research that have derived from new structural information.

【相分離 メカニズムと疾患"膜のないオルガネラ"はいかに機能するか?神経変性疾患・ウイルス感染とどう関わるか?】(第3章)相分離と生体機能・疾患 相分離で見直すオートファジー
藤岡 優子, 野田 展生
実験医学, 39, 10, 1628, 1633, (株)羊土社, 2021年06月
日本語, オートファジーは細胞内成分全般を対象とした分解系であり、オートファゴソームとよばれる二重膜オルガネラの新生を通して分解対象を隔離し、リソソームへと輸送することで分解を行う。液-液相分離はオートファジーの制御に重要な役割を担っており、オートファジー関連タンパク質を集めることでオートファゴソーム新生を推進するほか、分解基質となるタンパク質を集めることで効率的かつ選択的な隔離・分解を可能にしている。後者においては、分解基質が適度な液体度をもった会合体を形成することが重要であることもわかってきた。(著者抄録)

マルチモードオートファジー カーゴの流動性が選択的オートファジーでの分解を左右する　　　　　　　　　　　　　　　
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, 大隅 良典, 野田 展生
日本細胞生物学会大会講演要旨集, 73回, S10, 2, (一社)日本細胞生物学会, 2021年06月
日本語

Mutagenesis and homology modeling reveal a predicted pocket of lysophosphatidylcholine acyltransferase 2 to catch Acyl‐CoA
Fumie Hamano, Kazuaki Matoba, Tomomi Hashidate‐Yoshida, Tomoyuki Suzuki, Kiyotake Miura, Daisuke Hishikawa, Takeshi Harayama, Koichi Yuki, Yoshihiro Kita, Nobuo N. Noda, Takao Shimizu, Hideo Shindou
The FASEB Journal, 35, 6, e21501, Wiley, 2021年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological events, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory stimuli, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritima PlsC, an enzyme in the same gene family as LPCAT2. Here, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the three-dimensional (3D) structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases.

Biomolecular condensates in autophagy regulation
Yuko Fujioka, Nobuo N. Noda
Current Opinion in Cell Biology, 69, 23, 29, Elsevier BV, 2021年04月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Autophagy is an intracellular degradation system that contributes to cellular homeostasis. Autophagosome formation is a landmark event in autophagy, which sequesters and delivers cytoplasmic components to the lysosome for degradation. Based on selectivity, autophagy can be classified into bulk and selective autophagy, which are mechanistically distinct from each other, especially in the requirement of cargos for autophagosome formation. Recent studies revealed that liquid-like biomolecular condensates, which are formed through liquid-liquid phase separation, regulate the autophagosome formation of both bulk and selective autophagy. Here, we focus on recent findings on the involvement of biomolecular condensates in autophagy regulation and discuss their significance.

p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response.
Kageyama, S, Gudmundsson, S, Sou, Y.-S, Ichimura, Y, Tamura, N, Kazuno, S, Ueno, T, Miura, Y, Noshiro, D, Abe, M, Mizushima, T, Miura, N, Okuda, S, Motohashi, H, Lee, J.-A, Sakimura, K, Ohe, T, Noda, N. N, Waguri, S, Eskelinen, E.-L, Komatsu, M
Nature Communications, 12, 1, 16, Springer Science and Business Media LLC, 2021年01月04日, ［査読有り］
研究論文（学術雑誌）, 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.

Secret of Atg9: lipid scramblase activity drives de novo autophagosome biogenesis.
Kazuaki Matoba, Nobuo N Noda
Cell death and differentiation, 27, 12, 3386, 3388, 2020年12月, ［国際誌］
英語, 研究論文（学術雑誌）

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年12月, ［国際誌］
英語, An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes
Keisuke Mochida, Akinori Yamasaki, Kazuaki Matoba, Hiromi Kirisako, Nobuo N. Noda, Hitoshi Nakatogawa
Nature Communications, 11, 1, 3306, 3306, Springer Science and Business Media LLC, 2020年12月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.

Structural and dynamics analysis of intrinsically disordered proteins by high-speed atomic force microscopy.
Kodera, N, Noshiro, D, Dora, S. K, Mori, T, Habchi, J, Blocquel, D, Gruet, A, Dosnon, M, Salladini, E, Bignon, C, Fujioka, Y, Oda, T, Noda, N. N, Sato, M, Lotti, M, Mizuguchi, M, Longhi, S, Ando, T
Nature Nanotechnology, 16, 2, 181, 189, Springer Science and Business Media LLC, 2020年11月23日, ［査読有り］
研究論文（学術雑誌）, Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion.
Matoba, K, Kotani, T, Tsutsumi, A, Tsuji, T, Mori, T, Noshiro, D, Sugita, Y, Nomura, N, Iwata, S, Ohsumi, Y, Fujimoto, T, Nakatogawa, H, Kikkawa, M, Noda, N. N
Nature Structural & Molecular Biology, 27, 12, 1185, 1193, Springer Science and Business Media LLC, 2020年10月26日, ［査読有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）, 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.

Liquid–liquid phase separation in autophagy
Nobuo N. Noda, Zheng Wang, Hong Zhang
Journal of Cell Biology, 219, 8, Rockefeller University Press, 2020年08月03日, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）, Liquid–liquid phase separation (LLPS) compartmentalizes and concentrates biomacromolecules into distinct condensates. Liquid-like condensates can transition into gel and solid states, which are essential for fulfilling their different functions. LLPS plays important roles in multiple steps of autophagy, mediating the assembly of autophagosome formation sites, acting as an unconventional modulator of TORC1-mediated autophagy regulation, and triaging protein cargos for degradation. Gel-like, but not solid, protein condensates can trigger formation of surrounding autophagosomal membranes. Stress and pathological conditions cause aberrant phase separation and transition of condensates, which can evade surveillance by the autophagy machinery. Understanding the mechanisms underlying phase separation and transition will provide potential therapeutic targets for protein aggregation diseases.

【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, (株)羊土社, 2020年03月
日本語, 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)

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.e9, Elsevier BV, 2020年03月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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, Springer Science and Business Media LLC, 2020年02月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, 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.

Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4
Takuo Osawa, Yuki Ishii, Nobuo N. Noda
Genes to Cells, 25, 1, 65, 70, Wiley, 2020年01月, ［最終著者, 責任著者］
研究論文（学術雑誌）

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis
Takuo Osawa, Nobuo N. Noda
Protein Science, 28, 6, 1005, 1012, Wiley, 2019年06月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）

Evolution from covalent conjugation to non-covalent interaction in the ubiquitin-like ATG12 system
Yu Pang, Hayashi Yamamoto, Hirokazu Sakamoto, Masahide Oku, Joe Kimanthi Mutungi, Mayurbhai Himatbhai Sahani, Yoshitaka Kurikawa, Kiyoshi Kita, Nobuo N. Noda, Yasuyoshi Sakai, Honglin Jia, Noboru Mizushima
Nature Structural & Molecular Biology, 26, 4, 289, 296, Springer Science and Business Media LLC, 2019年04月, ［査読有り］
英語, 研究論文（学術雑誌）, Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12-ATG5 complex retains the ability to facilitate ATG8-phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.

Atg2 mediates direct lipid transfer between membranes for autophagosome formation
Takuo Osawa, Tetsuya Kotani, Tatsuya Kawaoka, Eri Hirata, Kuninori Suzuki, Hitoshi Nakatogawa, Yoshinori Ohsumi, Nobuo N. Noda
Nature Structural & Molecular Biology, 26, 4, 281, 288, Springer Science and Business Media LLC, 2019年04月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, A key event in autophagy is autophagosome formation, whereby the newly synthesized isolation membrane (IM) expands to form a complete autophagosome using endomembrane-derived lipids. Atg2 physically links the edge of the expanding IM with the endoplasmic reticulum (ER), a role that is essential for autophagosome formation. However, the molecular function of Atg2 during ER-IM contact remains unclear, as does the mechanism of lipid delivery to the IM. Here we show that the conserved amino-terminal region of Schizosaccharomyces pombe Atg2 includes a lipid-transfer-protein-like hydrophobic cavity that accommodates phospholipid acyl chains. Atg2 bridges highly curved liposomes, thereby facilitating efficient phospholipid transfer in vitro, a function that is inhibited by mutations that impair autophagosome formation in vivo. These results suggest that Atg2 acts as a lipid-transfer protein that supplies phospholipids for autophagosome formation.

A C4N4 Diaminopyrimidine Fluorophore
Hidetoshi Noda, Yasuko Asada, Tatsuro Maruyama, Naoki Takizawa, Nobuo N. Noda, Masakatsu Shibasaki, Naoya Kumagai
Chemistry – A European Journal, 25, 17, 4299, 4304, Wiley, 2019年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A new scaffold for producing efficient organic fluorescent materials was identified: 2,5-diamino-4,6-diary-lpyrimidine featuring a C4N4 elemental composition. Single-step installation of two aryl groups at the 4,6-positions of the pyrimidine core delivered fluorescent organic materials in a modular fashion. A range of fluorescent compounds with distinct absorption/emission properties was readily accessed by changing the aromatic attachments. A generally high absorption coefficient and quantum yield were observed, including C4N4 derivatives that could fluoresce even in the solid state. The two amino groups at the 2,5-positions of the pyrimidine were essential for intense fluorescence with a large Stokes shift, which was corroborated by structural relaxation to a p-iminoquinone-like structure in the excited state. Besides live-cell imaging capabilities, fluorescent labeling of a protein involved in autophagy elucidated a new protein protein interaction, supporting potential utility in bioimaging applications.

Structural Studies of Selective Autophagy in Yeast.
Yamasaki A, Watanabe Y, Noda NN
Methods in molecular biology (Clifton, N.J.), 1880, 77, 90, 2019年, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Budding yeast has been utilized as a model system for studying basic mechanisms of autophagy. The cytoplasm-to-vacuole targeting (Cvt) pathway, which delivers some vacuolar enzymes into the vacuole selectively and constitutively, is one of the most characterized examples of selective autophagy in budding yeast. Here we summarize the methods of X-ray crystallography, NMR, and other biophysical analyses to study the structural basis of the Cvt pathway.

Membrane-binding domains in autophagy
Takuo Osawa, Jahangir, Md. Alam, Nobuo N. Noda
Chemistry and Physics of Lipids, 218, 1, Elsevier {BV}, 2019年01月, ［査読有り］, ［最終著者, 責任著者］

Lipidation-independent vacuolar functions of Atg8 rely on its noncanonical interaction with a vacuole membrane protein
Xiao-Man Liu, Akinori Yamasaki, Xiao-Min Du, Valerie C Coffman, Yoshinori Ohsumi, Hitoshi Nakatogawa, Jian-Qiu Wu, Nobuo N Noda, Li-Lin Du
eLife, 7, eLife Sciences Publications, Ltd, 2018年11月19日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）, The ubiquitin-like protein Atg8, in its lipidated form, plays central roles in autophagy. Yet, remarkably, Atg8 also carries out lipidation-independent functions in non-autophagic processes. How Atg8 performs its moonlighting roles is unclear. Here we report that in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, the lipidation-independent roles of Atg8 in maintaining normal morphology and functions of the vacuole require its interaction with a vacuole membrane protein Hfl1 (homolog of human TMEM184 proteins). Crystal structures revealed that the Atg8-Hfl1 interaction is not mediated by the typical Atg8-family-interacting motif (AIM) that forms an intermolecular β-sheet with Atg8. Instead, the Atg8-binding regions in Hfl1 proteins adopt a helical conformation, thus representing a new type of AIMs (termed helical AIMs here). These results deepen our understanding of both the functional versatility of Atg8 and the mechanistic diversity of Atg8 binding.

Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein Atg8 through Multi-Step Recognition.
Masaya Yamaguchi, Kenji Satoo, Hironori Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki, Nobuo N Noda
Journal of molecular biology, 430, 3, 249, 257, 2018年02月02日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Atg8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for Atg8, and it activates the C-terminal Gly116 of Atg8 using ATP. Here, we report the crystal structure of Atg8 bound to the C-terminal domain of Atg7 in an unprecedented mode. Atg8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7-Atg8 structures. Instead, Atg8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes Atg8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.

Endosomal rab cycles regulate parkin-mediated mitophagy
Koji Yamano, Chunxin Wang, Shireen A. Sarraf, Christian Münch, Reika Kikuchi, Nobuo N. Noda, Yohei Hizukuri, Masato T. Kanemaki, Wade Harper, Keiji Tanaka, Noriyuki Matsuda, Richard J. Youle
eLife, 7, eLife Sciences Publications Ltd, 2018年01月23日, ［査読有り］
英語, 研究論文（学術雑誌）, Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Autophagy-regulating protease Atg4: Structure, function, regulation and inhibition
Tatsuro Maruyama, Nobuo N. Noda
Journal of Antibiotics, 71, 1, 72, 78, Nature Publishing Group, 2018年01月01日, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
英語, Autophagy is an intracellular degradation system that contributes to cellular homeostasis through degradation of various targets such as proteins, organelles and microbes. Since autophagy is related to various diseases such as infection, neurodegenerative diseases and cancer, it is attracting attention as a new therapeutic target. Autophagy is mediated by dozens of autophagy-related (Atg) proteins, among which Atg4 is the sole protease that regulates autophagy through the processing and deconjugating of Atg8. As the Atg4 activity is essential and highly specific to autophagy, Atg4 is a prospective target for developing autophagy-specific inhibitors. In this review article, we summarize our current knowledge of the structure, function and regulation of Atg4 including efforts to develop Atg4-specific inhibitors.

