Updated on 2024/03/11

写真a

 
FUKATA Masaki
 
Organization
Graduate School of Medicine Center for Neurological Diseases and Cance Division Professor
Graduate School
Graduate School of Medicine
Undergraduate School
School of Medicine Department of Medicine
Title
Professor
External link

Degree 1

  1. 博士(医学) ( 2000.3   広島大学 ) 

Research Interests 19

  1. 細胞内情報伝達

  2. 神経科学

  3. 生化学

  4. 低分子量G蛋白質

  5. パルミトイル化

  6. シナプス

  7. グルタミン酸受容体

  8. イメージング

  9. てんかん

  10. G蛋白質

  11. AMPA受容体

  12. 細胞生物学

  13. 酵素

  14. 超解像顕微鏡

  15. 質量分析

  16. 蛋白質輸送

  17. 自己免疫性脳炎

  18. 脂質修飾

  19. 統合脳・分子脳科学

Research Areas 3

  1. Life Science / Medical biochemistry

  2. Life Science / Cell biology

  3. Life Science / Neuroscience-general

Research History 9

  1. Nagoya University Graduate School of Medicine   Professor

    2023.4

  2. National Institute for Physiological Sciences (NIPS), National Institutes of Natural Sciences (NINS)   Division of Membrane Physiology   Professor

    2007.6 - 2024.3

  3. PRESTO, JST   Metabolism and Cellular Function

    2005.10 - 2009.3

  4. National Center for Geriatrics and Gerontology (NCGG), Research Institute   Laboratory of Genomics and Proteomics   Head

    2005.3 - 2007.5

  5. University of California at San Francisco (UCSF)   Department of Physiology   Overseas Research Fellowships

    2003.4 - 2005.2

  6. Nagoya University Graduate School of Medicine   Department of Cell Pharmacology   Assistant

    2001.4 - 2003.3

  7. Nagoya University Graduate School of Medicine   Department of Cell Pharmacology   JSPS (PD)

    2000.4 - 2001.3

  8. Hiroshima University School of Medicine   Department of Biochemistry   JSPS (DC2)

    1998.4 - 2000.3

  9. 神戸大学医学部附属病院   小児科学   医員

    1994.6 - 1995.3

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Education 2

  1. 広島大学大学院   医学系研究科   生理系専攻

    1996.4 - 2000.3

  2. Kobe University   School of Medicine   Faculty of Medicine

    1988.4 - 1994.3

Professional Memberships 3

  1. THE JAPAN NEUROSCIENCE SOCIETY

  2. THE JAPANESE BIOCHEMICAL SOCIETY

  3. Society for Neuroscience

Awards 1

  1. 平成20年度文部科学大臣表彰 若手科学者賞

    2008  

    深田 正紀

 

Papers 102

  1. Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity Reviewed

    Miyazaki Y, Otsuka T, Yamagata Y, Endo T, Sanbo M, Sano H, Kobayashi K, Inahashi H, Kornau H-C, Schmitz D, Prüss H, Meijer D, Hirabayashi M, Fukata Y#, Fukata M#

    Cell Reports   Vol. 43 ( 1 ) page: 113634 - 113634   2024.1

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.celrep.2023.113634

    Scopus

    PubMed

  2. <i>ADAM22</i> ethnic-specific variant reducing binding of membrane-associated guanylate kinases causes focal epilepsy and behavioural disorder Reviewed

    Nosková L*, Fukata Y*, Stránecký V, Šaligová J, Bodnárová O, Giertlová M, Fukata M#, Kmoch S#

    Brain Communications   Vol. 5 ( 6 ) page: fcad295   2023.10

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    Abstract

    Pathogenic variants of ADAM22 affecting either its biosynthesis and/or its interactions with either LGI1 and/or PSD-95 have been recently identified in individuals with developmental and epileptic encephalopathy. Here, we describe a girl with seizures, delayed psychomotor development, and behavioural disorder, carrying a homozygous variant in ADAM22 (NM_021723.5:c.2714C &amp;gt; T). The variant has a surprisingly high frequency in the Roma population of the Czech and Slovak Republic, with 11 of 213 (∼5.2%) healthy Roma individuals identified as heterozygous carriers. Structural in silico characterization revealed that the genetic variant encodes the missense variant p.S905F, which localizes to the PDZ-binding motif of ADAM22. Studies in transiently transfected mammalian cells revealed that the variant has no effect on biosynthesis and stability of ADAM22. Rather, protein–protein interaction studies showed that the p.S905F variant specifically impairs ADAM22 binding to PSD-95 and other proteins from a family of membrane-associated guanylate kinases, while it has only minor effect on ADAM22–LGI1 interaction. Our study indicates that a significant proportion of epilepsy in patients of Roma ancestry may be caused by homozygous c.2714C &amp;gt; T variants in ADAM22. The study of this ADAM22 variant highlights a novel pathogenic mechanism of ADAM22 dysfunction and reconfirms an essential role of interaction of ADAM22 with membrane-associated guanylate kinases in seizure protection in humans.

    DOI: 10.1093/braincomms/fcad295

    Web of Science

    Scopus

    PubMed

    Other Link: https://academic.oup.com/braincomms/article-pdf/5/6/fcad295/53898834/fcad295.pdf

  3. Anti-LGI4 Antibody Is a Novel Juxtaparanodal Autoantibody for Chronic Inflammatory Demyelinating Polyneuropathy. Reviewed International journal

    Xu Zhang, Jun-Ichi Kira, Hidenori Ogata, Tomohiro Imamura, Mikio Mitsuishi, Takayuki Fujii, Masaki Kobayashi, Kazuo Kitagawa, Yukihiro Namihira, Yusuke Ohya, Guzailiayi Maimaitijiang, Ryo Yamasaki, Yuko Fukata, Masaki Fukata, Noriko Isobe, Yuri Nakamura

    Neurology(R) neuroimmunology & neuroinflammation   Vol. 10 ( 2 )   2023.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    BACKGROUND AND OBJECTIVES: The objective of this study was to discover novel nodal autoantibodies in chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: We screened for autoantibodies that bind to mouse sciatic nerves and dorsal root ganglia (DRG) using indirect immunofluorescence (IFA) assays with sera from 113 patients with CIDP seronegative for anti-neurofascin 155 and anticontactin-1 antibodies and 127 controls. Western blotting, IFA assays using HEK293T cells transfected with relevant antigen expression plasmids, and cell-based RNA interference assays were used to identify target antigens. Krox20 and Periaxin expression, both of which independently control peripheral nerve myelination, was assessed by quantitative real-time PCR after application of patient and control sera to Schwann cells. RESULTS: Sera from 4 patients with CIDP, but not control sera, selectively bound to the nodal regions of sciatic nerves and DRG satellite glia (p = 0.048). The main immunoglobulin G (IgG) subtype was IgG4. IgG from these 4 patients stained a 60-kDa band on Western blots of mouse DRG and sciatic nerve lysates. These features indicated leucine-rich repeat LGI family member 4 (LGI4) as a candidate antigen. A commercial anti-LGI4 antibody and IgG from all 4 seropositive patients with CIDP showed the same immunostaining patterns of DRG and cultured rat Schwann cells and bound to the 60-kDa protein in Western blots of LGI4 overexpression lysates. IgG from 3 seropositive patients, but none from controls, bound to cells cotransfected with plasmids containing LGI4 and a disintegrin and metalloprotease domain-containing protein 22 (ADAM22), an LGI4 receptor. In cultured rat Schwann and human melanoma cells constitutively expressing LGI4, LGI4 siRNA effectively downregulated LGI4 and reduced patients' IgG binding compared with scrambled siRNA. Application of serum from a positive patient to Schwann cells expressing ADAM22 significantly reduced the expression of Krox20, but not Periaxin. Anti-LGI4 antibody-positive patients had a relatively old age at onset (mean age 58 years), motor weakness, deep and superficial sensory impairment with Romberg sign, and extremely high levels of CSF protein. Three patients showed subacute CIDP onset resembling Guillain-Barré syndrome. DISCUSSION: IgG4 anti-LGI4 antibodies are found in some elderly patients with CIDP who present subacute sensory impairment and motor weakness and are worth measuring, particularly in patients with symptoms resembling Guillain-Barré syndrome.

    DOI: 10.1212/NXI.0000000000200081

    PubMed

  4. S-palmitoylation of tyrosinase at cysteine500 regulates melanogenesis Reviewed International journal

    Yoko Niki, Naoko Adachi, Masaki Fukata, Yuko Fukata, Shinichiro Oku, Chieko Makino-Okamura, Seiji Takeuchi, Kazumasa Wakamatsu, Shosuke Ito, Lieve Declercq, Daniel B. Yarosh, Tomas Mammone, Chikako Nishigori, Naoaki Saito, Takehiko Ueyama

    Journal of Investigative Dermatology   Vol. 143 ( 2 ) page: 317 - 327   2022.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    Palmitoylation is a lipid modification involving the attachment of palmitic acid to a cysteine residue, thereby affecting protein function. We investigated the effect of palmitoylation of tyrosinase, the rate-limiting enzyme in melanin synthesis, using a human three-dimensional skin model system and melanocyte culture. The palmitoylation inhibitor, 2-bromopalmitate, increased melanin content and tyrosinase protein levels in melanogenic cells by suppressing tyrosinase degradation. The palmitoylation site was Cysteine500 in the C-terminal cytoplasmic tail of tyrosinase. The nonpalmitoylatable mutant, tyrosinase (C500A), was slowly degraded and less ubiquitinated than wild-type tyrosinase. Screening for the Asp-His-His-Cys (DHHC) family of proteins for tyrosinase palmitoylation suggested that DHHC2, 3, 7, and 15 are involved in tyrosinase palmitoylation. Knockdown of DHHC2, 3, or 15 increased tyrosinase protein levels and melanin content. Determination of their subcellular localization in primary melanocytes revealed that DHHC2, 3, and 15 were localized in the endoplasmic reticulum, Golgi apparatus, and/or melanosomes, whereas only DHHC2 was localized in the melanosomes. Immunoprecipitation showed that DHHC2 and DHHC3 predominantly bind to mature and immature tyrosinase, respectively. Taken together, tyrosinase palmitoylation at Cysteine500 by DHHC2, 3, and/or 15, especially DHHC2 in trans-Golgi apparatus and melanosomes and DHHC3 in the endoplasmic reticulum and cis-Golgi apparatus, regulate melanogenesis by modulating tyrosinase protein levels.

    DOI: 10.1016/j.jid.2022.08.040

    PubMed

  5. Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum. Reviewed International journal

    Takafumi Kawai, Hirotaka Narita, Kohtarou Konno, Sharmin Akter, Rizki Tsari Andriani, Hirohide Iwasaki, Shoji Nishikawa, Norihiko Yokoi, Yuko Fukata, Masaki Fukata, Pattama Wiriyasermkul, Pornparn Kongpracha, Shushi Nagamori, Keizo Takao, Tsuyoshi Miyakawa, Manabu Abe, Kenji Sakimura, Masahiko Watanabe, Atsushi Nakagawa, Yasushi Okamura

    The Biochemical journal   Vol. 479 ( 11 ) page: 1127 - 1145   2022.5

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    Language:English   Publishing type:Research paper (scientific journal)  

    Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.

    DOI: 10.1042/BCJ20220033

    PubMed

  6. Biallelic <i>ADAM22</i> pathogenic variants cause progressive encephalopathy and infantile-onset refractory epilepsy Reviewed

    *Marieke M. van, der Knoop, *Reza Maroofian, *Yuko Fukata, Yvette van Ierland, Ehsan G. Karimiani, Anna-Elina Lehesjoki, Mikko Muona, Anders Paetau, Yuri Miyazaki, Yoko Hirano, Laila Selim, Marina de França, Rodrigo Ambrosio Fock, Christian Beetz, Claudia A.L. Ruivenkamp, Alison J. Eaton, Francois D. Morneau-Jacob, Lena Sagi-Dain, Lilach Shemer-Meiri, Amir Peleg, Jumana Haddad-Halloun, Daan J. Kamphuis, Cacha M, P. C. D. Peeters-Scholte, Semra Hiz Kurul, Rita Horvath, Hanns Lochmüller, David Murphy, Stephan Waldmüller, Stephanie Spranger, David Overberg, Alison M. Muir, Aboulfazl Rad, Barbara Vona, Firdous Abdulwahad, Sateesh Maddirevula, Inna S. Povolotskaya, Victoria Y. Voinova, Vykuntaraju K. Gowda, Varunvenkat M. Srinivasan, Fowzan S. Alkuraya, Heather C. Mefford, Majid Alfadhel, Tobias B. Haack, Pasquale Striano, Mariasavina Severino, **Masaki Fukata, **Yvonne Hilhorst-Hofstee, **Henry Houlden

    Brain     2022.4

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    Authorship:Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Oxford University Press (OUP)  

    Abstract

    Pathogenic variants in A Disintegrin And Metalloproteinase (ADAM) 22, the postsynaptic cell membrane receptor for the glycoprotein leucine-rich repeat glioma-inactivated protein 1 (LGI1), have been recently associated with recessive developmental and epileptic encephalopathy. However, so far, only two affected individuals have been described and many features of this disorder are unknown. We refine the phenotype and report 19 additional individuals harboring compound heterozygous or homozygous inactivating ADAM22 variants, of whom 18 had clinical data available. Additionally, we provide follow-up data from two previously reported cases. All affected individuals exhibited infantile-onset, treatment-resistant epilepsy. Additional clinical features included moderate to profound global developmental delay/intellectual disability (20/20), hypotonia (12/20), delayed motor development (19/20). Brain MRI findings included cerebral atrophy (13/20), supported by post-mortem histological examination in patient-derived brain tissue, cerebellar vermis atrophy (5/20), and callosal hypoplasia (4/20). Functional studies in transfected cell lines confirmed the deleteriousness of all identified variants and indicated at least three distinct pathological mechanisms: defective cell membrane expression (1), impaired LGI1-binding (2), and/or impaired interaction with the postsynaptic density protein PSD-95 (3). We reveal novel clinical and molecular hallmarks of ADAM22 deficiency and provide knowledge that might inform clinical management and early diagnostics.

    DOI: 10.1093/brain/awac116

  7. Palmitoylation of the small GTPase Cdc42 by DHHC5 modulates spine formation and gene transcription Reviewed

    Wirth A, Labus J, Galil DA, Schill Y, Schmidt S, Bunke T, Gorinski N, Yokoi N, Fukata M, Ponimaskin E

    Journal of Biological Chemistry     2022

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.jbc.2022.102048

  8. 14-3-3 proteins stabilize LGI1-ADAM22 levels to regulate seizure thresholds in mice Reviewed International journal

    Norihiko Yokoi, Yuko Fukata, Kei Okatsu, Atsushi Yamagata, Yan Liu, Makoto Sanbo, Yuri Miyazaki, Teppei Goto, Manabu Abe, Hidetoshi Kassai, Kenji Sakimura, Dies Meijer, Masumi Hirabayashi, Shuya Fukai, Masaki Fukata

    Cell Reports   Vol. 37 ( 11 ) page: 110107 - 110107   2021.12

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    Authorship:Last author, Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    What percentage of the protein function is required to prevent disease symptoms is a fundamental question in genetic disorders. Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations or autoantibodies, cause epilepsy and amnesia. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, by genetic and structural analysis, we demonstrate that quantitative dual phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to dimerized 14-3-3. This interaction protects LGI1-ADAM22 from endocytosis-dependent degradation. Accordingly, forskolin-induced PKA activation increases ADAM22 levels. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we find that ∼50% of LGI1 and ∼10% of ADAM22 levels are sufficient to prevent lethal epilepsy. Furthermore, ADAM22 function is required in excitatory and inhibitory neurons. These results suggest strategies to increase LGI1-ADAM22 complexes over the required levels by targeting PKA or 14-3-3 for epilepsy treatment.