Biophysical characterization of Atg11, a scaffold protein essential for selective autophagy in yeast
Hironori Suzuki, Nobuo N. Noda
FEBS Open Bio, 8, 1, 110, 116, Wiley Blackwell, 2018年01月01日, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Autophagy is an intracellular degradation system in which the formation of an autophagosome is a key event. In budding yeast, autophagosomes are generated from the preautophagosomal structure (PAS), in which Atg11 and Atg17 function as scaffolds essential for selective and nonselective types of autophagy, respectively. Structural studies have been extensively performed on Atg17, but not on Atg11, preventing us from understanding the selective type of the PAS. Here, we purified and characterized Atg11. Biophysical analyses, including analytical ultracentrifugation and CD, showed that Atg11 behaves as an elongated homodimer abundant in α-helices in solution. Moreover, truncation analyses suggested that Atg11 has a parallel coiled-coil architecture, in contrast to the antiparallel dimeric architecture of Atg17.

Structural biology of the core autophagy machinery
Hironori Suzuki, Takuo Osawa, Yuko Fujioka, Nobuo N. Noda
CURRENT OPINION IN STRUCTURAL BIOLOGY, 43, 10, 17, CURRENT BIOLOGY LTD, 2017年04月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, In autophagy, which is an intracellular degradation system that is conserved among eukaryotes, degradation targets are sequestered through the de novo synthesis of a double-membrane organelle, the autophagosome, which delivers them to the lysosomes for degradation. The core autophagy machinery comprising 18 autophagy-related (Atg) proteins in yeast plays an essential role in autophagosome formation; however, the molecular role of each Atg factor and the mechanism of autophagosome formation remain elusive. Recent years have seen remarkable progress in structural biological studies on the core autophagy machinery, opening new avenues for autophagy research. This review summarizes recent advances in structural biological and mechanistic studies on the core autophagy machinery and discusses the molecular mechanisms of autophagosome formation.

Structural Biology of the Cvt Pathway
Akinori Yamasaki, Nobuo N. Noda
JOURNAL OF MOLECULAR BIOLOGY, 429, 4, 531, 542, ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2017年02月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
英語, Macroautophagy is a degradation process in which autophagosomes are generated to isolate and transport various materials, including damaged organelles and protein aggregates, as cargos to the lysosomes or vacuoles. Bulk autophagy is one of the two types of macroautophagy, which is triggered by starvation and targets non-specific cargos. The second type, that is, selective autophagy, identifies and preferentially degrades specific cargos via receptor recognition. Cytoplasm-to-vacuole targeting (Cvt) is a selective autophagy pathway that specifically transports vacuolar hydrolases into the vacuole in budding yeast cells and has been extensively studied as a model of selective autophagy. In the present review, we focused on the Cvt pathway, especially on the recent structural insights into cargo assembly, receptor recognition, and recruitment mechanisms of the Cvt machinery. Elucidating the Cvt pathway would help in understanding the basic molecular mechanisms of various types of selective autophagy. (C) 2017 The Authors. Published by Elsevier Ltd.

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, CELL PRESS, 2016年07月, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）, 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.

Structural Basis for Receptor-Mediated Selective Autophagy of Aminopeptidase I Aggregates
Akinori Yamasaki, Yasunori Watanabe, Wakana Adachi, Kuninori Suzuki, Kazuaki Matoba, Hiromi Kirisako, Hiroyuki Kumeta, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki, Nobuo N. Noda
CELL REPORTS, 16, 1, 19, 27, CELL PRESS, 2016年06月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2: 1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity.

Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions
Mariko Yokogawa, Takashi Tsushima, Nobuo N. Noda, Hiroyuki Kumeta, Yoshiaki Enokizono, Kazuo Yamashita, Daron M. Standley, Osamu Takeuchi, Shizuo Akira, Fuyuhiko Inagaki
SCIENTIFIC REPORTS, 6, 22324, NATURE PUBLISHING GROUP, 2016年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Small differences make a big impact: Structural insights into the differential function of the 2 Atg8 homologs in C-elegans
Fan Wu, Peng Wang, Yuxian Shen, Nobuo N. Noda, Hong Zhang
AUTOPHAGY, 12, 3, 606, 607, TAYLOR & FRANCIS INC, 2016年, ［査読有り］
英語, The 2 C. elegans homologs of Atg8, LGG-1 and LGG-2, show differential function in the degradation of protein aggregates during embryogenesis. LGG-1 is essential for the degradation of various protein aggregates, while LGG-2 has cargo-specific and developmental stage-specific roles. LGG-1 and LGG-2 differentially interact with autophagy substrates and ATG proteins. LGG-1 and LGG-2 possess 2 hydrophobic pockets, the W-site and the L-site, which recognize the LIR motif in Atg8-binding proteins. The plasticity of the W-site and the size and shape of the L-site differ between LGG-1 and LGG-2, thus determining their preferences for distinct LIR motifs. The N-terminal tails of LGG-1 and LGG-2 adopt unique closed and open conformations, respectively, which may result in distinct membrane tethering and fusion activities. LGG-1 and LGG-2 have different affinities for ATG-7 and ATG-3, and lipidation of LGG-2 is regulated by levels of lipidated LGG-1. Taken together, the structural differences between LGG-1 and LGG-2 provide insights into their differential functions in the aggrephagy pathway.

Atg101: Not Just an Accessory Subunit in the Autophagy-initiation Complex
Nobuo N. Noda, Noboru Mizushima
CELL STRUCTURE AND FUNCTION, 41, 1, 13, 20, JAPAN SOC CELL BIOLOGY, 2016年, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, The Saccharomyces cerevisiae autophagy-initiation complex, Atg1 kinase complex, consists of Atg1, Atg13, Atg17, Atg29, and Atg31, while the corresponding complex in most other eukaryotes, including mammals, is composed of ULK1 (or ULK2), Atg13, FIP200 (also known as RB1CC1), and Atg101. ULKs are homologs of Atg1, and FIP200 is partially homologous to Atg17. However, the sequence of Atg101 is not similar to that of Atg29 or Atg31. Although Atg101 is essential for autophagy and widely conserved in eukaryotes, its precise function and structure have remained largely unknown. Now, structural and cell biological analysis of Atg101 together with its binding partner Atg13 reveal that Atg101 is required for stabilization of "uncapped" Atg13 in most eukaryotes and also for recruitment of downstream Atg proteins through the newly identified WF motif. By contrast, S. cerevisiae has stable "capped" Atg13, which does not require Atg101 for its stabilization. Possible roles for other binding partners such as Atg29, Atg31, and Atg28 in different organisms are also discussed.

Structural Basis of the Differential Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy
Fan Wu, Yasunori Watanabe, Xiang-Yang Guo, Xin Qi, Peng Wang, Hong-Yu Zhao, Zheng Wang, Yuko Fujioka, Hui Zhang, Jin-Qi Ren, Tian-Cheng Fang, Yu-Xian Shen, Wei Feng, Jun-Jie Hu, Nobuo N. Noda, Hong Zhang
Molecular Cell, 60, 6, 914, 929, Elsevier BV, 2015年12月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.

The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy
Hayashi Yamamoto, Takayuki Shima, Masaya Yamaguchi, Yuh Mochizuki, Hisashi Hoshida, Soichiro Kakuta, Chika Kondo-Kakuta, Nobuo N. Noda, Fuyuhiko Inagaki, Takehiko Itoh, Rinji Akada, Yoshinori Ohsumi
JOURNAL OF BIOLOGICAL CHEMISTRY, 290, 49, 29506, U476, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Open and closed HORMAs regulate autophagy initiation
Hironori Suzuki, Takeshi Kaizuka, Noboru Mizushima, Nobuo N. Noda
AUTOPHAGY, 11, 11, 2123, 2124, TAYLOR & FRANCIS INC, 2015年11月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
英語, The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.

Atg1 family kinases in autophagy initiation
Nobuo N. Noda, Yuko Fujioka
CELLULAR AND MOLECULAR LIFE SCIENCES, 72, 16, 3083, 3096, SPRINGER BASEL AG, 2015年08月, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, Autophagosome formation, a landmark event in autophagy, is accomplished by the concerted actions of Atg proteins. Among all Atg proteins, Atg1 kinase in yeast and its counterpart in higher eukaryotes, ULK1 kinase, function as the most upstream factor in this process and mediate autophagy initiation. In this review, we summarize current knowledge of the structure, molecular function, and regulation of Atg1 family kinases in the initiation of autophagy.

Structure of the Atg101-Atg13 complex reveals essential roles of Atg101 in autophagy initiation
Hironori Suzuki, Takeshi Kaizuka, Noboru Mizushima, Nobuo N. Noda
Nature Structural & Molecular Biology, 22, 7, 572, +, NATURE PUBLISHING GROUP, 2015年07月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Atg101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes, but it is absent from the functionally equivalent Atg1 complex in budding yeast. Here, we report the crystal structure of the fission yeast Atg101-Atg13 complex. Atg101 has a Hop1, Rev7 and Mad2 (HORMA) architecture similar to that of Atg13. Mad2 HORMA has two distinct conformations (O-Mad2 and C-Mad2), and, intriguingly, Atg101 resembles O-Mad2 rather than the C-Mad2-like Atg13. Atg13 HORMA from higher eukaryotes possesses an inherently unstable fold, which is stabilized by Atg101 via interactions analogous to those between O-Mad2 and C-Mad2. Mutational studies revealed that Atg101 is responsible for recruiting downstream factors to the autophagosome-formation site in mammals via a newly identified WF finger. These data define the molecular functions of Atg101, providing a basis for elucidating the molecular mechanisms of mammalian autophagy initiation by the ULK complex.

Mechanisms of Autophagy
Nobuo N. Noda, Fuyuhiko Inagaki
ANNUAL REVIEW OF BIOPHYSICS, VOL 44, 44, 101, 122, ANNUAL REVIEWS, 2015年, ［査読有り］, ［招待有り］, ［筆頭著者, 責任著者］
英語, 論文集(書籍)内論文, The formation of the autophagosome, a landmark event in autophagy, is accomplished by the concerted actions of Atg proteins. The initial step of starvation-induced autophagy in yeast is the assembly of the Atg1 complex, which, with the help of other Atg groups, recruits Atg conjugation systems and initiates the formation of the autophagosome. In this review, we describe from a structural-biological point of view the structure, interaction, and molecular roles of Atg proteins, especially those in the Atg1 complex and in the Atg conjugation systems.

Structural basis of starvation-induced assembly of the autophagy initiation complex
Yuko Fujioka, Sho W. Suzuki, Hayashi Yamamoto, Chika Kondo-Kakuta, Yayoi Kimura, Hisashi Hirano, Rinji Akada, Fuyuhiko Inagaki, Yoshinori Ohsumi, Nobuo N. Noda
Nature Structural & Molecular Biology, 21, 6, 513, 521, NATURE PUBLISHING GROUP, 2014年06月, ［査読有り］, ［最終著者, 責任著者］
英語, 研究論文（学術雑誌）, Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established. Here we studied the interactions of yeast Atg13 with Atg1 and Atg17 by X-ray crystallography. Atg13 binds tandem microtubule interacting and transport domains in Atg1, using an elongated helix-loop-helix region. Atg13 also binds Atg17, using a short region, thereby bridging Atg1 and Atg17 and leading to Atg1-complex formation. Dephosphorylation of specific serines in Atg13 enhanced its interaction with not only Atg1 but also Atg17. These observations update the autophagy-initiation model as follows: upon starvation, dephosphorylated Atg13 binds both Atg1 and Atg17, and this promotes PAS assembly and autophagy progression.

Proteomic Profiling of Autophagosome Cargo in Saccharomyces cerevisiae
Kuninori Suzuki, Shingo Nakamura, Mayumi Morimoto, Kiyonaga Fujii, Nobuo N. Noda, Fuyuhiko Inagaki, Yoshinori Ohsumi
PLOS ONE, 9, 3, e91651, PUBLIC LIBRARY SCIENCE, 2014年03月, ［査読有り］
英語, 研究論文（学術雑誌）, Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.

Architecture of the Atg12-Atg5-Atg16 Complex and its Molecular Role in Autophagy
Nobuo N. Noda, Fuyuhiko Inagaki
Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging, 3, 57, 65, Elsevier Inc., 2014年01月31日, ［査読有り］
英語, 論文集(書籍)内論文, Atg5 is covalently modified with Atg12 via reactions that are similar to ubiquitination, and it noncovalently interacts with Atg16. Formation of the Atg12-Atg5-Atg16 complex is essential for its E3-like function: facilitation of Atg8 transfer from Atg3 to phosphatidylethanolamine at autophagic membranes. Structural studies on the Atg12-Atg5-Atg16 complex revealed that the unique architecture of this protein complex is totally distinct from the other E3 enzymes. The Atg12-Atg5-Atg16 complex interacts directly with Atg3 via Atg12, and enhances the conjugase activity of Atg3 by rearranging its catalytic center, while it is targeted to the membranes via Atg5 and Atg16, and promotes the transfer of Atg8 from Atg3 to the membranes.

Selective Autophagy: Role of Interaction between the Atg8 Family Interacting Motif and Atg8 Family Proteins
Nobuo N. Noda, Fuyuhiko Inagaki
Autophagy: Cancer, Other Pathologies, Inflammation, Immunity, Infection, and Aging, 39, 48, Elsevier Inc., 2013年10月, ［査読有り］
英語, 論文集(書籍)内論文, Autophagy mediates the selective degradation of various targets, such as aggregated proteins, damaged or superfluous organelles, and invading microbes. Specific adaptors, also known as receptors, play a critical role in target recognition in autophagy. Almost all adaptors possess at least one Atg8 family interacting motif (AIM) that interacts directly with Atg8 family proteins, as a prerequisite for selective packaging of targets into autophagosomes. Recent studies have revealed not only the divergence of AIM sequences and their interaction with Atg8 family proteins, but also the regulation of the AIM-Atg8 interaction through phosphorylation. © 2014 Elsevier Inc. All rights reserved.

Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors
Keiko Tsuganezawa, Yoshiyasu Shinohara, Naoko Ogawa, Shun Tsuboi, Norihisa Okada, Masumi Mori, Shigeyuki Yokoyama, Nobuo N. Noda, Fuyuhiko Inagaki, Yoshinori Ohsumi, Akiko Tanaka
Journal of Biomolecular Screening, 18, 9, 1103, 1109, 2013年10月, ［査読有り］
英語, 研究論文（学術雑誌）, The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 μM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors. © 2013 Society for Laboratory Automation and Screening.

Atg18 phosphoregulation controls organellar dynamics by modulating its phosphoinositide-binding activity
Naoki Tamura, Masahide Oku, Moemi Ito, Nobuo N. Noda, Fuyuhiko Inagaki, Yasuyoshi Sakai
JOURNAL OF CELL BIOLOGY, 202, 4, 685, 698, ROCKEFELLER UNIV PRESS, 2013年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven beta-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization.

Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site
MacHiko Sakoh-Nakatogawa, Kazuaki Matoba, Eri Asai, Hiromi Kirisako, Junko Ishii, Nobuo N Noda, Fuyuhiko Inagaki, Hitoshi Nakatogawa, Yoshinori Ohsumi
Nature Structural and Molecular Biology, 20, 4, 433, 439, 2013年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Two autophagy-related ubiquitin-like systems have unique features: the E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, and the other ubiquitin-like protein conjugate Atg12-Atg5 promotes that conjugase activity of Atg3. Here, we elucidate the mode of this action of Atg12-Atg5 as a new E3 enzyme by using Saccharomyces cerevisiae proteins. Biochemical analyses based on structural information suggest that Atg3 requires a threonine residue to catalyze the conjugation reaction instead of the typical asparagine residue used by other E2 enzymes. Moreover, the catalytic cysteine residue of Atg3 is arranged in the catalytic center such that the conjugase activity is suppressed
Atg12-Atg5 induces a reorientation of the cysteine residue toward the threonine residue, which enhances the conjugase activity of Atg3. Thus, this study reveals the mechanism of the key reaction that drives membrane biogenesis during autophagy. © 2013 Nature America, Inc. All rights reserved.

Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation.
Nobuo N Noda, Yuko Fujioka, Takao Hanada, Yoshinori Ohsumi, Fuyuhiko Inagaki
EMBO reports, 14, 2, 206, 11, 2013年02月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12-Atg5 conjugate functions as an E3-like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12-Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.

Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance
James Nyirenda, Shunsuke Matsumoto, Takashi Saitoh, Nobuo Maita, Nobuo N. Noda, Fuyuhiko Inagaki, Daisuke Kohda
STRUCTURE, 21, 1, 32, 41, CELL PRESS, 2013年01月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 N-15 NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide cross-link 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.

Noncanonical recognition and UBL loading of distinct E2s by autophagy-essential Atg7
Masaya Yamaguchi, Kazuaki Matoba, Ryoko Sawada, Yuko Fujioka, Hitoshi Nakatogawa, Hayashi Yamamoto, Yoshihiro Kobashigawa, Hisashi Hoshida, Rinji Akada, Yoshinori Ohsumi, Nobuo N. Noda, Fuyuhiko Inagaki
Nature Structural & Molecular Biology, 19, 12, 1250, +, NATURE PUBLISHING GROUP, 2012年12月, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）, 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.

Structure-based Analyses Reveal Distinct Binding Sites for Atg2 and Phosphoinositides in Atg18
Yasunori Watanabe, Takafumi Kobayashi, Hayashi Yamamoto, Hisashi Hoshida, Rinji Akada, Fuyuhiko Inagaki, Yoshinori Ohsumi, Nobuo N. Noda
JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 38, 31681, 31690, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年09月, ［査読有り］
英語, 研究論文（学術雑誌）, Autophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by an autophagosome and delivered to a lysosome/vacuole. Atg18 plays a critical role in autophagosome formation as a complex with Atg2 and phosphatidylinositol 3-phosphate (PtdIns(3) P). However, little is known about the structure of Atg18 and its recognition mode of Atg2 or PtdIns(3) P. Here, we report the crystal structure of Kluyveromyces marxianus Hsv2, an Atg18 paralog, at 2.6 angstrom resolution. The structure reveals a seven-bladed beta-propeller without circular permutation. Mutational analyses of Atg18 based on the K. marxianus Hsv2 structure suggested that Atg18 has two phosphoinositide-binding sites at blades 5 and 6, whereas the Atg2-binding region is located at blade 2. Point mutations in the loops of blade 2 specifically abrogated autophagy without affecting another Atg18 function, the regulation of vacuolar morphology at the vacuolar membrane. This architecture enables Atg18 to form a complex with Atg2 and PtdIns(3) P in parallel, thereby functioning in the formation of autophagosomes at autophagic membranes.

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans
Fan Wu, Yuping Li, Fuxin Wang, Nobuo N. Noda, Hong Zhang
JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 35, 29457, 29467, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans.

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation
Hitoshi Nakatogawa, Shiran Ohbayashi, Machiko Sakoh-Nakatogawa, Soichiro Kakuta, Sho W. Suzuki, Hiromi Kirisako, Chika Kondo-Kakuta, Nobuo N. Noda, Hayashi Yamamoto, Yoshinori Ohsumi
JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 34, 28503, 28507, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年08月, ［査読有り］
英語, 研究論文（学術雑誌）, In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif ( AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.

Structural Insights into Atg10-Mediated Formation of the Autophagy-Essential Atg12-Atg5 Conjugate
Masaya Yamaguchi, Nobuo N. Noda, Hayashi Yamamoto, Takayuki Shima, Hiroyuki Kumeta, Yoshihiro Kobashigawa, Rinji Akada, Yoshinori Ohsumi, Fuyuhiko Inagaki
STRUCTURE, 20, 7, 1244, 1254, CELL PRESS, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The 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 beta strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and cross-linking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two beta 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.

Tertiary Structure-Function Analysis Reveals the Pathogenic Signaling Potentiation Mechanism of Helicobacter pylori Oncogenic Effector CagA
Takeru Hayashi, Miki Senda, Hiroko Morohashi, Hideaki Higashi, Masafumi Horio, Yui Kashiba, Lisa Nagase, Daisuke Sasaya, Tomohiro Shimizu, Nagarajan Venugopalan, Hiroyuki Kumeta, Nobuo N. Noda, Fuyuhiko Inagaki, Toshiya Senda, Masanori Hatakeyama
CELL HOST & MICROBE, 12, 1, 20, 33, CELL PRESS, 2012年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The Helicobacter pylori type IV secretion effector CagA is a major bacterial virulence determinant and critical for gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA localizes to the inner face of the plasma membrane, where it acts as a pathogenic scaffold/hub that promiscuously recruits host proteins to potentiate oncogenic signaling. We find that CagA comprises a structured N-terminal region and an intrinsically disordered C-terminal region that directs versatile protein interactions. X-ray crystallographic analysis of the N-terminal CagA fragment (residues 1-876) revealed that the region has a structure comprised of three discrete domains. Domain I constitutes a mobile CagA N terminus, while Domain II tethers CagA to the plasma membrane by interacting with membrane phosphatidylserine. Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA. The present work provides a tertiary-structural basis for the pathophysiological/oncogenic action of H. pylori CagA.

Crystal Structure of the C-Terminal Globular Domain of Oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 angstrom Resolution
Shunsuke Matsumoto, Mayumi Igura, James Nyirenda, Masaki Matsumoto, Satoru Yuzawa, Nobuo Noda, Fuyuhiko Inagaki, Daisuke Kohda
BIOCHEMISTRY, 51, 20, 4157, 4166, AMER CHEMICAL SOC, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a beta-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

Structure of the Novel C-terminal Domain of Vacuolar Protein Sorting 30/Autophagy-related Protein 6 and Its Specific Role in Autophagy
Nobuo N. Noda, Takafumi Kobayashi, Wakana Adachi, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 20, 16256, 16266, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年05月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 beta-sheet-alpha-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 beta-alpha 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.

Autophagy-related Protein 32 Acts as Autophagic Degron and Directly Initiates Mitophagy
Noriko Kondo-Okamoto, Nobuo N. Noda, Sho W. Suzuki, Hitoshi Nakatogawa, Ikuko Takahashi, Miou Matsunami, Ayako Hashimoto, Fuyuhiko Inagaki, Yoshinori Ohsumi, Koji Okamoto
JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 13, 10631, 10638, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 Nand 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.

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b
Yoshihiro Kobashigawa, Akira Tomitaka, Hiroyuki Kumeta, Nobuo N. Noda, Masaya Yamaguchi, Fuyuhiko Inagaki
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 108, 51, 20579, 20584, NATL ACAD SCIENCES, 2011年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and non-receptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

Structural Basis of Atg8 Activation by a Homodimeric E1, Atg7
Nobuo N. Noda, Kenji Satoo, Yuko Fujioka, Hiroyuki Kumeta, Kenji Ogura, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki
Molecular Cell, 44, 3, 462, 475, CELL PRESS, 2011年11月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）, E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

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
Masaya Yamaguchi, Nobuo N. Noda, Hitoshi Nakatogawa, Hiroyuki Kumeta, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 38, 29599, 29607, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 similar to 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.

Selective Transport of alpha-Mannosidase by Autophagic Pathways STRUCTURAL BASIS FOR CARGO RECOGNITION BY Atg19 AND Atg34
Yasunori Watanabe, Nobuo N. Noda, Hiroyuki Kumeta, Kuninori Suzuki, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 39, 30026, 30033, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）, In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and alpha-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 atg19 Delta atg34 Delta cells expressing Atg19(Delta ABD), 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 beta-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.

Ser386 phosphorylation of transcription factor IRF-3 induces dimerization and association with CBP/p300 without overall conformational change
Kiyohiro Takahasi, Masataka Horiuchi, Kiyonaga Fujii, Shingo Nakamura, Nobuo N. Noda, Mitsutoshi Yoneyama, Takashi Fujita, Fuyuhiko Inagaki
GENES TO CELLS, 15, 8, 901, 910, WILEY-BLACKWELL, 2010年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The transcription factor IRF-3 is activated by microbial invasions and produces a variety of cytokines including type-I interferon. Upon microbial infection, IRF-3 is phosphorylated at its C-terminal regulatory domain, then oligomerized, translocated into the nucleus, and here it binds to CBP/p300. Although a number of studies have been reported investigating the activation mechanism of IRF-3, there are a number of unresolved issues, especially on the phosphorylation sites, the oligomerization process and the binding mechanism with CBP/p300. In this report, the phosphorylated IRF-3 regulatory domain (IRF-3 RD) was prepared using the kinase IKK-i, and the active form of phosphorylated IRF-3 RD was identified. The paper also reports the crystal structure of the active form of the phosphorylated IRF-3 RD. Furthermore, the phosphorylation of Ser386 was found to be essential for its dimerization and binding with CBP/p300 using mutational analysis and mass spectrometry. Thus, we conclude that the phosphorylation of Ser386 is essential for activation of IRF-3.