    DOI: 10.1016/j.celrep.2021.110107

    PubMed

  9. A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro. Reviewed International journal

    Chihiro Nakamoto, Yuhei Goto, Yoko Tomizawa, Yuko Fukata, Masaki Fukata, Kasper Harpsøe, David E Gloriam, Kazuhiro Aoki, Tomonori Takeuchi

    Molecular brain   Vol. 14 ( 1 ) page: 173 - 173   2021.12

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    Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed 'R-GenGAR-DA'. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = - 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

    DOI: 10.1186/s13041-021-00882-8

    PubMed

  10. Encephalitis patient-derived monoclonal GABAA receptor antibodies cause epileptic seizures Reviewed International journal

    Jakob Kreye, Sukhvir K. Wright, Adriana van Casteren, Laura Stöffler, Marie-Luise Machule, S. Momsen Reincke, Marc Nikolaus, Scott van Hoof, Elisa Sanchez-Sendin, Marie A. Homeyer, César Cordero Gómez, Hans-Christian Kornau, Dietmar Schmitz, Angela M. Kaindl, Philipp Boehm-Sturm, Susanne Mueller, Max A. Wilson, Manoj A. Upadhya, Divya R. Dhangar, Stuart Greenhill, Gavin Woodhall, Paul Turko, Imre Vida, Craig C. Garner, Jonathan Wickel, Christian Geis, Yuko Fukata, Masaki Fukata, Harald Prüss

    J Exp Med   Vol. 218 ( 11 ) page: e20210012   2021.9

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    Publisher:Rockefeller University Press  

    <title>Abstract</title>Autoantibodies targeting the GABAA receptor (GABAAR) hallmark an autoimmune encephalitis presenting with frequent seizures and psychomotor abnormalities. Their pathogenic role is still not well-defined, given the common overlap with further autoantibodies and the lack of patient-derived mAbs. Five GABAAR mAbs from cerebrospinal fluid cells bound to various epitopes involving the α1 and γ2 receptor subunits, with variable binding strength and partial competition. mAbs selectively reduced GABAergic currents in neuronal cultures without causing receptor internalization. Cerebroventricular infusion of GABAAR mAbs and Fab fragments into rodents induced a severe phenotype with seizures and increased mortality, reminiscent of encephalitis patients’ symptoms. Our results demonstrate direct pathogenicity of autoantibodies on GABAARs independent of Fc-mediated effector functions and provide an animal model for GABAAR encephalitis. They further provide the scientific rationale for clinical treatments using antibody depletion and can serve as tools for the development of antibody-selective immunotherapies.

    DOI: 10.1084/jem.20210012

  11. Trans-synaptic LGI1–ADAM22–MAGUK in AMPA and NMDA receptor regulation Invited Reviewed

    Yuko Fukata, Yoko Hirano, Yuri Miyazaki, Norihiko Yokoi, Masaki Fukata

    Neuropharmacology   Vol. 194   page: 108628 - 108628   2021.8

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    Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.neuropharm.2021.108628

    PubMed

  12. MAGUKs are essential, but redundant, in long-term potentiation Reviewed

    Xiumin Chen, Yuko Fukata, Masaki Fukata, Roger A. Nicoll

    Proceedings of the National Academy of Sciences   Vol. 118 ( 28 ) page: e2107585118 - e2107585118   2021.7

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    Publishing type:Research paper (scientific journal)   Publisher:Proceedings of the National Academy of Sciences  

    This study presents evidence that the MAGUK family of synaptic scaffolding proteins plays an essential, but redundant, role in long-term potentiation (LTP). The action of PSD-95, but not that of SAP102, requires the binding to the transsynaptic adhesion protein ADAM22, which is required for nanocolumn stabilization. Based on these and previous results, we propose a two-step process in the recruitment of AMPARs during LTP. First, AMPARs, via TARPs, bind to exposed PSD-95 in the PSD. This alone is not adequate to enhance synaptic transmission. Second, the AMPAR/TARP/PSD-95 complex is stabilized in the nanocolumn by binding to ADAM22. A second, ADAM22-independent pathway is proposed for SAP102.

    DOI: 10.1073/pnas.2107585118

    PubMed

  13. Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice. Reviewed International journal

    Tomoyuki Yoshida, Atsushi Yamagata, Ayako Imai, Juhyon Kim, Hironori Izumi, Shogo Nakashima, Tomoko Shiroshima, Asami Maeda, Shiho Iwasawa-Okamoto, Kenji Azechi, Fumina Osaka, Takashi Saitoh, Katsumi Maenaka, Takashi Shimada, Yuko Fukata, Masaki Fukata, Jumpei Matsumoto, Hisao Nishijo, Keizo Takao, Shinji Tanaka, Shigeo Okabe, Katsuhiko Tabuchi, Takeshi Uemura, Masayoshi Mishina, Hisashi Mori, Shuya Fukai

    Nature communications   Vol. 12 ( 1 ) page: 1848 - 1848   2021.3

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    Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.

    DOI: 10.1038/s41467-021-22059-6

    PubMed

  14. LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention Reviewed

    Yuko Fukata, Xiumin Chen, Satomi Chiken, Yoko Hirano, Atsushi Yamagata, Hiroki Inahashi, Makoto Sanbo, Hiromi Sano, Teppei Goto, Masumi Hirabayashi, Hans-Christian Kornau, Harald Prüss, Atsushi Nambu, Shuya Fukai, Roger A. Nicoll, Masaki Fukata

    Proceedings of the National Academy of Sciences   Vol. 118 ( 3 ) page: e2022580118 - e2022580118   2021.1

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    Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Proceedings of the National Academy of Sciences  

    Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv<sub>1</sub> channels, and LRRTM4–Neurexin adhesion molecules. <italic>Adam22</italic><sup><italic>ΔC5/ΔC5</italic></sup> knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

    DOI: 10.1073/pnas.2022580118

    Other Link: https://syndication.highwire.org/content/doi/10.1073/pnas.2022580118

  15. Forelimb movements evoked by optogenetic stimulation of the macaque motor cortex Reviewed

    Hidenori Watanabe, Hiromi Sano, Satomi Chiken, Kenta Kobayashi, Yuko Fukata, Masaki Fukata, Hajime Mushiake, Atsushi Nambu

    Nature Communications   Vol. 11 ( 1 )   2020.12

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    Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1038/s41467-020-16883-5

    Other Link: http://www.nature.com/articles/s41467-020-16883-5

  16. The extracellular domain of angulin-1 and palmitoylation of its cytoplasmic region are required for angulin-1 assembly at tricellular contacts Reviewed

    Yukako Oda, Taichi Sugawara, Yuko Fukata, Yasushi Izumi, Tetsuhisa Otani, Tomohito Higashi, Masaki Fukata, Mikio Furuse

    Journal of Biological Chemistry     2020.2

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1074/jbc.RA119.010491

  17. Human CSF monoclonal LGI1 autoantibodies increase neuronal excitability Reviewed

    Kornau HC, Kreye J, Stumpf A, Fukata Y, Parthier D, Sammons RP, Imbrosci B, Kurpjuweit S, Kowski AB, Fukata M, Prüss H, Schmitz D

    Ann Neurol     2020.1

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1002/ana.25666

  18. Dynamic palmitoylation controls the microdomain localization of the DKK1 receptors CKAP4 and LRP6. Reviewed International journal

    Ryota Sada, Hirokazu Kimura, Yuko Fukata, Masaki Fukata, Hideki Yamamoto, Akira Kikuchi

    Science signaling   Vol. 12 ( 608 ) page: eaat9519   2019.11

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    Dickkopf1 (DKK1) was originally identified as an antagonist of Wnt signaling that binds to and induces the clathrin-mediated endocytosis of the Wnt coreceptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6). DKK1 also binds to cytoskeleton-associated protein 4 (CKAP4), which was originally identified as an endoplasmic reticulum (ER) protein but also functions at the plasma membrane as a receptor for various ligands. The DKK1-CKAP4 pathway is activated in several human cancers and promotes cell proliferation by activating signaling through the kinases PI3K and AKT. We found that both CKAP4 and LRP6 primarily localized to detergent-resistant membrane (DRM) fractions of the plasma membrane in a palmitoylation-dependent manner and that palmitoylation of CKAP4 was required for it to promote cell proliferation. DKK1 induced the depalmitoylation of both CKAP4 and LRP6 by acylprotein thioesterases (APTs), resulting in their translocation to the non-DRM fractions. Moreover, DKK1-dependent depalmitoylation of both receptors required activation of the PI3K-AKT pathway. DKK1 simultaneously bound CKAP4 and LRP6, resulting in the formation of a ternary complex. LRP5/6 knockdown decreased DKK1-dependent AKT activation and cancer cell proliferation through CKAP4, whereas CKAP4 knockdown did not affect DKK1-dependent inhibition of Wnt signaling through LRP5/6. These results indicate that the palmitoylation states of CKAP4 and LRP6 play important roles in their signaling and that LRP5/6 enhance DKK1-CKAP4 signaling.

    DOI: 10.1126/scisignal.aat9519

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  19. ABHD10 is an S-depalmitoylase affecting redox homeostasis through peroxiredoxin-5. Reviewed

    Cao Y, Qiu T, Kathayat R, Azizi SA, Fukata Y, Fukata M, Rice P, Dickinson BC

      Vol. 15   page: 1232 - 1240   2019.11

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  20. Targeting CCR5 trafficking to inhibit HIV-1 infection. Reviewed

    Boncompain G, Herit F, Tessier S, Lescure A, Del Nery E, Gestraud P, Staropoli I, Fukata Y, Fukata M, Brelot A, Niedergang F, Perez F

    Science Advances   Vol. 5   page: eaax0821   2019.10

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    DOI: 10.1126/sciadv.aax0821

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  21. Deleted in colorectal cancer (netrin-1 receptor) antibodies and limbic encephalitis in a cat with hippocampal necrosis. Reviewed International journal

    Daisuke Hasegawa, Yumi Ohnishi, Eiji Koyama, Satoru Matsunaga, Shouhei Ohtani, Akio Nakanishi, Takanori Shiga, James K Chambers, Kazuyuki Uchida, Norihiko Yokoi, Yuko Fukata, Masaki Fukata

    Journal of veterinary internal medicine   Vol. 33 ( 3 ) page: 1440 - 1445   2019.5

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    A 7-year-old neutered female domestic shorthaired cat born in Poland and then moved to Japan presented to the local clinic with recent onset of convulsive cluster seizures and status epilepticus. Magnetic resonance imaging revealed bilateral swelling of the hippocampus with T2 hyperintensity and contrast enhancing image, suggesting hippocampal necrosis. The cat completely recovered after treatment with antiepileptic drugs (AED) and administration of prednisolone (1 mg/kg PO q24h for 4 days and tapered). However, cluster seizures reoccurred and developed into status epilepticus despite increasing doses of AED. Although the convulsions were resolved by other AEDs, stupor and renal failure developed, and the cat was euthanized. Pathological findings were consistent with hippocampal necrosis. Immunological analysis for leucine-rich glioma inactivated 1 (LGI1) autoantibodies was negative, but antibodies against DCC (deleted in colorectal carcinoma) known as netrin-1 receptor were found. This report describes a case of feline autoimmune limbic encephalitis and hippocampal necrosis that were presumably associated with DCC autoantibodies.

    DOI: 10.1111/jvim.15492

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  22. Systematic Screening of Depalmitoylating Enzymes and Evaluation of Their Activities by the Acyl-PEGyl Exchange Gel-Shift (APEGS) Assay. Invited Reviewed International journal

    Kanadome T, Yokoi N, Fukata Y, Fukata M

    Methods Mol Biol   Vol. 2009   page: 83 - 98   2019

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    Palmitoylation is a reversible posttranslational lipid modification of proteins involved in a wide range of cellular functions. More than a thousand proteins are estimated to be palmitoylated. In neurons, PSD-95, a major postsynaptic scaffold protein, requires palmitoylation for its specific accumulation at the synapse and dynamically cycles between palmitoylated and depalmitoylated states. Although palmitoylating enzymes of PSD-95 have been well characterized, little is known about the depalmitoylating enzymes (e.g., thioesterases for palmitoylated PSD-95). An elegant pharmacological analysis has suggested that subsets of α/β hydrolase domain (ABHD)-containing proteins of the metabolic serine hydrolase superfamily involve thioesterases for palmitoylated proteins. Here, we describe a systematic method to screen the ABHD serine hydrolase genes, which unveiled ABHD17 as the depalmitoylating enzyme for PSD-95. Furthermore, we introduce the acyl-PEGyl exchange gel-shift (APEGS) method that enables quantification of palmitoylation levels/stoichiometries on proteins in various biological samples and can be used to monitor the dynamic depalmitoylation process of proteins.

    DOI: 10.1007/978-1-4939-9532-5_7

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  23. Structural basis of epilepsy-related ligand–receptor complex LGI1–ADAM22 Reviewed

    Atsushi Yamagata, Yuri Miyazaki, Norihiko Yokoi, Hideki Shigematsu, Yusuke Sato, Sakurako Goto-Ito, Asami Maeda, Teppei Goto, Makoto Sanbo, Masumi Hirabayashi, Mikako Shirouzu, Yuko Fukata, Masaki Fukata, Shuya Fukai

    Nature Communications   Vol. 9 ( 1 ) page: 1546   2018.12

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    DOI: 10.1038/s41467-018-03947-w

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    Other Link: http://www.nature.com/articles/s41467-018-03947-w

  24. Long-term clinical follow-up of a patient with non-paraneoplastic cerebellar ataxia associated with anti-mGluR1 autoantibodies Reviewed

    Nobuaki Yoshikura, Akio Kimura, Masaki Fukata, Yuko Fukata, Norihiko Yokoi, Naoko Harada, Yuichi Hayashi, Takashi Inuzuka, Takayoshi Shimohata

    Journal of Neuroimmunology   Vol. 319   page: 63 - 67   2018.6

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    The clinical features of cerebellar ataxia associated with anti-metabotropic glutamate receptor 1 (mGluR1) autoantibodies, a rare autoimmune-mediated cerebellar ataxia, remain to be elucidated. Here, we describe a patient with non-paraneoplastic cerebellar ataxia associated with anti-mGluR1 autoantibodies, who was followed up over 5 years. She presented with relapses and remissions of subacute progressive cerebellar ataxia that were responsive to immunotherapy. Although serum anti-mGluR1 autoantibodies were continuously detected and cerebellar atrophy gradually progressed, repeated intravenous immunoglobulin therapy and oral immunosuppressants ensured cerebellar ataxia remained at almost the same level during the observation period.