The NMR structure of the autophagy-related protein Atg8
Hiroyuki Kumeta, Masahiro Watanabe, Hitoshi Nakatogawa, Masaya Yamaguchi, Kenji Ogura, Wakana Adachi, Yuko Fujioka, Nobuo N. Noda, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOMOLECULAR NMR, 47, 3, 237, 241, SPRINGER, 2010年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Kumeta H, Watanabe M, Nakatogawa H, Yamaguchi M, Ogura K, Adachi W, Fujioka Y, Noda NN, Ohsumi Y, Inagaki F, Journal of biomolecular NMR, 2010, vol. 47, no. 3, pp. 237-241

Atg8-family interacting motif crucial for selective autophagy
Nobuo N. Noda, Yoshinori Ohsumi, Fuyuhiko Inagaki
FEBS LETTERS, 584, 7, 1379, 1385, ELSEVIER SCIENCE BV, 2010年04月, ［査読有り］
英語, Autophagy is a bulk degradation system conserved among most eukaryotes. Recently, autophagy has been shown to mediate selective degradation of various targets such as aggregated proteins and damaged or superfluous organelles. Structural studies have uncovered the conserved specific interactions between autophagic receptors and Atg8-family proteins through WXXL-like sequences, which we term the Atg8-family interacting motif (AIM). AIM functions in various autophagic receptors such as Atg19 in the cytoplasm-to-vacuole targeting pathway, p62 and neighbor of BRCA1 gene 1 (NBR1) in autophagic degradation of protein aggregates, and Atg32 and Nix in mitophagy, and may link the target-receptor complex to autophagic membranes and/or their forming machineries. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Dimeric Coiled-coil Structure of Saccharomyces cerevisiae Atg16 and Its Functional Significance in Autophagy
Yuko Fujioka, Nobuo N. Noda, Hitoshi Nakatogawa, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 2, 1508, 1515, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5.Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 angstrom resolution and its CCD at 2.5 angstrom resolution. The CCD and full-length Atg16 each exhibit an extended alpha-helix, 90 and 130 angstrom, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.

Characterization of the Atg17-Atg29-Atg31 complex specifically required for starvation-induced autophagy in Saccharomyces cerevisiae
Yukiko Kabeya, Nobuo N. Noda, Yuko Fujioka, Kuninori Suzuki, Fuyuhiko Inagaki, Yoshinori Ohsumi
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 389, 4, 612, 615, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2009年11月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 97 kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198 kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation. (C) 2009 Elsevier Inc. All rights reserved.

Crystallization of Saccharomyces cerevisiae alpha-mannosidase, a cargo protein of the Cvt pathway
Yasunori Watanabe, Nobuo N. Noda, Kazuya Honbou, Kuninori Suzuki, Yasuyoshi Sakai, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 65, Pt 6, 571, 573, WILEY-BLACKWELL PUBLISHING, INC, 2009年06月, ［査読有り］
英語, 研究論文（学術雑誌）, Saccharomyces cerevisiae alpha-mannosidase (Ams1) is a cargo protein that is transported to the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ams1 functions as a resident hydrolase. Ams1 has been overexpressed in the methylotrophic yeast Pichia pastoris, purified and crystallized in two crystal forms. Form I belongs to space group P2(1), with unit-cell parameters a = 145.7, b = 127.7, c = 164.0 angstrom, beta = 101.5 degrees. Form II belongs to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 127.9, b = 163.7, c = 291.5 angstrom. Diffraction data were collected from these crystals to a resolution of 3.3 angstrom for form I and of 2.6 angstrom for form II using synchrotron radiation.

Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex
Masataka Horiuchi, Kosei Takeuchi, Nobuo Noda, Nobuyuki Muroya, Toru Suzuki, Takahisa Nakamura, Junko Kawamura-Tsuzuku, Kiyohiro Takahasi, Tadashi Yamamoto, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 284, 19, 13244, 13255, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

The structure of Atg4B-LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy
Kenji Satoo, Nobuo N. Noda, Hiroyuki Kumeta, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
EMBO JOURNAL, 28, 9, 1341, 1350, NATURE PUBLISHING GROUP, 2009年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg8 is conjugated to phosphatidylethanolamine (PE) by ubiquitin-like conjugation reactions. Atg8 has at least two functions in autophagy: membrane biogenesis and target recognition. Regulation of PE conjugation and deconjugation of Atg8 is crucial for these functions in which Atg4 has a critical function by both processing Atg8 precursors and deconjugating Atg8-PE. Here, we report the crystal structures of catalytically inert human Atg4B (HsAtg4B) in complex with processed and unprocessed forms of LC3, a mammalian orthologue of yeast Atg8. On LC3 binding, the regulatory loop and the N-terminal tail of HsAtg4B undergo large conformational changes. The regulatory loop masking the entrance of the active site of free HsAtg4B is lifted by LC3 Phe119, so that a groove is formed along which the LC3 tail enters the active site. At the same time, the N-terminal tail masking the exit of the active site of HsAtg4B in the free form is detached from the enzyme core and a large flat surface is exposed, which might enable the enzyme to access the membrane-bound LC3-PE. The EMBO Journal (2009) 28, 1341-1350. doi: 10.1038/emboj.2009.80; Published online 26 March 2009

ATG Systems from the Protein Structural Point of View
Nobuo N. Noda, Yoshinori Ohsumi, Fuyuhiko Inagaki
CHEMICAL REVIEWS, 109, 4, 1587, 1598, AMER CHEMICAL SOC, 2009年04月, ［査読有り］
英語, Noda NN, Ohsumi Y, Inagaki F, Chemical reviews, 2009, vol. 109, no. 4, pp. 1587-1598

Structural basis of target recognition by Atg8/LC3 during selective autophagy
Nobuo N. Noda, Hiroyuki Kumeta, Hitoshi Nakatogawa, Kenji Satoo, Wakana Adachi, Junko Ishii, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
GENES TO CELLS, 13, 12, 1211, 1218, WILEY-BLACKWELL, 2008年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Crystallization of the coiled-coil domain of Atg16 essential for autophagy
Yuko Fujioka, Nobuo N. Noda, Minako Matsushita, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 64, 1046, 1048, BLACKWELL PUBLISHING, 2008年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg16 is a scaffold protein that interacts with Atg12-Atg5 protein conjugates via its N-terminal domain and self-assembles via its coiled-coil domain, thus forming a multimeric Atg12-Atg5-Atg16 complex that is essential for autophagy. The coiled-coil domain of Atg16 was expressed, purified and crystallized. The crystal belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 127.7, c = 77.8 angstrom. Self-rotation functions and volume-to-weight ratio values suggested that the crystal may contain six molecules per asymmetric unit. Since the domain does not contain a methionine residue, selenomethionine-labelled crystals were prepared with a leucine-to-methionine substitution in the coiled-coil domain and these crystals were used for the collection of single-wavelength anomalous dispersion data to 2.5 angstrom resolution.

Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by the free-interface diffusion method
Nobuo N. Noda, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF SYNCHROTRON RADIATION, 15, Pt 3, 266, 268, WILEY-BLACKWELL, 2008年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12-Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12-Atg5-Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12-Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12-Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12-Atg5-Atg16N ternary complex was formed by mixing purified Atg12-Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 angstrom resolution.

In vitro reconstitution of plant ATG8 and ATG12 conjugation systems essential for autophagy
Yuko Fujioka, Nobuo N. Noda, Kiyonaga Fujii, Kohki Yoshimoto, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 283, 4, 1921, 1928, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2008年01月, ［査読有り］
英語, 研究論文（学術雑誌）, Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.

Crystallization and preliminary crystallographic analysis of the Tob-hCaf1 complex
Kinya Nishida, Masataka Horiuchi, Nobuo N. Noda, Kiyohiro Takahasi, Norimasa Iwasaki, Akio Minami, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 63, 1061, 1063, BLACKWELL PUBLISHING, 2007年12月, ［査読有り］
英語, 研究論文（学術雑誌）, The Tob/BTG family is a group of antiproliferative proteins that contain two highly homologous regions named Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D family of deadenylases. The antiproliferative region of human Tob (residues 1-138) and intact hCaf1 were co-expressed in Escherichia coli, purified and successfully cocrystallized. The crystal belongs to the tetragonal space group I422, with unit-cell parameters a = b = 150.9, c = 113.9 angstrom, and is estimated to contain one heterodimer per asymmetric unit. The crystal diffracted to around 2.6 angstrom resolution.

The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy
Takao Hanada, Nobuo N. Noda, Yoshinori Satomi, Yoshinobu Ichimura, Yuko Fujioka, Toshifumi Takao, Fuyuhiko Inagaki, Yoshinori Ohsumi
JOURNAL OF BIOLOGICAL CHEMISTRY, 282, 52, 37298, 37302, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2007年12月, ［査読有り］
英語, 研究論文（学術雑誌）, 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.

Crystallization and preliminary X-ray analysis of Atg10
Masaya Yamaguti, Nobuo N. Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 63, 443, 445, BLACKWELL PUBLISHING, 2007年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg10 is an E2-like enzyme that catalyzes the conjugation reaction between Atg12 and Atg5. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Microcrystals of Saccharomyces cerevisiae Atg10 were obtained by the free-interface diffusion method using polyethylene glycol and sodium acetate as precipitants. Using these precipitants, large crystals suitable for data collection were obtained using the sitting-drop vapour-diffusion method. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 51.61, c = 256.16 angstrom, and are estimated to contain two protein molecules per asymmetric unit. A native data set was collected to 2.3 angstrom resolution from a single crystal.

Crystallization of Saccharomyces cerevisiae aminopeptidase 1, the major cargo protein of the Cvt pathway
Wakana Adachi, Nobuo N. Suzuki, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 63, 3, 200, 203, BLACKWELL PUBLISHING, 2007年03月
英語, 研究論文（学術雑誌）, The vacuole hydrolase aminopeptidase 1 (Ape1) is a cargo protein transported to the vacuole by the cytosol-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ape1 is processed into mature Ape1 (mApe1). mApe1 has been expressed, purified and crystallized in two crystal forms. Form I belongs to space group P2(1), with unit-cell parameters a = 120.6, b = 219.5, c = 133.1 angstrom, beta = 116.5 degrees. Form II belongs to space group R3, with unit-cell parameters a = 141.2, c = 349.4 angstrom. Diffraction data were collected from these crystals to a resolution of 2.5 angstrom for form I and 1.83 angstrom for form II. Self-rotation functions and the volume-to-weight ratio values suggest that forms I and II contain 12 and four mApe1 molecules per asymmetric unit, respectively, and that mApe1 exists as a tetrahedral dodecamer in both crystal forms.

Structure of Atg5 center dot Atg16, a complex essential for autophagy
Minako Matsushita, Nobuo N. Suzuki, Keisuke Obara, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 282, 9, 6763, 6772, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2007年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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 center dot 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 1.97 angstrom 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.

The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation
Yuya Yamada, Nobuo N. Suzuki, Takao Hanada, Yoshinobu Ichimura, Hiroyuki Kumeta, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 282, 11, 8036, 8043, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2007年03月, ［査読有り］
英語, 研究論文（学術雑誌）, 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-angstrom 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 similar to 80 residues and has a random coil structure in solution and another with a long a-helical structure that protrudes from the core region as far as 30 angstrom. 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.

Crystallization and preliminary crystallographic analysis of human Atg4B-LC3 complex
Kenji Satoo, Nobuo N. Suzuki, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 63, 99, 102, BLACKWELL PUBLISHING, 2007年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The reversible modification of Atg8 with phosphatidylethanolamine (PE) is crucial for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Atg4 is a cysteine protease that is responsible for the processing and deconjugation of Atg8. Human Atg4B (HsAtg4B; a mammalian orthologue of yeast Atg4) and LC3 (a mammalian orthologue of yeast Atg8) were expressed and purified and two complexes, one consisting of HsAtg4B(1-354) and LC3(1-120) (complex I; the product complex) and the other consisting of HsAtg4B(1-354) and LC3(1-124) (complex II; the substrate complex), were crystallized using polyethylene glycol 3350 as a precipitant. In both complexes His280 of HsAtg4B was mutated to alanine. The crystals belong to the same space group P2(1)2(1)2(1), with unit-cell parameters a = 47.5, b = 91.8, c = 102.6 angstrom for complex I and a = 46.9, b = 90.9, c = 102.5 angstrom for complex II. Diffraction data were collected to a resolution of 1.9 angstrom from both crystals.

Full-length p40(phox) structure suggests a basis for regulation mechanism of its membrane binding
Kazuya Honbou, Reiko Minakami, Satoru Yuzawa, Ryu Takeya, Nobuo N. Suzuki, Sachiko Kamakura, Hideki Sumimoto, Fuyuhiko Inagaki
EMBO JOURNAL, 26, 4, 1176, 1186, NATURE PUBLISHING GROUP, 2007年02月, ［査読有り］
英語, 研究論文（学術雑誌）, The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40(phox) associates via the PB1 domain with the essential oxidase activator p67(phox), and is considered to function by recruiting p67(phox) to phagosomes; in this process, the PX domain of p40(phox) binds to phosphatidylinositol 3-phosphate [ PtdIns(3) P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3) P-binding activity of p40(phox) is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40(phox) PB1 domain for the PX domain localizes on the opposite side of that for the p67phox PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1-PB1 association with p67(phox).

Expression, purification and crystallization of the Atg5-Atg16 complex essential for autophagy
Minako Matsushita, Nobuo N. Suzuki, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 62, 1021, 1023, BLACKWELL PUBLISHING, 2006年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg5 is a novel 34 kDa protein that is covalently modified by Atg12, a ubiquitin-like modifier, and forms a complex with Atg16. The Atg12-Atg5-Atg16 complex localizes to autophagosome precursors and plays an essential role in autophagosome formation. Saccharomyces cerevisiae Atg5 in complex with the N-terminal regions of Atg16 was expressed, purified and crystallized in four crystal forms. Forms I, II and III belong to space group P2(1), with unit-cell parameters a = 66.3, b = 104.4, c = 112.1 angstrom, beta = 92.1 degrees (form I), a= 79.5, b = 101.4, c = 95.1 angstrom, beta = 98.6 degrees (form II) or a = 56.9, b = 101.2, c = 66.5 angstrom, beta = 100.6 degrees (form III). Form IV belongs to space group P4(2)2(1)2, with unit-cell parameters a = 73.3, c = 148.1 angstrom. Diffraction data were collected from all crystal forms and high-resolution data to beyond 2.0 angstrom resolution were obtained from a form IV crystal.

Crystallization and preliminary X-ray analysis of Atg3
Yuya Yamada, Nobuo N. Suzuki, Yuko Fujioka, Yoshinobu Ichimura, Yoshinori Ohsumi, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 62, 1016, 1017, BLACKWELL PUBLISHING, 2006年10月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Crystals of Saccharomyces cerevisiae Atg3 have been obtained by the sitting-drop vapour-diffusion method using ammonium sulfate and lithium sulfate as precipitants. A native data set was collected from a single crystal to 2.5 angstrom resolution. The crystals belong to space group P4(1) or P4(3), with unit-cell parameters a = 59.33, c = 115.22 angstrom, and are expected to contain one protein molecule per asymmetric unit.

Crystallization and preliminary crystallographic analysis of p40(phox), a regulatory subunit of NADPH oxidase
Kazuya Honbou, Satoru Yuzawa, Nobuo N. Suzuki, Yuko Fujioka, Hideki Sumimoto, Fuyuhiko Inagaki
ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 62, 1018, 1020, BLACKWELL PUBLISHING, 2006年10月, ［査読有り］
英語, 研究論文（学術雑誌）, p40(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for production of the superoxide that kills invasive microorganisms. Full-length p40(phox) was expressed in Escherichia coli, purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 3.0 angstrom resolution at 100 K using synchrotron radiation. The crystal belongs to space group C222(1), with unit-cell parameters a = 146.27, b = 189.81, c = 79.88 angstrom. This crystal was estimated to contain two or three protein molecules per asymmetric unit from the acceptable range of volume-to-weight ratio values.

Crystal structure of the human Atg4B-LC3 complex
Kenji Sato, Nobuo N. Suzuki, Kenji Sugawara, Yuko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki
AUTOPHAGY, 2, 4, 354, 354, LANDES BIOSCIENCE, 2006年10月, ［査読有り］
英語

Structural basis for the specificity and catalysis of human Atg4B responsible for mammalian autophagy
K Sugawara, NN Suzuki, Y Fujioka, N Mizushima, Y Ohsumi, F Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 280, 48, 40058, 40065, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005年12月, ［査読有り］
英語, 研究論文（学術雑誌）, Reversible modification of Atg8 with phosphatidylethanolamine is crucial for autophagy, the bulk degradation system conserved in eukaryotic cells. Atg4 is a novel cysteine protease that processes and deconjugates Atg8. Herein, we report the crystal structure of human Atg4B ( HsAtg4B) at 1.9-(A) over circle resolution. Despite no obvious sequence homology with known proteases, the structure of HsAtg4B shows a classical papain-like fold. In addition to the papain fold region, HsAtg4B has a small alpha/beta-fold domain. This domain is thought to be the binding site for Atg8 homologs. The active site cleft of HsAtg4B is masked by a loop ( residues 259 - 262), implying a conformational change upon substrate binding. The structure and in vitro mutational analyses provide the basis for the specificity and catalysis of HsAtg4B. This will enable the design of Atg4-specific inhibitors that block autophagy.

Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization
Y Kamada, Y Fujioka, NN Suzuki, F Inagaki, S Wullschleger, R Loewith, MN Hall, Y Ohsumi
MOLECULAR AND CELLULAR BIOLOGY, 25, 16, 7239, 7248, AMER SOC MICROBIOLOGY, 2005年08月, ［査読有り］
英語, 研究論文（学術雑誌）, The target of rapamycin (TOR) protein kinases, Tor1 and Tor2, form two distinct complexes (TOR complex I and 2) in the yeast Saccharomyces cerevisiae. TOR complex 2 (TORC2) contains Tor2 but not Tor1 and controls polarity of the actin cytoskeleton via the Rho1/Pkc1/MAPK cell integrity cascade. Substrates of TORC2 and how TORC2 regulates the cell integrity pathway are not well understood. Screening for multicopy suppressors of tor2, we obtained a plasmid expressing an N-terminally truncated Ypk2 protein kinase. This truncation appears to partially disrupt an autoinhibitory domain in Ypk2, and a point mutation in this region (Ypk2(D239A)) conferred upon full-length Ypk2 the ability to rescue growth of cells compromised in TORC2, but not TORCI, function. YPK2(D239A) also suppressed the lethality of tor2 Delta cells, suggesting that Ypks play an essential role in TORC2 signaling. Ypk2 is phosphorylated directly by Tor2 in vitro, and Ypk2 activity is largely reduced in tor2 Delta cells. In contrast, Ypk2(D239A) has increased and TOR2-independent activity in vivo. Thus, we propose that Ypk protein kinases are direct and essential targets of TORC2, coupling TORC2 to the cell integrity cascade.

The crystal structure of plant ATG12 and its biological implication in autophagy
NN Suzuki, K Yoshimoto, Y Fujioka, Y Ohsumi, F Inagaki
AUTOPHAGY, 1, 2, 119, 126, LANDES BIOSCIENCE, 2005年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG 12 at 1.8 angstrom resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.

Structure of a cell polarity regulator, a complex between atypical PKC and Par6 PB1 domains
Y Hirano, S Yoshinaga, R Takeya, NN Suzuki, M Horiuchi, M Kohjima, H Sumimoto, F Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 280, 10, 9653, 9661, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005年03月, ［査読有り］
英語, 研究論文（学術雑誌）, A complex of atypical PKC and Par6 is a common regulator for cell polarity- related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 Angstrom. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID ( OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40(phox) and p67(phox) PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.

Solution structure of the tandem src homology 3 domains of p47(phox) in an autoinhibited form
S Yuzawa, K Ogura, M Horiuchi, NN Suzuki, Y Fujioka, M Kataoka, H Sumimoto, F Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 279, 28, 29752, 29760, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004年07月, ［査読有り］
英語, 研究論文（学術雑誌）, The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the generation of superoxide anions (O-2(radical anion)) that kill invading microorganisms. p47(phox) is a cytosolic subunit of the phagocyte NADPH oxidase, which plays a crucial role in the assembly of the activated NADPH oxidase complex. The molecular shapes of the p47(phox) tandem SH3 domains either with or without a polybasic/autoinhibitory region (PBR/AIR) at the C terminus were studied using small angle x-ray scattering. The tandem SH3 domains with PBR/AIR formed a compact globular structure, whereas the tandem SH3 domains lacking the PBR/AIR formed an elongated structure. Alignment anisotropy analysis by NMR based on the residual dipolar couplings revealed that the tandem SH3 domains with PBR/AIR were in good agreement with a globular module corresponding to the split half of the intertwisted dimer in crystalline state. The structure of the globular module was elucidated to represent a solution structure of the tandem SH3 domain in the autoinhibited form, where the PBR/AIR bundled the tandem SH3 domains and the linker forming a closed structure. Once PBR/AIR is released by phosphorylation, rearrangements of the SH3 domains may occur, forming an open structure that binds to the cytoplasmic proline-rich region of membrane-bound p22(phox).

The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8
K Sugawara, NN Suzuki, Y Fujioka, N Mizushima, Y Ohsumi, F Inagaki
GENES TO CELLS, 9, 7, 611, 618, BLACKWELL PUBLISHING LTD, 2004年07月, ［査読有り］
英語, 研究論文（学術雑誌）, Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 Angstrom resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABA(A) receptor-associated protein (GABARAP), contains a ubiquitin core with two alpha helices, alpha1 and alpha2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among alpha1, alpha2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins.

Binding of FAD to cytochrome b(558) is facilitated during activation of the phagocyte NADPH oxidase, leading to superoxide production
S Hashida, S Yuzawa, NN Suzuki, Y Fujioka, T Takikawa, H Sumimoto, F Inagaki, H Fujii
JOURNAL OF BIOLOGICAL CHEMISTRY, 279, 25, 26378, 26386, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2004年06月, ［査読有り］
英語, 研究論文（学術雑誌）, The superoxide-producing phagocyte NADPH oxidase can be reconstituted in a cell-free system. The activity of NADPH oxidase is dependent on FAD, but the physiological status of FAD in the oxidase is not fully elucidated. To clarify the role of FAD in NADPH oxidase, FAD-free full-length recombinant p47(phox), p67(phox), p40(phox), and Rac were prepared, and the activity was reconstituted with these proteins and purified cytochrome b(558) (cyt b(558)) with different amounts of FAD. A remarkably high activity, over 100 mumol/s/mumol heme, was obtained in the oxidase with purified cyt b558, ternary complex (p47-p67-p40(phox)), and Rac. From titration with FAD of the activity of NADPH oxidase reconstituted with purified FAD-devoid cyt b(558), the dissociation constant K-d of FAD in cyt b(558) of reconstituted oxidase was estimated as nearly 1 nM. We also examined addition of FAD on the assembly process in reconstituted oxidase. The activity was remarkably enhanced when FAD was present during assembly process, and the efficacy of incorporating FAD into the vacant FAD site in purified cyt b(558) increased, compared when FAD was added after assembly processes. The absorption spectra of reconstituted oxidase under anaerobiosis showed that incorporation of FAD into cyt b(558) recovered electron flow from NADPH to heme. From both K-d values of FAD and the amount of incorporated FAD in cyt b(558) of reconstituted oxidase, in combination with spectra, we propose the model in which the K-d values of FAD in cyt b(558) is changeable after activation and FAD binding works as a switch to regulate electron transfer in NADPH oxidase.

A molecular mechanism for autoinhibition of the tandem SH3 domains of p47(phox), the regulatory subunit of the phagocyte NADPH oxidase
S Yuzawa, NN Suzuki, Y Fujioka, K Ogura, H Sumimoto, F Inagaki
GENES TO CELLS, 9, 5, 443, 456, BLACKWELL PUBLISHING LTD, 2004年05月, ［査読有り］
英語, 研究論文（学術雑誌）, The phagocyte NADPH oxidase is a multisubunit enzyme responsible for the production of reactive oxygen species. p47(phox) is a cytosolic component of the NADPH oxidase and plays an important role in the assembly of the activated complex. The structural determination of the tandem SH3 domains of p47(phox) is crucial for elucidation of the molecular mechanism of the activation of p47(phox). We determined the X-ray crystal structure of the tandem SH3 domains with the polybasic/autoinhibitory region (PBR/AIR) of p47(phox). The GAPPR sequence involved in PBR/AIR forms a left-handed polyproline type-II helix (PPII) and interacts with the conserved SH3 binding surfaces of the SH3 domains simultaneously. These SH3 domains are related by a 2-fold pseudosymmetry axis at the centre of the binding groove and interact with the single PPII helix formed by the GAPPR sequence with opposite orientation. In addition, a number of intra-molecular interactions among the SH3 domains, PBR/AIR and the linker tightly hold the architecture of the tandem SH3 domains into the compact structure and stabilize the autoinhibited form synergistically. Phosphorylation of the serine residues in PBR/AIR could destabilize and successively release the intra-molecular interactions. Thus, the overall structure could be rearranged from the autoinhibitory conformation to the active conformation and the PPII ligand binding surfaces on the SH3 domains are now unmasked, which enables their interaction with the target sequence in p22(phox).

Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase
NN Suzuki, K Koizumi, M Fukushima, A Matsuda, F Inagaki
STRUCTURE, 12, 5, 751, 764, CELL PRESS, 2004年05月, ［査読有り］
英語, 研究論文（学術雑誌）, Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the LICK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first LICK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.

X-ray crystal structure of IRF-3 and its functional implications
K Takahasi, NN Suzuki, M Horiuchi, M Mori, W Suhara, Y Okabe, Y Fukuhara, H Terasawa, S Akira, T Fujita, F Inagaki
NATURE STRUCTURAL BIOLOGY, 10, 11, 922, 927, NATURE PUBLISHING GROUP, 2003年11月, ［査読有り］
英語, 研究論文（学術雑誌）, Transcription factor IRF-3 is post-translationally activated by Toll-like receptor (TLR) signaling and has critical roles in the regulation of innate immunity. Here we present the X-ray crystal structure of the C-terminal regulatory domain of IRF-3(175-427) (IRF-3 175C) at a resolution of 2.3 Angstrom. IRF-3 175C is structurally similar to the Mad homology domain 2 of the Smad family. Structural and functional analyses reveal phosphorylation-induced IRF-3 dimerization, which generates an extensive acidic pocket responsible for binding with p300/CBP. Although TLR and Smad signaling are evolutionarily independent, our results suggest that IRF-3 originates from Smad and acquires its function downstream of TLR.