    DOI: 10.1016/j.jneuroim.2018.04.001

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  25. Neurobiology of autoimmune encephalitis Invited Reviewed

    Masaki Fukata, Norihiko Yokoi, Yuko Fukata

    Current Opinion in Neurobiology   Vol. 48   page: 1 - 8   2018.2

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    Autoimmune encephalitis presenting with amnesia, seizures, and psychosis is highly topical in basic and clinical neuroscience. Recent studies have identified numerous associated autoantibodies, targeting cell-surface synaptic proteins including neurotransmitter receptors (e.g. NMDA receptors (NMDARs)) and a secreted protein, LGI1. In vitro and in vivo analyses of the influence of the autoantibodies have begun to clarify their causal roles. Of particular interest is the generation of recombinant monoclonal antibodies from patients’ B cells with anti-NMDAR encephalitis. Patient monoclonal antibodies could be useful to reveal their direct, detailed pathogenicity. Such identification and characterization of autoantibodies could create new categories of neurological diseases and promote the understanding of patho-physiologic roles of target proteins in human brain function.

    DOI: 10.1016/j.conb.2017.07.012

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  26. In situ screening for postsynaptic cell adhesion molecules during synapse formation Reviewed

    Takeshi Uemura, Tomoko Shiroshima, Asami Maeda, Misato Yasumura, Takashi Shimada, Yuko Fukata, Masaki Fukata, Tomoyuki Yoshida

    JOURNAL OF BIOCHEMISTRY   Vol. 162 ( 4 ) page: 295 - 302   2017.10

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    Neuronal synapse formation is regulated by pre- and postsynaptic cell adhesion molecules. Presynaptic neurexins (NRXNs) and receptor protein tyrosine phosphatases (RPTPs; PTP delta, PTP sigma and LAR in mammals) can induce postsynaptic differentiation through the interaction with various postsynaptic cell adhesion molecules. Here, we developed a novel in situ screening method to identify postsynaptic membranous proteins involved in synaptogenesis. Magnetic beads coated with the extracellular domains of NRXN1 beta(-S4) and PTP delta-A6 variants preferentially induced excitatory postsynaptic differentiation on the beads' surface when co-cultured with cortical neurons. After inducing postsynaptic sites on these beads, protein complexes including NRXN1 beta(-S4)/PTP delta-A6 and their ligands on the neuronal membrane were chemically cross-linked and purified using a magnetic separator. Liquid chromatography-tandem mass spectrometry analysis of the complexes revealed two types of postsynaptic ligands for NRXN1 beta(-S4) and PTP delta-A6, one has an activity to induce presynaptic differentiation in a trans manner, whereas the other has no such activity. These results suggest that synapse formation is regulated by the interplay between presynaptic NRXN/PTP delta and their postsynaptic ligands with functionally different impacts on pre- and postsynaptic differentiation. Thus, our in situ screening method for identifying synapse-organizing complexes will help to understand the molecular basis for elaborate neuronal networks.

    DOI: 10.1093/jb/mvx030

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  27. Epilepsy and synaptic proteins Invited Reviewed

    Yuko Fukata, Masaki Fukata

    CURRENT OPINION IN NEUROBIOLOGY   Vol. 45   page: 1 - 8   2017.8

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    Throughout history, epilepsy affects about 1-2% of the population worldwide. Epilepsy can be caused by traumatic brain injury, exposure to certain toxins and drugs, and mutations of genes that often encode synaptic proteins. In addition to conventional linkage and association studies, the recent trio exome sequencing in epilepsy and proteomic analysis in autoimmune synaptopathies have accelerated identification of novel epilepsy-related proteins, most of which play critical roles in synaptic transmission. Furthermore, super resolution microscopy analysis has revealed subsynaptic nanoscale distribution of presynaptic and postsynaptic proteins and suggests a precise trans-synaptic alignment of neurotransmitter release to receptors. Such identification and characterization of epilepsy-related synaptic proteins have been promoting the development of anti-epileptic drugs and the understanding of mechanisms of synaptic transmission.

    DOI: 10.1016/j.conb.2017.02.001

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  28. Dynamic Palmitoylation Targets MAP6 to the Axon to Promote Microtubule Stabilization during Neuronal Polarization Reviewed

    Elena Tortosa, Youri Adolfs, Masaki Fukata, R. Jeroen Pasterkamp, Lukas C. Kapitein, Casper C. Hoogenraad

    NEURON   Vol. 94 ( 4 ) page: 809 - +   2017.5

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    Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediatedbythe familyofa/bHydrolasedomain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6' s palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.

    DOI: 10.1016/j.neuron.2017.04.042

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  29. Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin Reviewed

    Himari Ogino, Arisa Hisanaga, Takao Kohno, Yuta Kondo, Kyoko Okumura, Takana Kamei, Tempei Sato, Hiroshi Asahara, Hitomi Tsuiji, Masaki Fukata, Mitsuharu Hattori

    JOURNAL OF NEUROSCIENCE   Vol. 37 ( 12 ) page: 3181 - 3191   2017.3

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    The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo. Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.

    DOI: 10.1523/JNEUROSCI.3632-16.2017

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  30. The LGI1-ADAM22 protein complex in synaptic transmission and synaptic disorders Invited Reviewed

    Yuko Fukata, Norihiko Yokoi, Yuri Miyazaki, Masaki Fukata

    NEUROSCIENCE RESEARCH   Vol. 116   page: 39 - 45   2017.3

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    Physiological functioning of the brain requires fine-tuned synaptic transmission, and its dysfunction causes various brain disorders such as autism, dementia, and epilepsy. It is therefore extremely important to identify and characterize key regulators of synaptic function. In particular, disease-related synaptic proteins, such as autism-related neurexin-neuroligin and psychiatric disorder-related NMDA receptor, have attracted considerable attention. Recent basic and clinical research has highlighted critical roles of a ligand-receptor complex, LGI1-ADAM22, in synaptic transmission and brain function, as mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy and autoantibodies to LGI1 cause limbic encephalitis which is characterized by memory loss and seizures. Here, we will review our current knowledge about LGI1 and ADAM22, and discuss their patho-physiological roles in synaptic transmission and synaptic disorders. (C) 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

    DOI: 10.1016/j.neures.2016.09.011

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  31. Astrocyte-Mediated Infantile-Onset Leukoencephalopathy Mouse Model Reviewed

    Shouta Sugio, Koujiro Tohyama, Shinichiro Oku, Kanehiro Fujiyoshi, Takeshi Yoshimura, Keigo Hikishima, Ryutaro Yano, Takahiro Fukuda, Masaya Nakamura, Hideyuki Okano, Masahiko Watanabe, Masaki Fukata, Kazuhiro Ikenaka, Kenji F. Tanaka

    GLIA   Vol. 65 ( 1 ) page: 150 - 168   2017.1

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    Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile-onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte-specific Mlc1 overexpression causes infantile-onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na+/K+ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na+/K+ pump. In contrast, no changes in Na+/K+ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na+/K+ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile-onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile-onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting.

    DOI: 10.1002/glia.23084

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  32. Coupling of a voltage-gated Ca2+ channel homologue with a plasma membrane H+-ATPase in yeast Reviewed

    Toshihiko Cho, Aya Ishii-Kato, Yuko Fukata, Yoshitaka Nakayama, Kazuko Iida, Masaki Fukata, Hidetoshi Iida

    GENES TO CELLS   Vol. 22 ( 1 ) page: 94 - 104   2017.1

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    Yeast has a homologue of mammalian voltage-gated Ca2+ channels (VGCCs), enabling the efficient uptake of Ca2+. It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming (1) and auxiliary (2)/ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear. Therefore, we screened candidate proteins that interact with Mid1 by an unbiased proteomic approach and identified a plasma membrane H+-ATPase, Pma1, as a candidate. Mid1 coimmunoprecipitated with Pma1, and Mid1-EGFP colocalized with Pma1-mCherry at the plasma membrane. The physiological relevance of their interaction was determined using the temperature-sensitive mutant, pma1-10. At the nonpermissive temperature, the membrane potential was less negative and Ca2+ uptake was lower in pma1-10 than in wild-type cells. Increased extracellular H+ increased the rate of Ca2+ uptake. Therefore, H+ extrusion by Pma1 may be important for Ca2+ influx through Cch1/Mid1. These results suggest that Pma1 interacts physically with Cch1/Mid1 Ca2+ channels to enhance their activity via its H+-pumping activity.

    DOI: 10.1111/gtc.12458

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  33. Identification of PSD-95 Depalmitoylating Enzymes Reviewed

    Norihiko Yokoi, Yuko Fukata, Atsushi Sekiya, Tatsuro Murakami, Kenta Kobayashi, Masaki Fukata

    JOURNAL OF NEUROSCIENCE   Vol. 36 ( 24 ) page: 6431 - 6444   2016.6

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    Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. alpha/beta-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, G alpha(q), and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function.

    DOI: 10.1523/JNEUROSCI.0419-16.2016

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  34. Dysfunctional ADAM22 implicated in progressive encephalopathy with cortical atrophy and epilepsy. Reviewed

    Muona M, Fukata Y, Anttonen AK, Laari A, Palotie A, Pihko H, qvist T, Valanne L, Somer M, Fukata M, Lehesjoki AE

    Neurol Genet   Vol. 2 ( 1 ) page: e46   2016.2

  35. Local Palmitoylation Cycles and Specialized Membrane Domain Organization Invited Reviewed

    Yuko Fukata, Tatsuro Murakami, Norihiko Yokoi, Masaki Fukata

    Current Topics in Membranes   Vol. 77   page: 97 - 141   2016

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    Palmitoylation is an evolutionally conserved lipid modification of proteins. Dynamic and reversible palmitoylation controls a wide range of molecular and cellular properties of proteins including the protein trafficking, protein function, protein stability, and specialized membrane domain organization. However, technical difficulties in (1) detection of palmitoylated substrate proteins and (2) purification and enzymology of palmitoylating enzymes have prevented the progress in palmitoylation research, compared with that in phosphorylation research. The recent development of proteomic and chemical biology techniques has unexpectedly expanded the known complement of palmitoylated proteins in various species and tissues/cells, and revealed the unique occurrence of palmitoylated proteins in membrane-bound organelles and specific membrane compartments. Furthermore, identification and characterization of DHHC (Asp-His-His-Cys) palmitoylating enzyme–substrate pairs have contributed to elucidating the regulatory mechanisms and pathophysiological significance of protein palmitoylation. Here, we review the recent progress in protein palmitoylation at the molecular, cellular, and in vivo level and discuss how locally regulated palmitoylation machinery works for dynamic nanoscale organization of membrane domains.

    DOI: 10.1016/bs.ctm.2015.10.003

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  36. The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function Reviewed

    Kathryn L. Lovero, Yuko Fukata, Adam J. Granger, Masaki Fukata, Roger A. Nicoll

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 112 ( 30 ) page: E4129 - E4137   2015.7

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    Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set post-synaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

    DOI: 10.1073/pnas.1511910112

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  37. ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment Reviewed

    Michitaka Suzuki, Tatsuro Murakami, Jinglei Cheng, Hiroyuki Kano, Masaki Fukata, Toyoshi Fujimoto

    MOLECULAR BIOLOGY OF THE CELL   Vol. 26 ( 12 ) page: 2333 - 2342   2015.6

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    Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.

    DOI: 10.1091/mbc.E14-11-1504

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  38. BAI1 regulates spatial learning and synaptic plasticity in the hippocampus Reviewed

    Dan Zhu, Chenchen Li, Andrew M. Swanson, Rosa M. Villalba, Jidong Guo, Zhaobin Zhang, Shannon Matheny, Tatsuro Murakamis, Jason R. Stephenson, Sarah Daniel, Masaki Fukata, Randy A. Hall, Jeffrey J. Olson, Gretchen N. Neigh, Yoland Smith, Donald G. Rainnie, Erwin G. Van Meir

    JOURNAL OF CLINICAL INVESTIGATION   Vol. 125 ( 4 ) page: 1497 - 1508   2015.4

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    Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bail exhibit reduced protein levels of the canonical PO component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bail loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of DAM with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

    DOI: 10.1172/JCI74603

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  39. Postsynaptic nanodomains generated by local palmitoylation cycles Invited Reviewed

    Masaki Fukata, Atsushi Sekiya, Tatsuro Murakami, Norihiko Yokoi, Yuko Fukata

    BIOCHEMICAL SOCIETY TRANSACTIONS   Vol. 43 ( 2 ) page: 199 - 204   2015.4

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    Precise regulation of protein assembly at specialized membrane domains is essential for diverse cellular functions including synaptic transmission. However, it is incompletely understood how protein clustering at the plasma membrane is initiated, maintained and controlled. Protein palmitoylation, a common post-translational modification, regulates protein targeting to the plasma membrane. Such modified proteins are enriched in these specialized membrane domains. In this review, we focus on palmitoylation of PSD-95, which is a major postsynaptic scaffolding protein and makes discrete postsynaptic nanodomains in a palmitoylation-dependent manner and discuss a determinant role of local palmitoylation cycles in creating highly localized hotspots at the membrane where specific proteins concentrate to organize functional domains.

    DOI: 10.1042/BST20140238

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  40. Chemical corrector treatment ameliorates increased seizure susceptibility in a mouse model of familial epilepsy Reviewed

    Norihiko Yokoi, Yuko Fukata, Daisuke Kase, Taisuke Miyazaki, Martine Jaegle, Toshika Ohkawa, Naoki Takahashi, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Keiji Imoto, Dies Meijer, Masahiko Watanabe, Masaki Fukata

    NATURE MEDICINE   Vol. 21 ( 1 ) page: 19 - 26   2015.1

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    Epilepsy is one of the most common and intractable brain disorders. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the pathogenic mechanisms of LGI1 mutations remain unclear. We classified 22 reported LGI1 missense mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE encoding mutant proteins representative of the two groups. The secretion-defective LGI1(E383A) protein was recognized by the ER quality-control machinery and prematurely degraded, whereas the secretable LGI1(S473L) protein abnormally dimerized and was selectively defective in binding to one of its receptors, ADAM22. Both mutations caused a loss of function, compromising intracellular trafficking or ligand activity of LGI1 and converging on reduced synaptic LGI1-ADAM22 interaction. A chemical corrector, 4-phenylbutyrate (4PBA), restored LGI1(E383A) folding and binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI 1(E383A) model mice. This study establishes LGI1-related epilepsy as a conformational disease and suggests new therapeutic options for human epilepsy.

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  41. Non-Microtubular Localizations of Microtubule-Associated Protein 6 (MAP6) Reviewed

    Sylvie Gory-Faure, Vanessa Windscheid, Jacques Brocard, Sylvie Montessuit, Ryouhei Tsutsumi, Eric Denarier, Yuko Fukata, Christophe Bosc, Julie Delaroche, Nora Collomb, Masaki Fukata, Jean-Claude Martinou, Karin Pernet-Gallay, Annie Andrieux

    PLOS ONE   Vol. 9 ( 12 ) page: e114905   2014.12

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    MAP6 proteins (MAP6s), which include MAP6-N (also called Stable Tubule Only Polypeptide, or STOP) and MAP6d1 (MAP6 domain-containing protein 1, also called STOP-Like protein 21 kD, or SL21), bind to and stabilize microtubules. MAP6 deletion in mice severely alters integrated brain functions and is associated with synaptic defects, suggesting that MAP6s may also have alternative cellular roles. MAP6s reportedly associate with the Golgi apparatus through palmitoylation of their N-terminal domain, and specific isoforms have been shown to bind actin. Here, we use heterologous systems to investigate several biochemical properties of MAP6 proteins. We demonstrate that the three N-terminal cysteines of MAP6d1 are palmitoylated by a subset of DHHC-type palmitoylating enzymes. Analysis of the subcellular localization of palmitoylated MAP6d1, including electron microscopic analysis, reveals possible localization to the Golgi and the plasma membrane but no association with the endoplasmic reticulum. Moreover, we observed localization of MAP6d1 to mitochondria, which requires the N-terminus of the protein but does not require palmitoylation. We show that endogenous MAP6d1 localized at mitochondria in mature mice neurons as well as at the outer membrane and in the intermembrane space of purified mouse mitochondria. Last, we found that MAP6d1 can multimerize via a microtubule-binding module. Interestingly, most of these properties of MAP6d1 are shared by MAP6-N. Together, these results describe several properties of MAP6 proteins, including their intercellular localization and multimerization activity, which may be relevant to neuronal differentiation and synaptic functions.