Crystallization and preliminary X-ray analysis of human uridine-cytidine kinase 2
NN Suzuki, K Koizumi, M Fukushima, A Matsuda, F Inagaki
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 59, 1477, 1478, BLACKWELL MUNKSGAARD, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Uridine-cytidine kinase (UCK), which converts uridine and cytidine to their corresponding monophosphates, is a rate-limiting enzyme involved in the salvage pathway of pyrimidine- nucleotide biosynthesis. Two members of human UCK, named UCK1 and UCK2, were cloned recently and UCK2 was reported to play a crucial role in activating anti-tumour pro-drugs in human cancer cells. Human UCK2 was expressed, purified and crystallized alone or in complex with various ligands. Free UCK and UCK complexes were crystallized in six crystal forms. Form I (ligand-free) belongs to space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 93.7, c = 157.1 Angstrom. Forms IIa ( with CTP), IIb ( with UTP) and IIc ( with cytidine) belong to space group F222, with unit-cell parameters a = 133.3, b = 247.3, c = 91.6 Angstrom ( IIa), a = 132.1, b = 247.0, c = 91.5 Angstrom ( IIb) and a = 136.7, b = 246.3, c = 90.4 Angstrom (IIc), respectively. Form III (with ATPgammaS) belongs to space group C222(1), with unit-cell parameters a = 70.3, b = 149.9, c = 117.2 Angstrom. Form IV ( with cytidine and ATP) belongs to space group C2, with unit-cell parameters a = 89.0, b = 109.7, c = 64.8 Angstrom, beta = 95.3degrees. Diffraction data were collected from these crystals; form IV diffracted to 1.8 Angstrom resolution.

Crystallization and preliminary X-ray analysis of LC3-I
K Sugawara, NN Suzuki, Y Fujioka, N Mizushima, Y Ohsumi, F Inagaki
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 59, 1464, 1465, BLACKWELL MUNKSGAARD, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, Aut7/Apg8 is located in the intermediate structures of the autophagosome and plays an essential role in autophagosome formation. The processed form, cleaved at a C-terminus of Gly120 and called LC3-I, was expressed, purified and crystallized in two crystal forms. One form belongs to space group I4(1), with unit-cell parameters a = 84.39, c = 36.89 Angstrom. The other form belongs to space group P4(1) or P4(3), with unit-cell parameters a = 60.48, c = 35.28 Angstrom. From the latter form, a complete diffraction data set was collected to 2.1 Angstrom resolution.

Crystallization and preliminary crystallographic analysis of the autoinhibited form of the tandem SH3 domain of p47(phox)
S Yuzawa, NN Suzuki, Y Fujioka, K Ogura, H Sumimoto, F Inagaki
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 59, 1479, 1480, WILEY-BLACKWELL, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, p47(phox) is a cytosolic component of the phagocyte NADPH oxidase, which is responsible for the production of the superoxide which kills invasive microorgamisms. A recombinant form of a histidine-tagged tandem SH3 domain of the p47(phox)-containing polybasic autoinhibited region was expressed in Escherichia coli and purified and crystallized by the sitting-drop vapour-diffusion method at 293 K using polyethylene glycol 6000 as a precipitant. Diffraction data were collected to 2.15 Angstrom resolution at 100 K using synchrotron radiation. The crystal belongs to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 100.02, c = 44.94 Angstrom. The presence of one molecule per asymmetric unit gives a crystal volume per protein mass (V-M) of 2.6 Angstrom(3) Da(-1) and a solvent content of 52% by volume.

Crystallization and preliminary crystallographic analysis of DJ-1, a protein associated with male fertility and parkinsonism
K Honbou, NN Suzuki, M Horiuchi, T Taira, T Niki, H Ariga, F Inagaki
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 59, 1502, 1503, WILEY-BLACKWELL, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, DJ-1 was identified as a novel oncogene product that transformed mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 was also correlated with male infertility and parkinsonism. DJ-1 was crystallized using sodium citrate and HEPES at pH 7.5. The crystal belongs to space group P3(1) or P3(2), with unit-cell parameters a = 75.04, c = 74.88 Angstrom and contains two molecules in an asymmetric unit. An intensity data set was collected to 2.00 Angstrom resolution.

The crystal structure of DJ-1, a protein related to male fertility and bParkinson's disease
K Honbou, NN Suzuki, M Horiuchi, T Niki, T Taira, H Ariga, F Inagaki
JOURNAL OF BIOLOGICAL CHEMISTRY, 278, 33, 31380, 31384, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003年08月, ［査読有り］
英語, 研究論文（学術雑誌）, DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-Angstrom resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferase-like superfamily. However, DJ-1 contains an additional alpha-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress.

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年10月
(公社)日本生化学会, 日本語

相分離したp62bodyのULK1によるリン酸化は,酸化還元非依存的なストレス応答を活性化する
一村義信, 池田良, 池田良, 能代大輔, 森下英晃, 森下英晃, 高田周平, 蔭山俊, 藤岡優子, 船越智子, 小松(廣田)聡子, 荒井律子, RYZHII Elena, 阿部学, 古賀友紹, 本橋ほづみ, 中尾光善, 崎村建司, 堀井新, 和栗聡, 野田展生, 小松雅明, 日本生化学会大会(Web), 96th, 2023年

異常活性化型MEK1変異体による癌・先天性疾患の発症と薬剤耐性獲得機序の解明
久保田裕二, 藤岡優子, 高木祐輔, 松原大祐, 飯島正富, 百瀬功, 中井謙太, 野田展生, 武川睦寛, 日本癌学会学術総会抄録集(Web), 81st, 2022年

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年

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年

オートファジーにおける液-液相分離の機能
藤岡優子, 野田展生, 日本蛋白質科学会年会プログラム・要旨集, 22nd (Web), 2022年

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, 2046, 2051, 2021年08月, ［招待有り］, ［最終著者, 責任著者］
日本語

蛋白質の液－液相分離　　　　　　　　　　　　　　　
野田展生, 細胞, 53, 529, 532, 2021年08月, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

相分離で見直すオートファジー　　　　　　　　　　　　　　　
藤岡優子, 野田展生, 実験医学, 39, 10, 172, 177, 2021年06月, ［招待有り］, ［最終著者, 責任著者］
日本語

癌・先天性疾患を導くMEK1遺伝子変異体の異常活性化機構と薬剤耐性獲得機序の解明
久保田裕二, 藤岡優子, 野田展生, 武川睦寛, 日本癌学会学術総会抄録集(Web), 80th, 2021年

In vitro reconstitution of autophagic processes
Jahangir Md. Alam, Nobuo N. Noda, Biochemical Society Transactions, 48, 5, 2003, 2014, 2020年10月01日
Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations. Introduction Autophagy is an intracellular degradation system conserved among eukaryotes from yeast to human and mediates various cellular roles [1]. Autophagy is strongly induced by nutrient starvation, which contributes to cell survival by recycling amino acids, which are obtained by degrading non-essential proteins, for synthesizing essential proteins. Besides starvation, autophagy is induced by various stresses, cellular/organelle damages, and even invasive microbes, and contributes to cellular homeostasis through selective elimination of these harmful targets [2]. When autophagy is induced, an isolation membrane (IM), which is also known as a phagophore, abruptly appears in the cytoplasm, which expands and seals into a double-membrane organelle called an autophagosome [3]. During this process, cytoplasmic materials including proteins and nucleic acids as well as various organelles are sequestered into the autophagosome. Autophagosome then fuses with the lysosome (vacuole in the case of yeast and plant), and the inner membrane of the autophagosome, along with the sequestered materials, is degraded by lysosomal hydrolases. Because all the materials sequestered into autophagosomes are theoretically delivered to the lysosome for degradation, autophagic substrates are chosen during the process of autophagosome formation, indicating the critical importance of this process. Autophagosome formation in budding yeast involves around 20 Atg proteins that function by forming various supramolecular complexes [3,4]. Apart from these protein complexes, autophagosome formation also involves membrane structures including vesicles and organelles, mainly endoplasmic reticulum (ER), and even membraneless organelles, which form various interactions with each other and with Atg proteins [5–7]. Because these diverse higher-order structural components cooperate to mediate the de novo formation of autophagosomes, it is too complicated to be explained exclusively by cell biological approaches. In vitro reconstitution approach is powerful in explaining the molecular mechanisms of such a complicated process by simplification, though we must consistently be aware that the events observed in vitro do not necessarily mean that they also occur in cells. In this review article, we outline in vitro reconstitution trials of the steps involved in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations. Downloaded from http://portlandpress.com/biochemsoctrans/article-pdf/48/5/2003/896463/bst-2020-0130c.pdf by Elsevier user on 20 November 2020., Portland Press Ltd, 英語, 書評論文,書評,文献紹介等

Japanese author 液-液相分離でオートファジーが動きだす
野田 展生, 藤岡 優子, Natureダイジェスト, 17, 5, 28, 31, 2020年05月
ネイチャー・ジャパン ; 2010-, 日本語

液－液相分離によるオートファゴソームの形成部位の構築　　　　　　　　　　　　　　　
藤岡優子, 野田展生, 実験医学, 38, 8, 1354, 1357, 2020年05月, ［招待有り］, ［最終著者, 責任著者］
日本語

天然変性タンパク質によるオートファジー始動液滴の形成
藤岡優子, 野田展生, 生物物理, 60, 3, 171, 173, 2020年05月, ［査読有り］, ［招待有り］, ［最終著者, 責任著者］
一般社団法人 日本生物物理学会, 日本語

柔らかい構造の可視化①LLPSと膜動態を例に　　　　　　　　　　　　　　　
能代大輔, 野田展生, 実験医学, 38, 5, 84, 89, 2020年03月, ［招待有り］, ［最終著者, 責任著者］
日本語

選択的オートファジーの構造生物学的基盤　　　　　　　　　　　　　　　
野田展生, 医学のあゆみ, 272, 9, 769, 775, 2020年02月, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

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年

リゾホスファチジルコリンアシル転移酵素2(LPCAT2)の基質認識構造予測
浜野文三江, 浜野文三江, 的場一晃, 吉田(橋立)智美, 北芳博, 野田展生, 進藤英雄, 進藤英雄, 清水孝雄, 清水孝雄, 清水孝雄, 脂質生化学研究, 62, 2020年

オートファゴソーム形成に関与する膜タンパク質Atg9の膜中配向予測
森貴治, 的場一晃, 野田展生, 杉田有治, 杉田有治, 杉田有治, 分子シミュレーション討論会講演要旨集, 34th (CD-ROM), 2020年

オートファゴソームをつくるための脂質を供給する仕組み　　　　　　　　　　　　　　　
野田展生, FRAGRANCE JOURNAL, 47, 8, 26, 29, 2019年08月, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

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年

オートファジーの構造生物学第二章　　　　　　　　　　　　　　　
野田展生, 生化学, 91, 611, 619, 2019年, ［査読有り］, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

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年09月
(公社)日本生化学会, 日本語

オートファゴソーム形成場PASの液-液相分離を介した構築原理
藤岡優子, ALAM Jahangir Md, 野田展生, 日本分子生物学会年会プログラム・要旨集(Web), 41st, 2018年

オートファジーを制御するタンパク質群の構造と機能　　　　　　　　　　　　　　　
野田展生, バイオサイエンスとインダストリー, 76, 10, 11, 2018年, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

タンパク質を分解して再利用するオートファジーの仕組み　　　　　　　　　　　　　　　
野田展生, 日本の科学者, 53, 427, 433, 2018年, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語, 記事・総説・解説・論説等（商業誌、新聞、ウェブメディア）

オートファゴソーム形成の分子機構　　　　　　　　　　　　　　　
野田展生, 医学のあゆみ, 267, 13, 1014, 1018, 2018年, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

オートファジーの始動機構を観る (第5土曜特集 生命現象を観る : 革新的な構造生命科学が観せてくれる世界) -- (X線)
野田 展生, 医学のあゆみ, 262, 5, 373, 378, 2017年07月29日
医歯薬出版, 日本語

オートファジーの始動機構を観る　　　　　　　　　　　　　　　
野田展生, 医学のあゆみ, 262, 373, 378, 2017年, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語

天然変性タンパク質Atg13によるオートファジー始動複合体の高次集積機構
山本林, 山本林, 藤岡優子, 鈴木翔, 野田展生, 大隅良典, 日本生化学会大会(Web), 89th, 2016年

選択的オートファジー時における液胞加水分解酵素Ape1の認識機構の解明
山崎章徳, 渡邊康紀, 渡邊康紀, 足立わかな, 的場一晃, 桐浴裕巳, 鈴木邦律, 中戸川仁, 大隅良典, 稲垣冬彦, 野田展生, 日本生化学会大会(Web), 88th, 2015年

オートファジーの始動を制御する複合体の立体構造
藤岡優子, 野田展生, 日本結晶学会誌, 57, 3, 2015年

プレオートファゴソーム構造体の中核複合体のin vitro再構成と性状解析
藤岡優子, 山本林, 鈴木翔, 大隅良典, 野田展生, 野田展生, 日本生化学会大会(Web), 88th, 2015年

オートファゴソーム形成を駆動するユビキチン様タンパク質脂質化反応のメカニズム
中戸川万智子, 的場一晃, 野田展生, 稲垣冬彦, 中戸川仁, 大隅良典, 日本蛋白質科学会年会プログラム・要旨集, 14th, 2014年