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  42. Identification and Characterization of GABA(A) Receptor Autoantibodies in Autoimmune Encephalitis Reviewed

    Toshika Ohkawa, Shin'Ichiro Satake, Norihiko Yokoi, Yu Miyazaki, Tomohiko Ohshita, Gen Sobue, Hiroshi Takashima, Osamu Watanabe, Yuko Fukata, Masaki Fukata

    JOURNAL OF NEUROSCIENCE   Vol. 34 ( 24 ) page: 8151 - 8163   2014.6

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    Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA-and AMPA-type glutamate receptors, GABA(B) receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against the GABA(A) receptor in human sera from two patients diagnosed with encephalitis who presented with cognitive impairment and multifocal brain MRI abnormalities. Both patients had antibodies directed against the extracellular epitope of the beta 3 subunit of the GABA(A) receptor. The beta 3-subunit-containing GABA(A) receptor was a major target of the patients' serum antibodies in rat hippocampal neurons because the serum reactivity to the neuronal surface was greatly decreased by 80% when the beta 3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABA(A) receptor antibodies, one patient also had a low level of LGI1 antibodies, and the other also had CASPR2 antibodies. Application of the patients' serum at the time of symptom presentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABA(A) receptors. Furthermore, treatment of neurons with the patients' serum selectively reduced miniature IPSC amplitude and frequency without affecting miniature EPSCs. These results strongly suggest that the patients' GABA(A) receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABA(A) receptor encephalitis and expands the pathogenic roles of GABA(A) receptor autoantibodies.

    DOI: 10.1523/JNEUROSCI.4415-13.2014

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  43. Functional phylogenetic analysis of LGI proteins identifies an interaction motif crucial for myelination Reviewed

    Linde Kegel, Martine Jaegle, Siska Driegen, Eerik Aunin, Kris Leslie, Yuko Fukata, Masahiko Watanabe, Masaki Fukata, Dies Meijer

    DEVELOPMENT   Vol. 141 ( 8 ) page: 1749 - 1756   2014.4

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    The cellular interactions that drive the formation and maintenance of the insulating myelin sheath around axons are only partially understood. Leucine-rich glioma-inactivated (LGI) proteins play important roles in nervous system development and mutations in their genes have been associated with epilepsy and amyelination. Their function involves interactions with ADAM22 and ADAM23 cell surface receptors, possibly in apposing membranes, thus attenuating cellular interactions. LGI4-ADAM22 interactions are required for axonal sorting and myelination in the developing peripheral nervous system (PNS). Functional analysis revealed that, despite their high homology and affinity for ADAM22, LGI proteins are functionally distinct. To dissect the key residues in LGI proteins required for coordinating axonal sorting and myelination in the developing PNS, we adopted a phylogenetic and computational approach and demonstrate that the mechanism of action of LGI4 depends on a cluster of three amino acids on the outer surface of the LGI4 protein, thus providing a structural basis for the mechanistic differences in LGI protein function in nervous system development and evolution.

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  44. Neuronal major histocompatibility complex class I molecules are implicated in the generation of asymmetries in hippocampal circuitry Reviewed

    Aiko Kawahara, Shotaro Kurauchi, Yuko Fukata, Jose Martinez-Hernandez, Terumi Yagihashi, Yuya Itadani, Rui Sho, Taiichi Kajiyama, Nao Shinzato, Kenji Narusuye, Masaki Fukata, Rafael Lujan, Ryuichi Shigemoto, Isao Ito

    JOURNAL OF PHYSIOLOGY-LONDON   Vol. 591 ( 19 ) page: 4777 - 4791   2013.10

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    Key points center dot The molecular basis of left-right asymmetries in brain structure and function is a central question in neuroscience.
    center dot We have previously demonstrated that the neuronal circuitry composed of hippocampal pyramidal neurones is asymmetrical depending on the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone.
    center dot In this study, we analysed the hippocampus of 2-microglobulin (2m)-deficient mice lacking stable cell surface expression of major histocompatibility complex class I (MHCI), which is known to be important in cellular immunity.
    center dot We found that 2m-deficient mice lacked structural and functional asymmetries in hippocampal circuitry, suggesting that MHCI is critical for the generation of hippocampal asymmetry.
    center dot Our results provide a first step in elucidating the cellular process that generates brain asymmetries.
    Abstract Left-right asymmetry is a fundamental feature of higher-order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluR2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of 2 subunits affects the properties of NMDARs and generates two populations of synapses, 2-dominant' and 2-non-dominant' synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of 2-microglobulin (2m)-deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity-dependent refinement of neuronal connections and normal synaptic plasticity. We found that 2m proteins were localised in hippocampal synapses in wild-type mice. NMDA EPSCs in 2m-deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25-6981, an 2 subunit-selective antagonist, with those in 2-dominant' synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the 2m-deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of 2-dominant' synapses. These observations indicate that the 2m-deficient hippocampus lacks 2-non-dominant' synapses and circuit asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.

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  45. In Silico Screening for Palmitoyl Substrates Reveals a Role for DHHC1/3/10 (zDHHC1/3/11)-mediated Neurochondrin Palmitoylation in Its Targeting to Rab5-positive Endosomes Reviewed

    Shinichiro Oku, Naoki Takahashi, Yuko Fukata, Masaki Fukata

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 288 ( 27 ) page: 19816 - 19829   2013.7

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    Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) gamma-2, TARP gamma-8, cornichon-2, Ca2+/calmodulin-dependent protein kinase II alpha, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.

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  46. Local palmitoylation cycles define activity-regulated postsynaptic subdomains Reviewed

    Yuko Fukata, Ariane Dimitrov, Gaelle Boncompain, Ole Vielemeyer, Franck Perez, Masaki Fukata

    JOURNAL OF CELL BIOLOGY   Vol. 202 ( 1 ) page: 145 - 161   2013.7

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    Distinct PSD-95 clusters are primary landmarks of postsynaptic densities (PSDs), which are specialized membrane regions for synapses. However, the mechanism that defines the locations of PSD-95 clusters and whether or how they are reorganized inside individual dendritic spines remains controversial. Because palmitoylation regulates PSD-95 membrane targeting, we combined a conformation-specific recombinant antibody against palmitoylated PSD-95 with live-cell super-resolution imaging and discovered subsynaptic nanodomains composed of palmitoylated PSD-95 that serve as elementary units of the PSD. PSD-95 in nanodomains underwent continuous de/repalmitoylation cycles driven by local palmitoylating activity, ensuring the maintenance of compartmentalized PSD-95 clusters within individual spines. Plasma membrane targeting of DHHC2 palmitoyltransferase rapidly recruited PSD-95 to the plasma membrane and proved essential for postsynaptic nanodomain formation. Furthermore, changes in synaptic activity rapidly reorganized PSD-95 nano-architecture through plasma membrane-inserted DHHC2. Thus, the first genetically encoded antibody sensitive to palmitoylation reveals an instructive role of local palmitoylation machinery in creating activity-responsive PSD-95 nanodomains, contributing to the PSD (re)organization.

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  47. 2-Bromopalmitate Analogues as Activity-Based Probes To Explore Palmitoyl Acyltransferases Reviewed

    Baohui Zheng, Michael DeRan, Xinyan Li, Xuebin Liao, Masaki Fukata, Xu Wu

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 135 ( 19 ) page: 7082 - 7085   2013.5

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    Reversible S-palmitoylation is an important post-translational modification that regulates the trafficking, localization, and activity of proteins. Cysteine-rich Asp-His-His-Cys (DHHC) domain-containing enzymes are evolutionarily conserved protein palmitoyl acyltransferases (PATs). The human genome encodes 23 DHHC-PATs that regulate diverse cellular functions. Although chemical probes and proteomic methods to detect palmitoylated protein substrates have been reported, no probes for direct detection of the activity of PATs are available. Here we report the synthesis and characterization of 2-bromohexadec-15-ynoic acid and 2-bromooctadec-17-ynoic acid, which are analogues of 2-bromopalmitate (2-BP), as activity-based probes for PATs as well as other palmitoylating and 2-BP-binding enzymes. These probes will serve as new chemical tools for activity-based protein profiling to explore PATs, to dissect the functions of PATs in cell signaling and diseases, and to facilitate the identification of their inhibitors.

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  48. Autoantibodies to epilepsy-related LGI1 in limbic encephalitis neutralize LGI1-ADAM22 interaction and reduce synaptic AMPA receptors Reviewed

    Toshika Ohkawa, Yuko Fukata, Miwako Yamasaki, Taisuke Miyazaki, Norihiko Yokoi, Hiroshi Takashima, Masahiko Watanabe, Osamu Watanabe, Masaki Fukata

    Journal of Neuroscience   Vol. 33 ( 46 ) page: 18161 - 18174   2013

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    More than 30 mutations in LGI1, a secreted neuronal protein, have been reported with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although LGI1 haploinsufficiency is thought to cause ADLTE, the underlying molecular mechanism that results in abnormal brain excitability remains mysterious. Here, we focused on a mode of action of LGI1 autoantibodies associated with limbic encephalitis (LE), which is one of acquired epileptic disorders characterized by subacute onset of amnesia and seizures.Wecomprehensively screened human sera from patients with immune-mediated neurological disorders for LGI1 autoantibodies, which also uncovered novel autoantibodies against six cell surface antigens including DCC, DPP10, and ADAM23. Our developed ELISA arrays revealed a specific role for LGI1 antibodies in LE and concomitant involvement of multiple antibodies, including LGI1 antibodies in neuromyotonia, a peripheral nerve disorder. LGI1 antibodies associated with LE specifically inhibited the ligand-receptor interaction between LGI1 and ADAM22/23 by targeting the EPTP repeat domain of LGI1 and reversibly reduced synaptic AMPA receptor clusters in rat hippocampal neurons. Furthermore, we found that disruption of LGI1-ADAM22 interaction by soluble extracellular domain ofADAM22was sufficient to reduce synapticAMPAreceptors in rat hippocampal neurons and that levels ofAMPAreceptor were greatly reduced in the hippocampal dentate gyrus in the epileptic LGI1 knock-out mouse. Therefore, either genetic or acquired loss of the LGI1-ADAM22 interaction reduces theAMPAreceptor function, causing epileptic disorders. These results suggest that by finely regulating the synapticAMPAreceptors, the LGI1-ADAM22 interaction maintains physiological brain excitability throughout life. © 2013 the authors.

    DOI: 10.1523/JNEUROSCI.3506-13.2013

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  49. Leucine-rich glioma inactivated 1 (Lgi1), an epilepsy-related secreted protein, has a nuclear localization signal and localizes to both the cytoplasm and the nucleus of the caudal ganglionic eminence neurons Reviewed

    Sayaka Kusuzawa, Takao Honda, Yuko Fukata, Masaki Fukata, Shigeaki Kanatani, Daisuke H. Tanaka, Kazunori Nakajima

    EUROPEAN JOURNAL OF NEUROSCIENCE   Vol. 36 ( 3 ) page: 2284 - 2292   2012.8

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    Leucine-rich glioma inactivated 1 (Lgi1) is a secreted synaptic protein that organizes a transsynaptic protein complex throughout the brain. Mutations in the Lgi1 gene have been found in patients with autosomal dominant lateral temporal lobe epilepsy (ADLTE). Although a large number of studies have focused on the expression and function of Lgi1 in the postnatal brain, information regarding its functions and distribution during development remains sparse. Here we report that Lgi1 mRNA is preferentially expressed in the caudal ganglionic eminence (CGE) of the early embryonic telencephalon, and LGI1 protein is unexpectedly localized in the nucleus of dissociated CGE neurons. Using bioinformatics analysis, we found that LGI1 contains a putative nuclear localization signal (NLS) in its leucine-rich repeat C-terminal domain. Furthermore, we show that the transient expression of Lgi1 in CGE neurons resulted in nuclear translocation of the LGI1 protein, and a mutation in the NLS led to the retention of LGI1 in the cytoplasm. We also confirmed that the NLS sequence of LGI1 had the ability to mediate the nuclear localization by using the NLS-containing fusion protein. Interestingly, when Lgi1 was expressed in neurons obtained from the medial ganglionic eminence or cerebral cortex, almost no nuclear localization of LGI1 was observed. These results raise the possibility of a novel role of Lgi1 within embryonic neurons through nuclear translocation and may provide insight into its potential effects on the development of the central nervous system and ADLTE pathogenesis.

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  50. Phosphatidylinositol 4-Kinase II alpha Is Palmitoylated by Golgi-localized Palmitoyltransferases in Cholesterol-dependent Manner Reviewed

    Dongmei Lu, Hui-qiao Sun, Hanzhi Wang, Barbara Barylko, Yuko Fukata, Masaki Fukata, Joseph P. Albanesi, Helen L. Yin

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 287 ( 26 ) page: 21856 - 21865   2012.6

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    Phosphatidylinositol 4-kinase II alpha (PI4KII alpha) is predominantly Golgi-localized, and it generates &gt;50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KII alpha and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (CCPCC177)-C-173 motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KII alpha and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KII alpha and were colocalized with PI4KII alpha in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KII alpha palmitoylation by &gt;3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KII alpha palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KII alpha, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KII alpha (CCPCC177)-C-173 palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-beta-cyclodextrin reversibly altered PI4KII alpha association with these DHHCs as well as PI4KII alpha localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KII alpha behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KII alpha to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.

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  51. SYNAPTIC PLASTICITY REGULATED BY PROTEIN-PROTEIN INTERACTIONS AND POSTTRANSLATIONAL MODIFICATIONS Invited Reviewed

    Norihiko Yokoi, Masaki Fukata, Yuko Fukata

    INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY, VOL 297   Vol. 297   page: 1 - 43   2012

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    alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) mediate the majority of fast excitatory synaptic transmission in the brain. AMPARs dynamically cycle in and out of the postsynaptic membrane in an activity-dependent manner. Because the number and functional properties of AMPARs at the postsynapse determine the efficacy of synaptic transmission, molecular mechanisms underlying AM PAR trafficking and gating are considered to have a central role in synaptic plasticity, a basic mechanism for learning and memory. In this chapter, we review the current knowledge about the regulatory mechanisms for AM PAR trafficking and channel gating by protein-protein interactions and posttranslational modifications. Especially, we focus on the recently established mode of action of the AMPAR auxiliary subunit, stargazin/TARPs, and PSD-95 scaffold. Furthermore, we introduce novel players in AMPAR regulation, PSD-95 palmitoylating enzymes and epilepsy-related ligand, LGI1.

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  52. LGI2 Truncation Causes a Remitting Focal Epilepsy in Dogs Reviewed

    Eija H. Seppala, Tarja S. Jokinen, Masaki Fukata, Yuko Fukata, Matthew T. Webster, Elinor K. Karlsson, Sami K. Kilpinen, Frank Steffen, Elisabeth Dietschi, Tosso Leeb, Ranja Eklund, Xiaochu Zhao, Jennifer J. Rilstone, Kerstin Lindblad-Toh, Berge A. Minassian, Hannes Lohi

    PLOS GENETICS   Vol. 7 ( 7 ) page: e1002194   2011.7

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    One quadrillion synapses are laid in the first two years of postnatal construction of the human brain, which are then pruned until age 10 to 500 trillion synapses composing the final network. Genetic epilepsies are the most common neurological diseases with onset during pruning, affecting 0.5% of 2-10-year-old children, and these epilepsies are often characterized by spontaneous remission. We previously described a remitting epilepsy in the Lagotto romagnolo canine breed. Here, we identify the gene defect and affected neurochemical pathway. We reconstructed a large Lagotto pedigree of around 34 affected animals. Using genome-wide association in 11 discordant sib-pairs from this pedigree, we mapped the disease locus to a 1.7 Mb region of homozygosity in chromosome 3 where we identified a protein-truncating mutation in the Lgi2 gene, a homologue of the human epilepsy gene LGI1. We show that LGI2, like LGI1, is neuronally secreted and acts on metalloproteinase-lacking members of the ADAM family of neuronal receptors, which function in synapse remodeling, and that LGI2 truncation, like LGI1 truncations, prevents secretion and ADAM interaction. The resulting epilepsy onsets at around seven weeks (equivalent to human two years), and remits by four months (human eight years), versus onset after age eight in the majority of human patients with LGI1 mutations. Finally, we show that Lgi2 is expressed highly in the immediate post-natal period until halfway through pruning, unlike Lgi1, which is expressed in the latter part of pruning and beyond. LGI2 acts at least in part through the same ADAM receptors as LGI1, but earlier, ensuring electrical stability (absence of epilepsy) during pruning years, preceding this same function performed by LGI1 in later years. LGI2 should be considered a candidate gene for common remitting childhood epilepsies, and LGI2-to-LGI1 transition for mechanisms of childhood epilepsy remission.

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  53. Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases Reviewed

    Aurore D. Levy, Veronique Devignot, Yuko Fukata, Masaki Fukata, Andre Sobel, Stephanie Chauvin

    MOLECULAR BIOLOGY OF THE CELL   Vol. 22 ( 11 ) page: 1930 - 1942   2011.6

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    Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.

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  54. Protein palmitoylation in neuronal development and synaptic plasticity Invited Reviewed

    Yuko Fukata, Masaki Fukata

    NATURE REVIEWS NEUROSCIENCE   Vol. 11 ( 3 ) page: 161 - 175   2010.3

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    Protein palmitoylation, a classical and common lipid modification, regulates diverse aspects of neuronal protein trafficking and function. The reversible nature of palmitoylation provides a potential general mechanism for protein shuttling between intracellular compartments. The recent discovery of palmitoylating enzymes-a large DHHC (Asp-His-His-Cys) protein family-and the development of new proteomic and imaging methods have accelerated palmitoylation analysis. It is becoming clear that individual DHHC enzymes generate and maintain the specialized compartmentalization of substrates in polarized neurons. Here, we discuss the regulatory mechanisms for dynamic protein palmitoylation and the emerging roles of protein palmitoylation in various aspects of pathophysiology, including neuronal development and synaptic plasticity.

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  55. Disruption of LGI1-linked synaptic complex causes abnormal synaptic transmission and epilepsy Reviewed

    Yuko Fukata, Kathryn L. Lovero, Tsuyoshi Iwanaga, Atsushi Watanabe, Norihiko Yokoi, Katsuhiko Tabuchi, Ryuichi Shigemoto, Roger A. Nicoll, Masaki Fukata

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 107 ( 8 ) page: 3799 - 3804   2010.2

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    Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1(-/-)) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1(+/-)) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor-mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy.

    DOI: 10.1073/pnas.0914537107

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  56. Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity Reviewed

    Anat Shmueli, Michal Segal, Tamar Sapir, Ryouhei Tsutsumi, Jun Noritake, Avi Bar, Sivan Sapoznik, Yuko Fukata, Irit Orr, Masaki Fukata, Orly Reiner

    EMBO JOURNAL   Vol. 29 ( 1 ) page: 107 - 119   2010.1

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    Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1-Ndel1-Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein. The EMBO Journal (2010) 29, 107-119. doi: 10.1038/emboj.2009.325; Published online 19 November 2009

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  57. Differential Activity-Dependent Secretion of Brain-Derived Neurotrophic Factor from Axon and Dendrite Reviewed

    Naoto Matsuda, Hui Lu, Yuko Fukata, Jun Noritake, Hongfeng Gao, Sujay Mukherjee, Tomomi Nemoto, Masaki Fukata, Mu-ming Poo

    JOURNAL OF NEUROSCIENCE   Vol. 29 ( 45 ) page: 14185 - 14198   2009.11

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    Brain-derived neurotrophic factor (BDNF) is essential for neuronal survival and differentiation during development and for synaptic function and plasticity in the mature brain. BDNF-containing vesicles are widely distributed and bidirectionally transported in neurons, and secreted BDNF can act on both presynaptic and postsynaptic cells. Activity-dependent BDNF secretion from neuronal cultures has been reported, but it remains unknown where the primary site of BDNF secretion is and whether neuronal activity can trigger BDNF secretion from axons and dendrites with equal efficacy. Using BDNF fused with pH-sensitive green fluorescent protein to visualize BDNF secretion, we found that BDNF-containing vesicles exhibited markedly different properties of activity-dependent exocytic fusion at the axon and dendrite of cultured hippocampal neurons. Brief spiking activity triggered a transient fusion pore opening, followed by immediate retrieval of vesicles without dilation of the fusion pore, resulting in very little BDNF secretion at the axon. On the contrary, the same brief spiking activity induced "full-collapse" vesicle fusion and substantial BDNF secretion at the dendrite. However, full vesicular fusion with BDNF secretion could occur at the axon when the neuron was stimulated by prolonged high-frequency activity, a condition neurons may encounter during epileptic discharge. Thus, activity-dependent axonal secretion of BDNF is highly restricted as a result of incomplete fusion of BDNF-containing vesicles, and normal neural activity induces BDNF secretion from dendrites, consistent with the BDNF function as a retrograde factor. Our study also revealed a novel mechanism by which differential exocytosis of BDNF-containing vesicles may regulate BDNF-TrkB signaling between connected neurons.

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  58. Palmitoylation Regulates Epidermal Homeostasis and Hair Follicle Differentiation Reviewed

    Pleasantine Mill, Angela W. S. Lee, Yuko Fukata, Ryouhei Tsutsumi, Masaki Fukata, Margaret Keighren, Rebecca M. Porter, Lisa McKie, Ian Smyth, Ian J. Jackson

    PLOS GENETICS   Vol. 5 ( 11 ) page: e1000748   2009.11

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    Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.

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  59. Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95 Reviewed

    Jun Noritake, Yuko Fukata, Tsuyoshi Iwanaga, Naoki Hosomi, Ryouhei Tsutsumi, Naoto Matsuda, Hideki Tani, Hiroko Iwanari, Yasuhiro Mochizuki, Tatsuhiko Kodama, Yoshiharu Matsuura, David S. Bredt, Takao Hamakubo, Masaki Fukata

    JOURNAL OF CELL BIOLOGY   Vol. 186 ( 1 ) page: 147 - 160   2009.7

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    Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

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  60. Dynamic protein palmitoylation in cellular signaling Invited Reviewed

    Tsuyoshi Iwanaga, Ryouhei Tsutsumi, Jun Noritake, Yuko Fukata, Masaki Fukata

    PROGRESS IN LIPID RESEARCH   Vol. 48 ( 3-4 ) page: 117 - 127   2009.5

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    Protein S-palmitoylation, the most common lipid modification with the 16-carbon fatty acid palmitate, provides an important mechanism for regulating protein trafficking and function. The unique reversibility of protein palmitoylation allows proteins to rapidly shuttle between intracellular membrane compartments. Importantly, this palmitate cycling can be regulated by some physiological stimuli, contributing to cellular homeostaisis and plasticity. Although the enzyme responsible for protein palmitoylation had been long elusive, DHHC family proteins, conserved from plants to mammals, have recently emerged as palmitoyl acyl transferases. Integrated approaches including advanced proteomics, live-cell imaging, and molecular genetics are beginning to clarify the molecular machinery for palmitoylation reaction in diverse aspects of cellular functions. (C) 2009 Elsevier Ltd. All rights reserved.

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  61. The Hydrophobic Cysteine-rich Domain of SNAP25 Couples with Downstream Residues to Mediate Membrane Interactions and Recognition by DHHC Palmitoyl Transferases Reviewed

    Jennifer Greaves, Gerald R. Prescott, Yuko Fukata, Masaki Fukata, Christine Salaun, Luke H. Chamberlain

    MOLECULAR BIOLOGY OF THE CELL   Vol. 20 ( 6 ) page: 1845 - 1854   2009.3

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    SNAP25 is synthesized as a soluble protein but must associate with the plasma membrane to function in exocytosis; however, this membrane-targeting pathway is poorly defined. SNAP25 contains a palmitoylated cysteine-rich domain with four cysteines, and we show that coexpression of specific DHHC palmitoyl transferases is sufficient to promote SNAP25 membrane association in HEK293 cells. siRNA-mediated knockdown of its SNARE partner, syntaxin 1A, does not affect membrane interaction of SNAP25 in PC12 cells, whereas specific cysteine-to-alanine mutations perturb membrane binding, which is restored by leucine substitutions. These results suggest a role for cysteine hydrophobicity in initial membrane interactions of SNAP25, and indeed other hydrophobic residues in the cysteine-rich domain are also important for membrane binding. In addition to the cysteine-rich domain, proline-117 is also essential for SNAP25 membrane binding, and experiments in HEK293 cells revealed that mutation of this residue inhibits membrane binding induced by coexpression with DHHC17, but not DHHC3 or DHHC7. These results suggest a model whereby SNAP25 interacts autonomously with membranes via its hydrophobic cysteine-rich domain, requiring only sufficient expression of partner DHHC proteins for stable membrane binding. The role of proline-117 in SNAP25 palmitoylation is one of the first descriptions of elements within substrate proteins that modulate DHHC specificity.

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  62. Alzheimer Disease A beta Production in the Absence of S-Palmitoylation-dependent Targeting of BACE1 to Lipid Rafts Reviewed

    Kulandaivelu S. Vetrivel, Xavier Meckler, Ying Chen, Phuong D. Nguyen, Nabil G. Seidah, Robert Vassar, Philip C. Wong, Masaki Fukata, Maria Z. Kounnas, Gopal Thinakaran

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 284 ( 6 ) page: 3793 - 3803   2009.2

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    Alzheimer disease beta-amyloid (A beta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of A beta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of A beta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

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  63. Identification of G Protein alpha Subunit-Palmitoylating Enzyme Reviewed

    Ryouhei Tsutsumi, Yuko Fukata, Jun Noritake, Tsuyoshi Iwanaga, Franck Perez, Masaki Fukata

    MOLECULAR AND CELLULAR BIOLOGY   Vol. 29 ( 2 ) page: 435 - 447   2009.1

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    The heterotrimeric G protein alpha subunit (G alpha) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as G alpha palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated G alpha(q), G alpha(s), and G alpha(i2) in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased G alpha(q/11) palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that G alpha(q) rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous G alpha(q) shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the alpha(1A)-adrenergic receptor/G alpha(q/11)-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling G alpha localization to the plasma membrane.

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  64. Discovery of protein-palmitoylating enzymes Invited Reviewed

    Ryouhei Tsutsumi, Yuko Fukata, Masaki Fukata

    PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY   Vol. 456 ( 6 ) page: 1199 - 1206   2008.9

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    Posttranslational modification provides proteins with additional function and regulatory control beyond genomic information, allowing cells to maintain homeostasis and respond to extracellular signals. Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Palmitoylation is unique in that it is reversible and dynamically regulated by specific extracellular signals. The reversible nature of protein palmitoylation enables proteins to shuttle between intracellular compartments upon extracellular signals. However, the molecular mechanisms of protein palmitoylation have long been elusive, mostly because the enzymes responsible for protein palmitoylation were unknown. Recently, genetically conserved DHHC family proteins have emerged as palmitoyl-acyl transferases. With the identification of specific enzymes for palmitoylated proteins, including H-Ras, PSD-95, and eNOS, the specificity and regulatory mechanism of DHHC enzymes are beginning to be clarified.

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  65. Fibroblast growth factor-regulated palmitoylation of the neural cell adhesion molecule determines neuronal morphogenesis Reviewed

    Evgeni Ponimaskin, Galina Dityateva, Mika O. Ruonala, Masaki Fukata, Yuko Fukata, Fritz Kobe, Fred S. Wouters, Markus Delling, David S. Bredt, Melitta Schachner, Alexander Dityatev

    JOURNAL OF NEUROSCIENCE   Vol. 28 ( 36 ) page: 8897 - 8907   2008.9

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    During development of the nervous system, short- and long-range signals cooperate to promote axonal growth, guidance, and target innervation. Particularly, a short- range signal transducer, the neural cell adhesion molecule ( NCAM), stimulates neurite outgrowth via mechanisms that require posttranslational modification of NCAM and signaling via receptors to a long-range messenger, the fibroblast growth factor ( FGF). In the present study we further characterized a mechanism which regulates the functional interplay between NCAM and FGF receptor(s). We show that activation of FGF receptor( s) by FGF2 leads to palmitoylation of the two major transmembrane NCAM isoforms, NCAM140 and NCAM180, translocation of NCAM to GM1 ganglioside-containing lipid rafts, and stimulation of neurite outgrowth of hippocampal neurons. Ablation of NCAM, mutation of NCAM140 or NCAM180 palmitoylation sites, or pharmacological suppression of NCAM signaling inhibited FGF2-stimulated neurite outgrowth. Of the 23 members of the aspartate-histidine-histidine-cysteine (DHHC) domain containing proteins, DHHC-7 most strongly stimulated palmitoylation of NCAM, and enzyme activity was enhanced by FGF2. Thus, our study uncovers a molecular mechanism by which a growth factor regulates neuronal morphogenesis via activation of palmitoylation, which in turn modifies subcellular location and thus signaling via an adhesion molecule.

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  66. Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein Reviewed

    Jennifer Greaves, Christine Salaun, Yuko Fukata, Masaki Fukata, Luke H. Chamberlain

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 283 ( 36 ) page: 25014 - 25026   2008.9

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    Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum ( ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.

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  67. Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice Reviewed

    Olav Olsen, Lars Funke, Jia-fu Long, Masaki Fukata, Toshinari Kazuta, Jonathan C. Trinidad, Kimberly A. Moore, Hidemi Misawa, Paul A. Welling, Alma L. Burlingame, Mingjie Zhang, David S. Bredt

    JOURNAL OF CELL BIOLOGY   Vol. 179 ( 1 ) page: 151 - 164   2007.10

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    Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.