栄養飢餓に応答してオートファジーを誘導する分子メカニズムの解析
鈴木翔, 藤岡優子, 山本林, 稲垣冬彦, 野田展生, 大隅良典, 日本細胞生物学会大会要旨集, 66th, 2014年

オートファジー関連タンパク質のリン酸化による機能制御
山本林, 鈴木翔, 藤岡優子, 木村弥生, 平野久, 野田展生, 大隅良典, 日本プロテオーム学会大会プログラム・抄録集, 2014 (Web), 2014年

オートファジーの構造生物学
野田 展生, 生化學, 85, 9, 762, 774, 2013年09月25日
日本生化学会, 日本語

Atg13を介したオートファジー始動機構の構造基盤
藤岡優子, 鈴木翔, 山本林, 星田尚司, 赤田倫治, 稲垣冬彦, 大隅良典, 野田展生, 日本生化学会大会(Web), 86th, 2013年

Atg13を介したオートファジー始動機構の構造基盤
藤岡優子, 鈴木翔, 山本林, 角田(近藤)千香, 木村弥生, 平野久, 赤田倫治, 稲垣冬彦, 大隅良典, 野田展生, 日本分子生物学会年会プログラム・要旨集(Web), 36th, 2013年

Structure Analysis of Ultramarine Fluorescent Protein Sirius
Tomoki Matsuda, Nobuo Noda, Fuyuhiko Inagaki, Takeharu Nagai, BIOPHYSICAL JOURNAL, 104, 2, 72A, 72A, 2013年01月
CELL PRESS, 英語, 研究発表ペーパー・要旨（国際会議）

Atg7とそのAtg8結合型の立体構造
野田 展生, 日本結晶学会誌, 54, 3, 166, 171, 2012年06月30日
Atg7 is a noncanonical E1 enzyme that activates Atg8 and transfers it to Atg3 (E2 enzyme), thus playing an essential role in conjugating Atg8 with phosphatidylethanolamine and thus in autophagy. Atg7 protomer is comprised of two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), and forms a homodimer through CTD. Atg7-Atg8 complex structures and biochemical analyses revealed that Atg8 is initially recognized by the C-terminal tail of CTD and is then transferred to the adenylation domain in CTD, where Atg8 Gly116 is adenylated and thioester-linked to the catalytic cysteine of Atg7. Atg8 is then transferred to Atg3 bound to the NTD of the opposite protomer within an Atg7 dimer via a trans mechanism., The Crystallographic Society of Japan, 日本語

植物Atg7-Atg3複合体構造:Atg8結合系におけるE1-E2酵素反応の洞察
的場一晃, 藤岡優子, 中戸川万智子, 中戸川仁, 稲垣冬彦, 大隅良典, 野田展生, 日本生化学会大会(Web), 85th, 2012年

PpAtg18のリン酸化によるペキソファジー膜動態の制御
田村直輝, 奥公秀, 伊藤萌美, 野田展生, 稲垣冬彦, 阪井康能, 生化学, 84回, ROMBUNNO.4T9A-12, 12, 2011年09月
(公社)日本生化学会, 日本語

群青色蛍光タンパク質SiriusのX線結晶構造解析
松田知己, 野田展生, 友杉亘, 稲垣冬彦, 永井健治, 日本蛋白質科学会年会プログラム・要旨集, 11th, 52, 2011年05月27日
日本語

オートファジーの構造生物学
野田展生, 大隅良典, 稲垣冬彦, 日本蛋白質科学会年会プログラム・要旨集, 10th, 24, 2010年05月15日
日本語

Atg8‐family interacting motifを介したAtg3‐Atg8相互作用の解析とその生物学的意義の解明
山口雅也, 野田展生, 中戸川仁, 久米田博之, 大隅良典, 稲垣冬彦, 日本蛋白質科学会年会プログラム・要旨集, 10th, 87, 2010年05月15日
日本語

選択的オートファジー受容体の立体構造から明らかとなった積荷認識の特異性
渡邊康紀, 野田展生, 久米田博之, 鈴木邦律, 大隅良典, 稲垣冬彦, 日本蛋白質科学会年会プログラム・要旨集, 10th, 105, 2010年05月15日
日本語

オートファジー関連タンパク質Atg18の構造解析
伊藤萌美, 野田展生, 田村直輝, 中村新伍, 藤井清永, 藤岡優子, 本坊和也, 阪井康能, 大隅良典, 稲垣冬彦, 日本蛋白質科学会年会プログラム・要旨集, 10th, 104, 2010年05月15日
日本語

Kluyveromyces marxianus Atg10ホモログの構造解析
山口雅也, 野田展生, 久米田博之, 山本林, 赤田倫治, 大隅良典, 稲垣冬彦, 生化学, ROMBUNNO.4P-0580, 2010年
日本語

群青色蛍光タンパク質SiriusのX線結晶構造解析
松田知己, 野田展生, 友杉亘, 稲垣冬彦, 永井健治, 生化学, ROMBUNNO.2P-0011, 2010年
日本語

オートファジーによる選択的タンパク質分解の分子機構と生理的意義
鈴木邦律, 中村新伍, 陰山卓哉, 森本真弓, 野田展生, 稲垣冬彦, 大隅良典, 生化学, 82回, ROMBUNNO.2S2A-4, 4, 2009年09月25日
(公社)日本生化学会, 日本語

Atg8の溶液構造解析及びAtg3との相互作用様式の解析
山口雅也, 野田展生, 久米田博之, 中戸川仁, 渡部正博, 藤岡優子, 大隅良典, 稲垣冬彦, 生化学, ROMBUNNO.3T5P-2, 2009年09月25日
日本語

Structural basis of cargo recognition during selective autophagy
Nobuo N. Noda, Yoshinori Ohsumi, Fuyuhiko Inagaki, AUTOPHAGY, 5, 6, 896, 896, 2009年08月
LANDES BIOSCIENCE, 英語, 研究発表ペーパー・要旨（国際会議）

細胞増殖抑制機能を持つTobN138‐hCaf1タンパク質複合体の結晶化およびそのX線回折像
西田欽也, 堀内正隆, 野田展生, 高橋清大, 岩崎倫政, 三浪明男, 稲垣冬彦, 日本整形外科学会雑誌, 82, 8, S1223, 2008年08月25日
日本語

Cvt pathwayのレセプタータンパク質Atg19の構造生物学的研究
渡辺康紀, 野田展生, 久米田博之, 藤岡優子, 鈴木邦律, 大隅良典, 稲垣冬彦, 生化学, 4P-0636, 2008年
日本語

Cvt経路関連タンパク質Atg8とAtg19 C末の複合体構造解析
久米田博之, 足立わかな, 小椋賢治, 小椋賢治, 野田展生, 野田展生, 藤岡優子, 中戸川仁, 大隅良典, 稲垣冬彦, 稲垣冬彦, Abstr Annu Meet NMR Soc Jpn, 46th, 194, 195, 2007年09月11日
日本語

NMR法を用いたLC3と標的分子の相互作用解析
佐藤健次, 久米田博之, 野田展生, 野田展生, 小椋賢治, 小椋賢治, 藤岡優子, 水島昇, 大隅良典, 稲垣冬彦, 稲垣冬彦, Abstr Annu Meet NMR Soc Jpn, 46th, 234, 235, 2007年09月11日
日本語

Saccharomyces cerevisiae Atg8の立体構造解析
渡部正博, 野田展生, 横地政志, 久米田博之, 小橋川敬博, 藤岡優子, 中戸川仁, 大隅良典, 稲垣冬彦, Abstr Annu Meet NMR Soc Jpn, 46th, 184, 185, 2007年09月11日
日本語

Atg8およびその哺乳類ホモログLC3の標的認識
久米田博之, 野田展生, 藤岡優子, 足立わかな, 佐藤健次, 小椋賢治, 中戸川仁, 水島昇, 大隈良典, 稲垣冬彦, 生化学, 1P-0041, 2007年
日本語

Structural biology of autophagy
Nobuo N. Suzuki, Minako Matsushita, Kenji Sugawara, Kenji Sato, Wakana Adachi, Yuya Yamada, Yuko Fujioka, Yoshinori Ohsumi, Fuyuhiko Inagaki, AUTOPHAGY, 2, 4, 335, 335, 2006年10月
LANDES BIOSCIENCE, 英語, 研究発表ペーパー・要旨（国際会議）

Structural basis of Atg conjugation systems: Crystal structures of Saccharomyces cerevisiae Atg3 and Atg5-Atg16 complex
Nobuo N. Suzuki, Minako Matsushita, Yuya Yamada, Yuko Fujioka, Keisuke Obara, Takao Hanada, Yoshinobu Ichimuro, Yoshinori Ohsumi, Fuyuhiko Inagaki, AUTOPHAGY, 2, 4, 353, 354, 2006年10月
LANDES BIOSCIENCE, 英語, 研究発表ペーパー・要旨（国際会議）

Crystal structure of aminopeptidase 1, the major cargo protein of the Cvt pathway
Wakang Adachi, Nobuo N. Suzuki, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Fuyuhiko Inagaki, AUTOPHAGY, 2, 4, 354, 355, 2006年10月, ［査読有り］
LANDES BIOSCIENCE, 英語, 研究発表ペーパー・要旨（国際会議）

A molecular mechanism for autoinhibition of the tandem SH3 domains of p47(phox), the regulatory subunit of the phagocyte NADPH oxidase (vol 9, pg 443, 2004)
S Yuzawa, NN Suzuki, Y Fujioka, K Ogura, H Sumimoto, F Inagaki, GENES TO CELLS, 9, 6, 609, 609, 2004年06月
BLACKWELL PUBLISHING LTD, 英語, その他

ヒトuridine kinase2の結晶学的研究
野田展生, 小泉克久, 福島正和, 稲垣冬彦, 生化学, 74, 8, 879, 2002年08月25日
日本語

食細胞NADPH oxidaseのFAD結合領域に関する定量的解析
橋田修吉, 滝川喬之, 湯沢聡, 野田展生, 住本秀樹, 稲垣冬彦, 藤井博匡, 生化学, 74, 8, 1070, 2002年08月25日
日本語

IRF‐3のX線結晶構造解析
高橋清大, 野田展生, 堀内正隆, 藤田尚志, 稲垣冬彦, 生化学, 74, 8, 946, 2002年08月25日
日本語

新規細胞増殖抑制因子TobとCaf1蛋白質複合体の結晶構造
堀内正隆, 野田展生, 高橋清大, 室屋伸行, 吉田富, 山本雅, 稲垣冬彦, 生化学, 74, 8, 669, 2002年08月25日
日本語

食細胞NADPH Oxidaseの活性化に必須であるTPRドメイン(N‐p67^phox)とRacとの相互作用 NMRによる解析
吉田慎一, 野田展生, 小椋賢治, 横地政志, 湯沢聡, 堀内正隆, 川村多聞, 住本英樹, 稲垣冬彦, 生化学, 73, 8, 955, 2001年08月25日
日本語

オートファゴソーム新生の分子機構　　　　　　　　　　　　　　　
野田展生
令和4 年度「感染・免疫・がん・炎症」全国共同研究拠点シンポジウム, 2023年03月29日, 日本語
［招待講演］

オートファゴソーム膜の伸展・成形機構　　　　　　　　　　　　　　　
野田展生
タンパク質研究シンポジウム～タンパク質研究はいま新たなステージに入ろうとしている！～, 2022年12月12日, 日本語
［招待講演］

Molecular mechanisms underlying autophagosome biogenesis　　　　　　　　　　　　　　　
Nobuo N. Noda
The 17th International Symposium of the Institute Network for Biomedical Sciences, 2022年10月13日, 英語, 口頭発表（招待・特別）
［招待講演］

Mechanisms of autophagy initiation by liquid-liquid phase separation　　　　　　　　　　　　　　　
Nobuo N. Noda
3rd Frankfurt Conference on Quality Control in Life Processes, 2022年10月06日, 英語, 口頭発表（招待・特別）
［招待講演］

液－液相分離によるオートファジー制御機構の解明　　　　　　　　　　　　　　　
野田展生
第１回生理学研究所—遺伝子病制御研究所連携シンポジウム, 2022年08月12日, 日本語
［招待講演］

Membrane dynamics regulated by autophagy-related proteins　　　　　　　　　　　　　　　
Nobuo N. Noda
FASEB The Protein Folding in the Cell Conference, 2022年07月13日, 英語
［招待講演］

液－液相分離によるオートファジー制御　　　　　　　　　　　　　　　
野田展生
第59回 日本生化学会北海道支部例会, 2022年07月09日, 日本語
［招待講演］

蛍光イメージングを駆使したオートファジー膜動態と液－液相分離研究　　　　　　　　　　　　　　　
野田展生
第74回 日本細胞生物学会大会, 2022年06月29日, 日本語
［招待講演］

Regulation of autophagic membrane dynamics by Atg8 lipidation system　　　　　　　　　　　　　　　
Tatsuro Maruyama, Alam Md. Jahangir, Nobuo N. Noda
Ubiquitin & Friends Symposium 2022, 2022年04月29日, 英語
［招待講演］