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  68. Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition Reviewed

    Masaki Mishima, Ryoko Maesaki, Miyuki Kasa, Takashi Watanabe, Masaki Fukata, Kozo Kaibuchi, Toshio Hakoshima

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 104 ( 25 ) page: 10346 - 10351   2007.6

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    Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

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  69. GM1-ganglioside-induced A beta assembly on synaptic membranes of cultured neurons Reviewed

    Naoki Yamamoto, Yuko Fukata, Masaki Fukata, Katsuhiko Yanagisawa

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES   Vol. 1768 ( 5 ) page: 1128 - 1137   2007.5

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    The cell-surface expression of GM1 ganglioside was studied using various cultured cells, including brain-derived endothelial cells, astrocytes, neuroblastoma cells (SH-SY5Y), and pheochromocytoma cells (PC12). GM1 ganglioside was detected only on the surface of native and nerve-growth-factor (NGF)-treated PC12 cells. We investigated whether GM1 ganglioside on the surface of these cells is sufficiently potent to induce the assembly of an exogenous soluble amyloid beta-protein (A beta). A marked A beta assembly was observed in the culture of NGF-treated PC12 cells. Notably, immunocytochemical study revealed that, despite the ubiquitous surface expression of GM1 ganglioside throughout cell bodies and neurites, A assembly initially occurred at the terminals of SNAP25-immunopositive neurites. A beta assembly in the culture was completely suppressed by the coincubation of A beta with the subunit B of cholera toxin, a natural ligand for GM1 ganglioside, or 4396C, a monoclonal antibody specific to GMI-ganglioside-bound A beta (GA beta). In primary neuronal cultures, A beta assembly initially occurred at synaptophysin-positive sites. These results suggest that the cell-surface expression of GM1 ganglioside is strictly cell-type-specific, and that expression of GM1 ganglioside on synaptic membranes is unique in terms of its high potency to induce A beta assembly through the generation of GA beta, which is an endogenous seed for A assembly in Alzheimer brain. (c) 2007 Elsevier B.V. All rights reserved.

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  70. IQGAP3, a novel effector of Rac1 and Cdc42, regulates neurite outgrowth Reviewed

    Shujie Wang, Takashi Watanabe, Jun Noritake, Masaki Fukata, Takeshi Yoshimura, Norimichi Itoh, Takumi Harada, Masato Nakagawa, Yoshiharu Matsuura, Nariko Arimura, Kozo Kaibuchi

    JOURNAL OF CELL SCIENCE   Vol. 120 ( 4 ) page: 567 - 577   2007.2

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    Rac1 and Cdc42, members of the Rho family GTPases, control diverse cellular processes such as cell migration and morphogenesis through their effectors. Among the effectors, IQGAP1 plays pivotal roles in the establishment of cytoskeletal architecture and intercellular adhesions in various cells. However, its roles remain to be clarified, especially in neuronal cells. We have identified IQGAP3 as a novel member of the IQGAP family, which is highly expressed in brain. We found that IQGAP3, an effector of Rac1 and Cdc42, associates directly with actin filaments and accumulates asymmetrically at the distal region of axons in hippocampal neurons. The depletion of IQGAP3 impairs neurite or axon outgrowth in neuronal cells with the disorganized cytoskeleton, but depletion of IQGAP1 does not. Furthermore, IQGAP3 is indispensable for Rac1/Cdc42-promoted neurite outgrowth in PC12 cells. Taken together, these results indicate that IQGAP3 can link the activation of Rac1 and Cdc42 with the cytoskeletal architectures during neuronal morphogenesis.

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  71. GODZ-mediated palmitoylation of GABA(A) receptors is required for normal assembly and function of GABAergic inhibitory synapses Reviewed

    Cheng Fang, Lunbin Deng, Cheryl A. Keller, Masaki Fukata, Yuko Fukata, Gong Chen, Bernhard Luscher

    JOURNAL OF NEUROSCIENCE   Vol. 26 ( 49 ) page: 12758 - 12768   2006.12

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    Golgi-specific DHHC (Asp-His-His-Cys) zinc finger protein (GODZ) is a DHHC family palmitoyl acyltransferase that is implicated in palmitoylation and regulated trafficking of diverse substrates that function either at inhibitory or excitatory synapses. Of particular interest is the gamma 2 subunit of GABAA receptors, which is required for targeting these receptors to inhibitory synapses. Here, we report that GODZ and, to a lesser extent, its close paralog sertoli cell gene with a zinc finger domain-beta (SERZ-beta) are the main members of the DHHC family of enzymes that are able to palmitoylate the gamma 2 subunit in heterologous cells. Yeast two-hybrid and colocalization assays in human embryonic kidney 293T (HEK293T) cells indicate that GODZ and SERZ-beta show indistinguishable palmitoylation-dependent interaction with the gamma 2 subunit. After coexpression in HEK293T cells, they form homomultimers and heteromultimers, as shown by coimmunoprecipitation and in vivo cross-linking experiments. Analyses in neurons transfected with dominant-negative GODZ (GODZ(C157S)) or plasmid-based GODZ-specific RNAi indicate that GODZ is required for normal accumulation of GABA(A) receptors at synapses, for normal whole-cell and synaptic GABAergic inhibitory function and, indirectly, for GABAergic innervation. Unexpectedly, GODZ was found to be dispensable for normal postsynaptic AMPA receptor-mediated glutamatergic transmission. We conclude that GODZ-mediated palmitoylation of GABAA receptors and possibly other substrates contributes selectively to the formation and normal function of GABAergic inhibitory synapses.

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  72. Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells Invited Reviewed

    Yuko Fukata, Tsuyoshi Iwanaga, Masaki Fukata

    METHODS   Vol. 40 ( 2 ) page: 177 - 182   2006.10

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    Posttranslational modifications, including phosphorylation, ubiquitination and lipid modifications, provide proteins with additional functions and regulation beyond genomic information. Palmitoylation is a reversible lipid modification with palmitic acid that plays critical roles in protein trafficking and function. However, the enzymes that mediate palmitoyl acyl transferase (PAT) have been elusive. Recent genetic analysis in yeast revealed that members of cysteine-rich DHHC domain containing proteins (DHHC proteins) mediate palmitoylation. In mammalian genomes, 23 DHHC proteins are predicted raising the possibility of a large family of PAT enzymes. Here, we describe a systematic method to examine which of the DHHC family members is responsible for palmitoylation of a substrate. (c) 2006 Elsevier Inc. All rights reserved.

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  73. Epilepsy-related ligand/receptor complex LGI1 and ADAM22 regulate synaptic transmission Reviewed

    Yuko Fukata, Hillel Adesnik, Tsuyoshi Iwanaga, David S. Bredt, Roger A. Nicoll, Masaki Fukata

    SCIENCE   Vol. 313 ( 5794 ) page: 1792 - 1795   2006.9

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    Abnormally synchronized synaptic transmission in the brain causes epilepsy. Most inherited forms of epilepsy result from mutations in ion channels. However, one form of epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF), is characterized by mutations in a secreted neuronal protein, LGI1. We show that ADAM22, a transmembrane protein that when mutated itself causes seizure, serves as a receptor for LGI1. LGI1 enhances AMPA receptor-mediated synaptic transmission in hippocampal slices. The mutated form of LGI1 fails to bind to ADAM22. ADAM22 is anchored to the postsynaptic density by cytoskeletal scaffolds containing stargazin. These studies in rat brain indicate possible avenues for understanding human epilepsy.

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  74. Identication of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase Reviewed

    Carlos Fernandez-Hernando, Masaki Fukata, Pascal N. Bernatchez, Yuko Fukata, Michelle I. Lin, David S. Bredt, William C. Sessa

    JOURNAL OF CELL BIOLOGY   Vol. 174 ( 3 ) page: 369 - 377   2006.7

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    Lipid modi. cations mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation deficient mutants of eNOS release less nitric oxide ( NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [H-3]palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

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  75. Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect Reviewed

    Matthias Hundt, Hiroki Tabata, Myung-Shin Jeon, Keitaro Hayashi, Yoshihiko Tanaka, Roma Krishna, Lauren De Giorgio, Yun-Cai Liu, Masaki Fukata, Amnon Altman

    IMMUNITY   Vol. 24 ( 5 ) page: 513 - 522   2006.5

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    The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4(+) T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLC gamma 1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3 zeta and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy.

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  76. Protein palmitoylation by DHHC protein family Invited Reviewed

    Fukata Y, Bredt DS, Fukata M

    The dynamic synapse: molecular methods in ionotropic receptor biology     page: 81 - 87   2006

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  77. Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex Reviewed

    O Olsen, KA Moore, M Fukata, T Kazuta, JC Trinidad, FW Kauer, M Streuli, H Misawa, AL Burlingame, RA Nicoll, DS Bredt

    JOURNAL OF CELL BIOLOGY   Vol. 170 ( 7 ) page: 1127 - 1134   2005.9

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    Synapses are highly specialized intercellular junctions organized by adhesive and scaffolding molecules that align presynaptic vesicular release with postsynaptic neurotransmitter receptors. The MALS/Veli-CASK-Mint-1 complex of PDZ proteins occurs on both sides of the synapse and has the potential to link transsynaptic adhesion molecules to the cytoskeleton. In this study, we purified the MALS protein complex from brain and found liprin-alpha as a major component. Liprin proteins organize the presynaptic active zone and regulate neurotransmitter release. Fittingly, mutant mice lacking all three MALS isoforms died perinatally with difficulty breathing and impaired excitatory synaptic transmission. Excitatory postsynaptic currents were dramatically reduced in autaptic cultures from MALS triple knockout mice due to a presynaptic deficit in vesicle cycling. These findings are consistent with a model whereby the MALS-CASK-liprin-alpha complex recruits components of the synaptic release machinery to adhesive proteins of the active zone.

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  78. Molecular constituents of neuronal AMPA receptors Reviewed

    Y Fukata, AV Tzingounis, JC Trinidad, M Fukata, AL Burlingame, RA Nicoll, DS Bredt

    JOURNAL OF CELL BIOLOGY   Vol. 169 ( 3 ) page: 399 - 404   2005.5

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    Dynamic regulation of &alpha;-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immuno-precipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins ( TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope- tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.

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  79. Identification of PSD-95 palmitoylating enzymes Reviewed

    M Fukata, Y Fukata, H Adesnik, RA Nicoll, DS Bredt

    NEURON   Vol. 44 ( 6 ) page: 987 - 996   2004.12

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    Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.

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  80. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration Reviewed

    T Watanabe, SJ Wang, J Noritake, K Sato, M Fukata, M Takefuji, M Nakagawa, N Izumi, T Akiyama, K Kaibuchi

    DEVELOPMENTAL CELL   Vol. 7 ( 6 ) page: 871 - 883   2004.12

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    Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.

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  81. Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses Reviewed

    S Tomita, M Fukata, RA Nicoll, DS Bredt

    SCIENCE   Vol. 303 ( 5663 ) page: 1508 - 1511   2004.3

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    Activity-dependent plasticity in the brain arises in part from changes in the number of synaptic AMPA receptors. Synaptic trafficking of AMPA receptors is controlled by stargazin and homologous transmembrane AMPA receptor regulatory proteins (TARPs). We found that TARPs were stable at the plasma membrane, whereas AMPA receptors were internalized in a glutamate-regulated manner. Interaction with AMPA receptors involved both extra- and intracellular determinants of TARPs. Upon binding to glutamate, AMPA receptors detached from TARPs. This did not require ion flux or intracellular second messengers. This allosteric mechanism for AMPA receptor dissociation from TARPs may participate in glutamate-mediated internalization of receptors in synaptic plasticity.

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  82. Positive role of IQGAP1, an effector of Rac1, in actin-meshwork formation at sites of cell-cell contact Reviewed

    J Noritake, M Fukata, K Sato, M Nakagawa, T Watanabe, N Izumi, SJ Wang, Y Fukata, K Kaibuchi

    MOLECULAR BIOLOGY OF THE CELL   Vol. 15 ( 3 ) page: 1065 - 1076   2004.3

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    The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and beta-catenin at sites of cell-cell contact in Madin-Darby canine kidney 11 epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and beta-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of "E-cadherin-mediated cell-cell adhesion--&gt;Rac1 activation--&gt;actin-meshwork formation by IQGAP1--&gt;increasing E-cadherin-mediated cell-cell adhesion."

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  83. Roles of Rho-family GTPases in cell polarisation and directional migration Invited Reviewed

    Masaki Fukata, Masato Nakagawa, Kozo Kaibuchi

    Current Opinion in Cell Biology   Vol. 15 ( 5 ) page: 590 - 597   2003

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    Polarised cell migration is a tightly regulated process that occurs in tissue development, chemotaxis and wound healing. Rho-family GTPases, including Cdc42, Rac1 and RhoA, play a central role in establishing cell polarisation, which requires asymmetric and ordered distribution of the signalling molecules and the cytoskeleton. Recent advances reveal that Rho GTPases, together with phosphatidylinositol 3-kinase, contribute to asymmetric phosphatidylinositol 3,4,5-trisphosphate distribution via a positive-feedback loop. Phosphatidylinositol 3,4,5-trisphosphate thereby activates the signalling cascades to the cytoskeleton as a second messenger. Rho GTPases also capture and stabilise microtubules through their effectors (e.g. IQGAP1, mDia and Par6) near the cell cortex, leading to polarised cell morphology and directional cell migration. Thus, elucidation of the signal transduction cascades from receptors to Rho GTPases and, subsequently, from Rho GTPases to microtubules has begun.

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  84. Rac1 and Cdc42 capture microtubules through IQGAP1 and CLIP-170 Reviewed

    M Fukata, T Watanabe, J Noritake, M Nakagawa, M Yamaga, S Kuroda, Y Matsuura, A Iwamatsu, F Perez, K Kaibuchi

    CELL   Vol. 109 ( 7 ) page: 873 - 885   2002.6

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    Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of 1Q-GAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.

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  85. Effects of Rho family GTPases on cell-cell adhesion. Invited Reviewed International journal

    Fukata M, Nakagawa M, Kuroda S, Kaibuchi K

    Methods Mol Biol   Vol. 189   page: 121 - 128   2002

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    DOI: 10.1385/1-59259-281-3:121

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  86. Rho-family GTPases in cadherin-mediated cell-cell adhesion Invited Reviewed

    Masaki Fukata, Kozo Kaibuchi

    Nature Reviews Molecular Cell Biology   Vol. 2 ( 12 ) page: 887 - 897   2001.12

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    Cell-cell adhesions are rearranged dynamically during tissue development and tumour metastasis. Recently, Rho-family GTPases, including RhoA, Rac1 and Cdc42, have emerged as key regulators of cadherin-mediated cell-cell adhesion. Following the identification and characterization of regulators and effectors of Rho GTPases, signal transduction pathways from cadherin to Rho GTPases and, in turn, from Rho GTPases to cadherin, are beginning to be clarified.