オートファジーに関する学際的研究：動作原理から病態生理まで
科学研究費助成事業
2023年11月17日 - 2030年03月31日
小松 雅明, 笹澤 有紀子, 野田 展生, 中戸川 仁, 綿田 裕孝, 安藤 美樹, 服部 信孝, 洲崎 悦生, 日置 寛之
日本学術振興会, 国際共同研究加速基金(国際先導研究), 順天堂大学, 23K20044

液―液相分離とオートファジーによる生体防御機構の解明
科学研究費助成事業
2024年04月 - 2029年03月
小松 雅明, 野田 展生, 和栗 聡, 森下 英晃, 三浦 芳樹
日本学術振興会, 基盤研究(S), 順天堂大学, 24H00060

オートファジーを標的とする膵がんの新規治療法の開発
科学研究費助成事業
2023年04月01日 - 2026年03月31日
大塩 貴子, 園下 将大, 野田 展生
日本学術振興会, 基盤研究(C), 北海道大学, 23K06667

液－液相分離による「ねむり」制御の分子基盤の解明
科学研究費助成事業
2022年04月01日 - 2025年03月31日
野田 展生, 戸田 浩史
日本学術振興会, 基盤研究(A), 北海道大学, 22H00411

パーキンソン病発症に関わるアグリソームの形成とクリアランスの制御機構
科学研究費助成事業
2021年04月01日 - 2024年03月31日
井本 正哉, 斉木 臣二, 野田 展生
日本学術振興会, 基盤研究(B), 順天堂大学, 21H02072

オートファジーシステムの構造学的解明
科学研究費助成事業
2019年06月28日 - 2024年03月31日
野田 展生, 福田 善之
脂質化Atg8は隔離膜に強く結合し、効率的なオートファゴソーム形成において重要な役割を担う。昨年度NMR法を用いることで同定した、膜との相互作用に関わるAtg8内の２つのフェニルアラニンについて変異体解析を行った結果、これらはin vitroにおいて巨大リポソーム膜の変形に重要な役割を担うこと、膜変形は脂質二重層の２つの層の間で面積差が生じることで引き起こされることを見出した。そしてこれらの残基は酵母および哺乳類における効率的なオートファジーに重要であることを明らかにし、Atg8が持つ膜摂動活性がオートファゴソーム形成に重要な役割を担うという新しいモデルを提唱した（Maruyama et al., Nat Struct Mol Biol 2021）。
これまでの研究で我々はAtg2が脂質輸送活性を持つこと、N末端領域に脂質を結合する疎水性ポケットを持つことを明らかにしてきたが、最近公開されたAtg2全長のAlphaFold2予測構造に基づき変異体解析を行なった結果、分子全体にわたって存在する疎水性ポアがAtg2の脂質輸送活性および酵母におけるオートファゴソーム形成に重要であることを明らかにした。
オートファゴソーム形成機構を理解するためには、実際にオートファジーが進行している細胞における膜形態を詳細に明らかにすることが重要である。ラパマイシン処理したAtg8-ｍNeonGreen発現酵母株を急速凍結したものを試料として、cryo-CLEMによる観察対象の局在の可視化、cryo-FIBで細胞のラメラ（薄片）作製を行った。cryo-TEMでラメラを観察したところ、一般的に知られている形状の隔離膜だけでなく、多層の膜構造を有する隔離膜と思われる構造が観察された。
日本学術振興会, 新学術領域研究(研究領域提案型), 19H05707

オートファゴソーム形成場のin vitro再構成と作動機構の解明
科学研究費助成事業
2018年04月01日 - 2021年03月31日
野田 展生
PASはオートファジーの進行に必須な構造体であるが、その実体は長らく不明であった。本研究ではまず出芽酵母におけるPASの性状を解析し、その実体は流動性の高い液滴であることを明らかにした。さらに精製タンパク質を用いた解析により、Atg1複合体が液－液相分離して液滴を形成すること、液滴内ではAtg17がランダムな配向で局在していること、この液滴がPASの構築に働くことを明らかにするとともに、Atg13のリン酸化状態がAtg1複合体の相分離を制御し、それがPASの構築自体を制御していることを明らかにした。さらに液胞膜上に局在するPASを人工膜を用いてin vitro再構成することに成功した。
日本学術振興会, 基盤研究(A), 公益財団法人微生物化学研究会, 18H03989

パーキンソン疾患に挑むケミカルバイオロジー
科学研究費助成事業
2018年04月01日 - 2021年03月31日
井本 正哉, 斉木 臣二, 野田 展生
パーキンソン疾患(PD)のメタボローム 解析およびタンパク質凝集クリアランスを指標にしたスクリーニングでヒットした３種類の治療薬シーズについて，創薬への展開を目指してその作用機構解析研究をおこなった．それらは，それぞれKeap1-Nrf2の結合阻害，PKCを介したTFEB活性化，標的タンパク質の液-液相分離の促進活性を有することでPD患者の脳内で観察されるタンパク質凝集をクリアランスすることを明らかにした．また，いずれの化合物もPDモデル系で顕著な神経保護活性を示した．
日本学術振興会, 基盤研究(B), 18H02099

オートファジー研究の国際活動支援
科学研究費助成事業
2015年11月06日 - 2018年03月31日
水島 昇, 吉森 保, 小松 雅明, 中戸川 仁, 野田 展生, 斉木 臣二
本新学術領域研究は、オートファジーの研究を推進するために、無細胞系構成生物学、構造生物学、細胞生物学、マウス等モデル生物学、ヒト遺伝学、疾患研究を有機的に連携させた集学的研究体制を構築することを目的として設置された。本国際活動支援班では、相互派遣企画委員会と国際共同推進委員会を設置し、領域の研究に関する、国際共同研究や国際連携を推進することを目的に、日本人研究者の海外派遣や海外研究者の招聘や雇用を中心に活動を行った。
日本学術振興会, 新学術領域研究(研究領域提案型), 東京大学, 15K21749

オートファジーを担うAtgタンパク質群の構造基盤
科学研究費助成事業
2013年06月28日 - 2018年03月31日
野田 展生
オートファジーの始動を司るAtg1複合体およびAtg101、オートファゴソーム形成と積荷認識に関わるAtg8の線虫ホモログ群、酵母における選択的オートファジーの積荷Ape1およびその選択的アダプターAtg19等の立体構造をX線結晶構造解析法により決定した。構造情報に基づいた機能解析を行なうことで、飢餓によるオートファジー始動のメカニズム、高等生物におけるAtg8ホモログ間の機能分担、蛋白質凝集体の選択的オートファジーにおける凝集体認識機構などを明らかにした。
日本学術振興会, 新学術領域研究(研究領域提案型), 公益財団法人微生物化学研究会, 25111004

オートファジーの集学的研究：分子基盤から疾患まで
科学研究費助成事業
2013年06月28日 - 2018年03月31日
水島 昇, 斉木 臣二, 野田 展生, 吉森 保, 小松 雅明, 中戸川 仁, 岩井 一宏, 内山 安男, 大隅 良典, 大野 博司, 木南 英紀, 田中 啓二, 佐藤 栄人, 菅原 秀明
本新学術領域研究は、オートファジーの研究を推進するために、無細胞系構成生物学、構造生物学、細胞生物学、マウス等モデル生物学、ヒト遺伝学、疾患研究を有機的に連携させた集学的研究体制を構築することを目的として設置された。本総括班では、領域における計画研究および公募研究の推進（企画調整）と支援を行うとともに、班会議・シンポジウムの開催、領域活動の成果の発信、「Autophagy Forum」の開設と運営、プロトコール集公開などを行った。
日本学術振興会, 新学術領域研究(研究領域提案型), 東京大学, 25111001

天然変性タンパク質Ａｔｇ１３によるオートファジー始動の制御機構
科学研究費助成事業
2012年04月01日 - 2014年03月31日
野田 展生
天然変性タンパク質Atg13は、飢餓時速やかに脱リン酸化され、Atg1およびAtg17と結合することでAtg1複合体を形成し、オートファジーの始動を引き起こす。しかしAtg13がその天然変性領域を用いてどのように他のAtg因子と相互作用するのか、リン酸化によるそれら相互作用の制御はどのように行われているのか等、分子機構の詳細ほほとんどわかっていなかった。
昨年度、Atg1-Atg13複合体の構造決定に成功したが、本年度はAtg13-Atg17複合体の結晶構造解析を行った。その結果、Atg13は天然変性領域内の短い領域二ヶ所を用いて、Atg17のN末端付近とC末端付近の互いに遠く離れた２つの酸性ポケットに結合することが明らかとなった。In vitro、in vivo両面での機能解析の結果、Atg13の２つのAtg17結合領域内に含まれるセリン残基のリン酸化により、Atg17との結合が直接負に制御されることを示した。リン酸化によりAtg13に導入される負電荷が、Atg17の酸性ポケットの負電荷と反発することで結合が減弱されると考えられる。さらにLC-MS/MSおよびリン酸化抗体を用いた解析により、これら結合を負に制御するリン酸化セリンが飢餓により脱リン酸化されることを明らかにした。すなわち飢餓によりAtg13の天然変性領域内の特定のセリン残基が脱リン酸化されると、Atg1およびAtg17両方への高い親和性を獲得し、その結果Atg1複合体の形成およびオートファジーの始動が起きることが明らかとなった。
日本学術振興会, 新学術領域研究(研究領域提案型), 公益財団法人微生物化学研究会, 24113725

選択的オートファジーの積荷を予測するアルゴリズムの作製とスクリーニングへの応用
科学研究費助成事業
2012年04月01日 - 2014年03月31日
鈴木 邦律, 野田 展生
本研究では、タンパク質分解に関わるオートファゴソーム（以下AP）というオルガネラを単離し、内容物を網羅的に同定した後、統計処理を行うことで、AP内に選択的に積み込まれるタンパク質を網羅的に同定しようとしたものである。本研究結果により、非選択的と考えられてきたオートファジーの積荷タンパク質の多くに偏りのあることが明らかとなった。今後はこの偏りを生み出す分子機構を明らかにし、真核生物における選択的オートファジーの生理的役割を解明したい。
日本学術振興会, 挑戦的萌芽研究, 東京大学, 24657083

オートファジー特異的Ｅ１酵素Ａｔｇ７の構造的基盤
科学研究費助成事業
2011年04月01日 - 2014年03月31日
野田 展生
オートファジーに特異的に機能するE1酵素Atg7の立体構造を，単独あるいは他のAtg因子群との複合体として初めて明らかにした．得られた立体構造に基づいた機能解析を進めた結果，Atg7は他のE1酵素に見られない特徴的な構造を用いてユビキチン様因子およびE2酵素を認識し，両者の間の結合反応をこれまで知られていない新規なメカニズムで担うことを明らかにした．Atg7が担う反応はオートファジーにのみ必須であることから，今回得られた知見はオートファジー特異的な制御剤開発への応用が期待できる．
日本学術振興会, 若手研究(A), 公益財団法人微生物化学研究会, 23687012

細胞内分解システムの構造学的解析
科学研究費助成事業
2006年 - 2010年
杤尾 豪人, 栗本 英治, 野田 展生, 山口 芳樹
ユビキチン-プロテアソームシステム及びオートファジーに関与する種々のタンパク質および超分子複合体を対象としてX線結晶構造解析、核磁気共鳴法、中性子小角散乱法等を用い、構造生物学的研究を行った。ユビキチン受容体やユビキチンリガーゼの機能発現およびプロテアソーム複合体の形成、調節のメカニズムやポリユビキチン鎖の構造に関する知見を得ることに成功したほか、脱脂質化酵素Atg4BとLC3の複合体、E1酵素Atg7、E3様酵素Atg12-Atg5結合体,Atg8と受容体Atg19の複合体,そしてLC3と受容体p62の複合体の立体構造を決定し、Atg8/LC3の可逆的脂質修飾機構および選択的オートファジーに関わる受容体との相互作用機構を明らかにした。
日本学術振興会, 特定領域研究, 京都大学, 18076003

自然免疫の構造生物学
科学研究費助成事業
2005年 - 2009年
稲垣 冬彦, 野田 展生, 小椋 賢治, 堀内 正隆
(1)NADPH酸化酵素の活性化に細胞質因子(p47,p67,p40)と膜因子(gp91,p22)の相互作用は必須である。p47のタンデムSH3と膜因子p22のプロリンに富む配列との相互作用およびPXドメインによるp40の膜への繋ぎ止めの制御機構を構造に基づいて明らかにした。(2)リン酸化IRF-3を調製し、IRF-3活性化機構について検討した。(3)インターフェロン産生に関わる細胞質内レセプターRIG like receptorファミリーのC末端ドメインがウィルス特異的なRNA二重鎖の認識に関わること、二重鎖RNAの認識様式を明らかにした。(4)インターフェロン産生に関わるTLR3,TLR4と相互作用するTRIF,TRAMのTIRドメインの構造を明らかにし、活性化に必要なオリゴマー形成機構について議論した。(5)カイコβGRPによる真菌特有の分子であるβ1-3グルカンの認識機構を明らかにした。
日本学術振興会, 基盤研究(S), 北海道大学, 17109002