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  87. Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites Reviewed

    M Nakagawa, M Fukata, M Yamaga, N Itoh, K Kaibuchi

    JOURNAL OF CELL SCIENCE   Vol. 114 ( 10 ) page: 1829 - 1838   2001.5

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    Rac1, a member of the Rho family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of Rad are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged Rad, we demonstrate that EGFP-Rac1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca2+ chelation, Re-establishment of cell-cell adhesion by restoration of Ca2+ caused EGFP-Rac1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-Rac1, We also investigated whether E-cadherin-mediated cell-cell adhesion induced Rad activation by measuring the amounts of GTP-bound Rad based on its specific binding to the Cdc42/Rac1 interactive binding region of p21-activated kinase, The formation of E-cadherin-mediated cell-cell adhesion induced Rad activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), IQGAP1, an effector of Rad, and EGFP-Rac1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. Rad activation was also confirmed by measuring the amounts of coimmunoprecipitated Rad with IQGAP1 during the establishment of cell-cell adhesion, Taken together, these results suggest that Rad is recruited at sites of E-cadherin-mediated cell-cell adhesion acid then activated, possibly through PI 3-kinase.

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  88. Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering Reviewed

    M Fukata, M Nakagawa, N Itoh, A Kawajiri, M Yamaga, S Kuroda, K Kaibuchi

    MOLECULAR AND CELLULAR BIOLOGY   Vol. 21 ( 6 ) page: 2165 - 2183   2001.3

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    We have previously proposed that IQGAP1, an effector of Rad and Cdc42, negatively regulates cadherin-mediated cell-cell adhesion by interacting with beta -catenin and by causing the dissociation of alpha -catenin from cadherin-beta -catenin-alpha -catenin complexes and that activated Rad and Cdc42 positively regulate cadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with beta -catenin. However, it remains to be clarified in which physiological processes the Rac1-Cdc42-IQGAP1 system is involved. We here examined whether the Rac1-IQGAP1 system is involved in the cell-cell dissociation of Madin-Darby canine kidney II cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)- or hepatocyte growth factor (HGF)-induced cell scattering. By using enhanced green fluorescent protein (EGFP)-tagged alpha -catenin, we found that EGFP-alpha -catenin decreased prior to cell-cell dissociation during cell scattering. We also found that the Rac1-GTP level decreased after stimulation with TPA and that the Rac1-IQGAP1 complexes decreased, while the IQGAP1-beta -catenin complexes increased during action of TPA. Constitutively active Rad and IQGAP1 carboxyl terminus, a putative dominant-negative mutant of IQGAP1, inhibited the disappearance of alpha -catenin from sites of cell-cell contact induced by TPA. Taken together, these results indicate that alpha -catenin is delocalized from cell-cell contact sites prior to cell-cell dissociation induced by TPA or HGF and suggest that the Rac1-IQGAP1 system is involved in cell-cell dissociation through alpha -catenin relocalization.

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  89. Synaptic development is controlled in the periactive zones of Drosophila synapses Reviewed

    M Sone, E Suzuki, M Hoshino, DM Hou, H Kuromi, M Fukata, S Kuroda, K Kaibuchi, Y Nabeshima, C Hama

    DEVELOPMENT   Vol. 127 ( 19 ) page: 4157 - 4168   2000.10

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    A cell-adhesion molecule fasciclin 2 (FAS2), which is required for synaptic growth and still life (SIF), an activator of RAC, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. beta PS integrin and discs large (DLG), both involved in synaptic development, also decorated the zone. However, shibire (SHI), the Drosophila dynamin that regulates endocytosis, was found in the distinct region. Mutant analyses showed that sif genetically interacted with Fas2 in synaptic growth and that the proper localization of SIF required FAS2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. We propose that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively.

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  90. Identification of a novel beta-catenin-interacting protein Reviewed

    A Kawajiri, N Itoh, M Fukata, M Nakagawa, M Yamaga, A Iwamatsu, K Kaibuchi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 273 ( 2 ) page: 712 - 717   2000.7

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    Cadherin is a well-known cell-cell adhesion molecule, and it binds to beta-catenin, which in turn binds to alpha-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell-cell adhesion. Here we purified two novel beta-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)-beta-catenin affinity column chromatography. Mass spectral analysis revealed p180 to be identical to KIAA0313 which has a putative Rap1 guanine nucleotide exchange factor (GEF) domain and p150 to be the same as KIAA0705 which has a high degree of sequence similarity to the synaptic scaffolding molecule (S-SCAM), which binds beta-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively (Ide et al., Biochem. Biophys. Res. Commun. 256, 456-461, 1999; Ohtsuka et al., Biochem. Biophys. Res. Commun. 265, 38-44, 1999). beta-Catenin was coimmunoprecipitated with KIAA0313 in Madin-Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and beta-catenin were partly colocalized at sites of cell-cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with beta-catenin through KIAA0705 in vivo at sites of cell-cell contact. (C) 2000 Academic Press.

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  91. Phosphorylation of ERM proteins at filopodia induced by Cdc42 Reviewed

    N Nakamura, N Oshiro, Y Fukata, M Amano, M Fukata, S Kuroda, Y Matsuura, T Leung, L Lim, K Kaibuchi

    GENES TO CELLS   Vol. 5 ( 7 ) page: 571 - 581   2000.7

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    Background: ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures. We have shown that Rho-associated kinase (Rho-kinase)/ROK alpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures. J. Biol. Chem. 273, 34663- 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.
    Results: Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42(V12)), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42(V12).
    Conclusion: The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.

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  92. Cell adhesion and Rho small GTPases Invited Reviewed

    M Fukata, M Nakagawa, S Kuroda, K Kaibuchi

    JOURNAL OF CELL SCIENCE   Vol. 112 ( 24 ) page: 4491 - 4500   1999.12

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    The Rho small GTPases, Cdc42, Ra1 and Rho, are implicated in regulation of integrin-mediated cell-substratum adhesion and cadherin-mediated cell-cell adhesion. Identification and characterization of effectors of these GTPases have provided insights into their modes of action. Rho-kinase, an effector of Rho, regulates integrin-mediated cell-substratum adhesion (focal adhesion) by regulating the phosphorylation state of myosin light chain (MLC): it directly phosphorylates MLC and also inactivates myosin phosphatase, IQGAP1, an effector of Cdc42 and Ra1, regulates cadherin-mediated cell-cell adhesion by interacting with beta-catenin and dissociating alpha-catenin from the cadherin-catenins complex. Activated Cdc4.2 and Rad inhibit IQGAP1, thereby stabilizing the cadherin-catenins complex. Cdc42/Rac1 and IQGAP1 thus appear to constitute a switch that regulates cadherin-mediated cell-cell adhesion.

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  93. Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases Invited Reviewed

    K Kaibuchi, S Kuroda, M Fukata, M Nakagawa

    CURRENT OPINION IN CELL BIOLOGY   Vol. 11 ( 5 ) page: 591 - 596   1999.10

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    Reports in the past two years have shown that Cdc42, Rad, and Rho - belonging to the Rho small GTPase family - participate in the regulation of cadherin-mediated cell-cell adhesion. IQGAP1, an effector of Cdc42 and Rad, interacts with cadherin and beta-catenin and induces the dissociation of alpha-catenin from the cadherin-catenins complex leading to disruption of cell-cell adhesion: activated Cdc42 and Rad counteract the effect of IQGAP1. Thus, Cdc42 and Rad appear to regulate cadherin-mediated cell-cell adhesion acting through IQGAP1.

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  94. Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin Reviewed

    M Fukata, S Kuroda, M Nakagawa, A Kawajiri, N Itoh, Shoji, I, Y Matsuura, S Yonehara, H Fujisawa, A Kikuchi, K Kaibuchi

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 274 ( 37 ) page: 26044 - 26050   1999.9

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    IQGAP1, a target of Cdc42 and Bad small GTPases, directly interacts with beta-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating alpha-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, R., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I,, Matsuura, Y., Yonehara, S,, and Kaibuchi, IT. (1998) Science 281, 832-855). Here we investigated how Cdc42 and Rad regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of beta-catenin, which contains the alpha-catenin-binding domain. IQGAP1 dissociated alpha-catenin from the beta-catenin-alpha-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) glutathione S-transferase (GST)Cdc42 and GTP gamma S.GST-Rac1 inhibited the binding of IQGAP1 to beta-catenin in a dose-dependent manner vitro, whereas neither GDP.GST-Cdc42, GDP.GST-Rac1, in nor GTP gamma S GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of alpha-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rad resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rad negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with beta-catenin, leading to stabilization of the cadherin-catenin complex.

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  95. Cdc42, Rac1, and their effector IQGAP1 as molecular switches for cadherin-mediated cell-cell adhesion Invited Reviewed

    S Kuroda, M Fukata, M Nakagawa, K Kaibuchi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 262 ( 1 ) page: 1 - 6   1999.8

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    Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca2+-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rad, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rad, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rad as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved. (C) 1999 Academic Press.

    DOI: 10.1006/bbrc.1999.1122

    Web of Science

  96. 低分子量GTP結合タンパク質の機能 低分子量G蛋白質Cdc42/Rac1と標的蛋白質IQGAP1による細胞間接着の制御機構の解析

    深田 正紀, 黒田 真也, 中川 誠人, 貝淵 弘三

    生化学   Vol. 71 ( 8 ) page: 690 - 690   1999.8

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  97. Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1 Reviewed

    M Hoshinoso, M Sone, M Fukata, S Kurodad, K Kaibuchi, Y Nabeshima, C Hama

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 274 ( 25 ) page: 17837 - 17844   1999.6

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    The Rho family GTPases are involved in a variety of cellular events by changing the organization of actin cytoskeletal networks in response to extracellular signals. However, it is not clearly known how their activities are spatially and temporally regulated. Here we report the identification of a novel guanine nucleotide exchange factor for Rad, STEF, which is related in overall amino acid sequence and modular structure to mouse Tiam1 and Drosophila SIF proteins. STEF protein contains two pleckstrin homology domains, a PDZ domain and a Dbl homology domain. The in vitro assay showed that STEF protein specifically enhanced the dissociation of GDP from Rac1 but not that from either RhoA or Cdc42. Expression of a truncated STEF protein in culture cells induced membrane ruffling with altered actin localization, which implies that this protein also activates Rad in vivo. The stef transcript was observed in restricted parts of mice, including cartilaginous tissues and the cortical plate of the central nervous system during embryogenesis. These findings suggested that STEF protein participates in the control of cellular events in several developing tissues, possibly changing the actin cytoskeletal network by activating Rad.

    DOI: 10.1074/jbc.274.25.17837

    Web of Science

  98. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin-mediated cell-cell adhesion Reviewed

    S Kuroda, M Fukata, M Nakagawa, K Fujii, T Nakamura, T Ookubo, Izawa, I, T Nagase, N Nomura, H Tani, Shoji, I, Y Matsuura, S Yonehara, K Kaibuchi

    SCIENCE   Vol. 281 ( 5378 ) page: 832 - 835   1998.8

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    The small guanosine triphosphatases (GTPases) Cdc42 and Rad regulate E-cadherin-mediated cell-cell adhesion. IQGAP1, a target: of Cdc42 and Rac1,was localized with E-cadherin and beta-catenin at sites of cell-cell contact in mouse L Fibroblasts expressing E-cadherin (EL cells), and interacted with E-cadherin and beta-catenin both in vivo and in vitro. IQGAP1 induced the dissociation of alpha-catenin from a cadherin-catenin complex in vitro and in vivo. Overexpression of IQGAP1 in EL cells, but not in L cells expressing an E-cadherin-alpha-catenin chimeric protein, resulted in a decrease in E-cadherin-mediated cell-cell adhesive activity. Thus, IQGAP1, acting downstream of Cdc42 and Rac1, appears to regulate cell-cell adhesion through the cadherin-catenin pathway.

    DOI: 10.1126/science.281.5378.832

    Web of Science

  99. p140Sra-1 (specifically Rac1-associated protein) is a novel specific target for Rac1 small GTPase Reviewed

    K Kobayashi, S Kuroda, M Fukata, T Nakamura, T Nagase, N Nomura, Y Matsuura, N Yoshida-Kubomura, A Iwamatsu, K Kaibuchi

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 273 ( 1 ) page: 291 - 295   1998.1

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    Rac1 small GTPase plays pivotal roles in various cell functions such as cell morphology, cell polarity, and cell proliferation, We have previously identified IQGAP1 from bovine brain cytosol as a target for Rad by an affinity purification method, By using the same method, we purified a specifically Rac1-associated protein with a molecular mass of about 140 kDa (p140) from bovine brain cytosol, This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S).glutathione S-transferase (GST)-Rac1 but not with the GDP.GST-Rac1, GTP gamma S.GST-Cdc42, or GTP gamma S.GST-RhoA. The amino acid sequences of this protein revealed that p140 is identified as a product of KIAA0068 gene, We denoted this protein as Sra-1 (Specifically Rad-associated protein), Recombinant Sra-1 interacted with GTP gamma S.GST-Rac1 and weakly with GDP.Rac1 but not with GST-Cdc42 or GST-RhoA, The N-terminal domain of Sra-1 (1-407 amino acids) was responsible for the interaction with Rad. Myc-tagged Sra-1 and the deletion mutant capable of interacting with Rad, but not the mutants unable to bind Rad, were colocalized with dominant active Rac1(Val-12) and cortical actin filament at the Rac1(Val-12)-induced membrane ruffling area in KB cells, Sra-1 was cosedimented with filamentous actin (F-actin), indicating that Sra-1 directly interacts with F-actin, These results suggest that Sra-1 is a novel and specific target for Rac1.

    DOI: 10.1074/jbc.273.1.291

    Web of Science

  100. Regulation of cell-cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases Reviewed

    S Kuroda, M Fukata, K Fujii, T Nakamura, Izawa, I, K Kaibuchi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   Vol. 240 ( 2 ) page: 430 - 435   1997.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

    Rad, a member of the Rho small GTPases family, has recently been shown to be involved in the regulation of cell-cell adhesion mediated by cadherin. Here we showed that Cdc42, another member of Rho family, accumulated at cell-cell contact sites. Microinjection of Rho GDI, a negative regulator of the Rho family members, into Madin-Darby canine kidney (MDCK) cells resulted in perturbation of epithelial cell. morphology and of cell-cell and cell-substratum adhesions, and comicroinjection of dominant active Cdc42 or Rad reversed the action of Rho GDI, suggesting that the active form of Cdc42 or Rad is required for maintaining the cell-cell and cell-substratum adhesions. These observations suggest that Cdc42, in addition to Rad, can regulate the cell-cell adhesion. (C) 1997 Academic Press.

    DOI: 10.1006/bbrc.1997.7675

    Web of Science

  101. Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42 Reviewed

    M Fukata, S Kuroda, K Fujii, T Nakamura, Shoji, I, Y Matsuura, K Okawa, A Iwamatsu, A Kikuchi, K Kaibuchi

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 272 ( 47 ) page: 29579 - 29583   1997.11

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    We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rad small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M,, Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 28363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) GST-Cdc42 but not by GDP-GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTP gamma S-GST-Cdc42 but not GDP GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.

    DOI: 10.1074/jbc.272.47.29579

    Web of Science

  102. Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1 Reviewed

    S Kuroda, M Fukata, K Kobayashi, M Nakafuku, N Nomura, A Iwamatsu, K Kaibuchi

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 271 ( 38 ) page: 23363 - 23367   1996.9

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    Cdc42 and Rac1 have been implicated in the regulation of various cell functions such as cell morphology, polarity, and cell proliferation. We have partially purified a Cdc42- and Rac1-associated protein with molecular mass of about 170 kDa (p170) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-glutathione S-transferase (GST)-Cdc42 and GTP gamma-GST-Rac1, or GTP gamma S-GST-RhoA). We identified p170 as an IQGAP, which is originally identified as putative Ras GTPase-activating protein. Recombinant IQGAP specifically interacted with GTP gamma S-Cdc42 and GTP gamma S-Rac1. The C-terminal fragment of IQGAP was specifically immunoprecipitated with dominant-active Cdc42(Val12) or Rac1(Val12) from the COS7 cells expressing Cdc42(Val12) or Rac1(Val12), respectively, Immunofluorescence analysis revealed that IQGAP was accumulated at insulin- or Rac1-induced membrane ruffling areas. This accumulation of IQGAP was blocked by the microinjection of the dominant-negative Rac1(Asn17) or Cdc42(Asn17). Moreover, IQGAP was accumulated at the cell-cell junction in MDCK cells, where alpha-catenin and ZO-1 were localized. These results suggest that IQGAP is a novel target molecule for Cdc42 and Rac1.

    DOI: 10.1074/jbc.271.38.23363

    Web of Science

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MISC 20

  1. てんかん発症を抑制するためのLGI1–ADAM22タンパク質複合体の量的制御機構 Invited Reviewed

    横井紀彦, 深田優子, 深田正紀

    生化学   Vol. 95 ( 3 ) page: 384 - 388   2023.6

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    Authorship:Last author   Language:Japanese  

    DOI: 10.14952/SEIKAGAKU.2023.950384

  2. Acyl-PEGyl exchange gel-shift (APEGS) assay for palmitoylation quantification Invited Reviewed

    Masaki Fukata, Norihiko Yokoi, Yuko Fukata

    Electrophoresis Letters   Vol. 65 ( 2 ) page: 41 - 45   2021

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    Authorship:Lead author, Last author, Corresponding author   Publisher:Japanese Electrophoresis Society  

    DOI: 10.2198/electroph.65.41

  3. zDHHC パルミトイル化酵素と ABHD17 脱パルミトイル化酵素 Invited Reviewed

    深田正紀, 横井紀彦, 平田哲也, 深田優子

    膜タンパク質工学ハンドブック     page: 316 - 323   2020.4

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  4. てんかん関連リガンド・受容体 LGI1・ADAM22 Invited Reviewed

    平野瑶子, 宮﨑裕理, 深田正紀, 深田優子

    膜タンパク質工学ハンドブック     page: 357 - 365   2020.4

  5. パルミトイル化修飾酵素を軸とした神経機能研究 Invited Reviewed

    平田哲也, 深田優子, 深田正紀

    生化学   Vol. 90 ( 2 ) page: 125 - 137   2018

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  6. 記憶・学習に関わる メンブレントラフィック Invited Reviewed

    深田優子, 横井紀彦, 宮﨑裕理, 深田正紀

    メンブレントラフィック, 化学同仁   ( 19 ) page: 157 - 175   2016

  7. ケミカルシャペロンを用いたタンパク質構造異常の修復はてんかんモデルマウスの上昇した痙攣感受性を軽減する Invited Reviewed

    横井紀彦, 深田優子, 深田正紀

    細胞工学   Vol. 85   page: 512 - 513   2015

  8. シナプス伝達修飾分子LGI1の 機能異常による“てんかん”発症 Invited Reviewed

    横井紀彦, 深田優子, 深田正紀

    メディカルバイオ   Vol. 7 ( 3 ) page: 40 - 47   2010.5

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    Authorship:Corresponding author   Language:Japanese   Publisher:オーム社  

    CiNii Books

    Other Link: http://search.jamas.or.jp/link/ui/2010195866

  9. LGI1が仲介するタンパク質複合体の破綻はシナプス伝達異常と てんかんを引き起こす Invited Reviewed

    岩永剛, 深田正紀, 深田優子

    細胞工学   Vol. 29 ( 6 )   2010

  10. The Molecular Mechanisms for AMPA Receptor Trafficking Invited Reviewed

    FUKATA Masaki, FUKATA Yuko

      Vol. 28 ( 3 ) page: 131 - 134   2008.6

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    CiNii Books

  11. PSD-95パルミトイル化脂質修飾酵素によるAMPA受容体動態制御メカニズム Invited Reviewed

    則竹淳, 深田優子, 深田正紀

    蛋白質核酸酵素   Vol. 53 ( 4 ) page: 430 - 435   2008.4

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  12. Protein palmitoylating enzymes Invited Reviewed

    TSUTSUMI Ryouhei, FUKATA Yoko, FUKATA Masaki

      Vol. 80 ( 12 ) page: 1119 - 1123   2008

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    Language:Japanese  

    CiNii Books

  13. Epilepsy-related LGI1 functions as a synaptic modulator through ADAM22 Invited Reviewed

    Protein, nucleic acid and enzyme   Vol. 52 ( 5 ) page: 449 - 455   2007.5

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    CiNii Books

    Other Link: http://search.jamas.or.jp/link/ui/2007209543

  14. パルミトイル化脂質修飾をつかさどる新規DHHC蛋白質ファミリー Invited Reviewed

    深田優子, 深田正紀

    蛋白質核酸酵素   Vol. 50 ( 13 ) page: 1666 - 1673   2005.11

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    Authorship:Last author, Corresponding author   Language:Japanese   Publisher:共立出版  

    CiNii Books

  15. 遺伝子導入と遺伝子ノックダウン : 新しい時代を迎える生細胞観察(I.遺伝子操作法と組織化学)

    渡辺 崇, 深田 正紀, 貝淵 弘三

    組織細胞化学   Vol. 2003   page: 9 - 15   2003.7

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    Language:Japanese   Publisher:日本組織細胞化学会  

    CiNii Books

    CiNii Research

  16. Mechanisms of Cell Adhesion and Cell Polarization in Rho Family GTPases

      Vol. 51 ( 1 ) page: 151 - 160   2003.7

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  17. 低分子量GTP結合タンパク質Rac1,Cdc42と標的タンパク質IQGAP1による微小管捕捉機構 Invited

    深田 正紀, 渡辺 崇, 則竹 淳, 中川 誠人, 貝淵 弘三

    細胞工学   Vol. 21 ( 9 ) page: 1064 - 1065   2002.8

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  18. 低分子量GTP結合タンパク質Rac1,Cdc42と標的タンパク質IQGAP1による細胞間接着の制御 Invited Reviewed

    深田 正紀, 中川 誠人, 山鹿 真幸, 貝淵 弘三

    細胞工学   Vol. 20 ( 4 ) page: 548 - 551   2001.4

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    Authorship:Lead author   Language:Japanese   Publisher:学研メディカル秀潤社  

    CiNii Books

    Other Link: http://search.jamas.or.jp/link/ui/2001189016

  19. Rhoファミリーによる細胞骨格、細胞接着の再構築 Invited Reviewed

    深田正紀, 黒田真也, 中川誠人, 貝淵弘三

    細胞工学   Vol. 17 ( 12 ) page: 1844 - 1855   1998.12

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  20. Cdc42、Rac1と標的タンパク質による細胞接着シグナル Invited Reviewed

    深田正紀, 黒田真也, 小林憲太, 貝淵弘三

    細胞工学   Vol. 15 ( 12 ) page: 1724 - 1731   1996.12

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Presentations 1

  1. Synaptic function and synaptic disorders Invited

    2023.5.10 

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    Event date: 2023.5

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

KAKENHI (Grants-in-Aid for Scientific Research) 23

  1. 抗体エンジニアリングによる自己免疫性脳炎の病態解明

    Grant number:23K18228  2023.6 - 2026.3

    科学研究費助成事業  挑戦的研究(萌芽)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

  2. 神経細胞の膜ナノドメイン構築原理とナノ病態機構の解明

    Grant number:23H00374  2023.4 - 2026.3

    科学研究費助成事業  基盤研究(A)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\47190000 ( Direct Cost: \36300000 、 Indirect Cost:\10890000 )

  3. 臨界期におけるシナプス成熟の分子メカニズムの解明

    Grant number:23H04243  2023.4 - 2025.3

    科学研究費助成事業  学術変革領域研究(A)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\12480000 ( Direct Cost: \9600000 、 Indirect Cost:\2880000 )

  4. 神経シナプスの成熟機構とその破綻による“てんかん"の分子病態

    Grant number:20H00459  2020.4 - 2023.3

    科学研究費助成事業  基盤研究(A)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\45240000 ( Direct Cost: \34800000 、 Indirect Cost:\10440000 )

    脳の生理と病態を理解するには、神経細胞間の情報伝達を担う“シナプス”の理解が必要不可欠である。シナプス伝達の効率は常に一定ではなく、外界刺激によって柔軟に変化し、これが記憶や学習などの脳高次機能の基盤となる。本研究では、申請者らが独自に見出した (1)パルミトイル化脂質修飾関連酵素と(2)分泌リガンド・受容体LGI1-ADAM22を起点として、シナプスの成熟機構とその破綻によるシナプス疾患の分子病態を解明する。
    本研究では、(1)分泌リガンド・受容体LGI1-ADAM22と(2)パルミトイル化脂質修飾関連酵素を起点として、シナプス前・後部の協調機構とシナプス-ナノドメインの形成機構を明らかにする。2021年度は、「LGI1-ADAM22によるシナプス-ナノドメイン間の連結制御機構の解明」を中心に進めた。私共は、前年度(2020年度)にLGI1-ADAM22-PSD-95(およびMAGUKファミリー)複合体が、シナプス前後部のナノドメインを対面整列させる中心的な構成因子として機能し、シナプス伝達を精緻に制御することを報告した(Fukata Y et al, PNAS 2021)。
    2021年度はこの成果をさらに発展させ、米国UCSFのNicoll博士との共同研究により、LGI1-ADAM22-PSD-95経路が、他のMAGUKタンパク質SAP102経路と協調して、海馬の長期増強(LTP)に必須の役割を果たしていることを見出した(Chen X et al, PNAS 2021)。さらに、私共はADAM22の合成経路と分解経路に関する研究を行い、ADAM22の832番目のセリン残基がPKAにより高度にリン酸化され、14-3-3というタンパク質と強固に結合することで安定にシナプス膜に局在することを見出した。実際、ADAM22のリン酸化欠損マウスでは、ADAM22の脳内での発現量は野生型マウスの40%にまで減少していた。さらに、ADAM22とLGI1の発現量が異なる7種類のマウスを作製し、ADAM22の量が健常マウスの約10%あればてんかん発症を抑えられることを明らかにした。一方、LGI1の量は健常の30%まで低下すると致死性てんかんが発症し、自発性てんかんを抑止するには50%は必要であることを見出した。(Yokoi et al. Cell Reports 2021)。
    (1) LGI1-ADAM22-PSD-95経路がSAP102経路と協調して海馬における長期増強に必須な役割を果たすことを見出した(Chen X et al, PNAS 2021、Fukata Y et al, Neuropharmacology 2021)。
    (2) ADAM22の合成経路と分解経路を明らかにし、マウス脳において、てんかん発症を抑止するのに十分なADAM22の量を明らかにした(Yokoi et al. Cell Reports 2021)。
    (1) LGI1-ADAM22-PSD-95複合体によるシナプス-ナノドメイン間の連結制御機構の解明
    ADAM22とPSD-95の結合破綻により、シナプス後部膜におけるPSD-95集積が減少する分子基盤の詳細を明らかにする。また、LGI1自己抗体を活用することにより、抗LGI1抗体脳炎の病態の詳細を明らかにする。
    (2) パルミトイル化サイクルによるシナプス-ナノドメインの形成・再編機構
    脱パルミトイル化酵素ABHD17ノックアウトマウスの神経細胞における、PSDナノドメインのサイズや大きさを解析する。また、パルミトイル化サイクルを受けないPSD-95変異体の性状解析を進める。

  5. S-アシル化修飾ゾーンによるシナプス機能制御

    Grant number:20H04915  2020.4 - 2022.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Grant amount:\7150000 ( Direct Cost: \5500000 、 Indirect Cost:\1650000 )

    S-アシル化脂質修飾は、ウイルスから動物、植物まで保存された普遍的なタンパク質の修飾反応である。S-アシル化は、タンパク質に飽和脂肪酸であるパルミチン酸を可逆的に付加することでタンパク質の細胞内での輸送や局在、機能を制御する。本研究で私共は、“細胞内局所で、S-アシル化酵素と脱S-アシル化酵素が協調して形成する「S-アシル化修飾ゾーン」が、タンパク質の特異的かつ動的な局在制御を担う”という仮説を検証する。
    本研究では、神経細胞の高度に特殊化した細胞膜領域であるシナプス膜領域をモデルとして、その形成と機能発現における「S-アシル化修飾ゾーン」の役割に着目してきた。とりわけ、シナプス後部膜に濃縮する代表的な足場タンパク質“PSD-95”の集積機構について解析を進めてきた。最近、私共は、PSD-95がシナプス後部膜に濃縮するには、(1) S-アシル化修飾に加えて、(2) PSD-95結合タンパク質として見出していたADAM22との結合が重要であることを見出した(Fukata et al. PNAS 2021)。2021年度は、米国UCSFのNicoll博士との共同研究にて、ADAM22-PSD-95経路が海馬の長期増強(LTP, long term potentiation)と呼ばれるシナプス可塑性に関与しているかを検討した。その結果、ADAM22-PSD-95経路は、SAP102経路と協調して、LTP誘導に必須の役割を果たしていることを明らかにした(Chen X, Fukata et al. PNAS 2021)。さらに、私共はADAM22の輸送ゾーン(生合成経路と分解経路)について解析を進め、ADAM22はPKAによりリン酸化され、14-3-3タンパク質と強固に結合することで安定にシナプス膜に発現することを見出した。一方、このリン酸化を受けない遺伝子改変マウスを作製したところ、ADAM22は14-3-3と結合できずに分解されてしまい、脳内のADAM22の量が約40%にまで減少することを見出した(Yokoi et al. Cell Rep 2021)。以上の結果より、シナプス後部へのPSD-95の集積およびそのシナプス機能には、S-アシル化と共に膜タンパク質ADAM22との結合が重要であることが明らかになった。
    令和3年度が最終年度であるため、記入しない。
    令和3年度が最終年度であるため、記入しない。

  6. Clarify the pathological mechanism for autoimmune encephalitis using recombinant autoantibodies

    Grant number:19K22548  2019.6 - 2022.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Research (Exploratory)

    Fukata Masaki

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    Authorship:Principal investigator 

    Grant amount:\6500000 ( Direct Cost: \5000000 、 Indirect Cost:\1500000 )

    Autoimmune encephalitis presenting with amnesia, seizures, and disorientation is highly topical in basic and clinical neuroscience. Recent studies have identified numerous associated autoantibodies, targeting synaptic proteins including neurotransmitter receptors and a secreted protein, LGI1. Here, by collaborating with Germany group, we isolated various recombinant monoclonal antibodies from patients' B cells with autoimmune encephalitis. Taking advantage of these antibodies, we elucidated patho-physiological mechanisms for autoimmune encephalitis.

  7. 数理解析を目指したパルミトイル化シグナル伝達研究

    Grant number:19H04974  2019.4 - 2021.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\10790000 ( Direct Cost: \8300000 、 Indirect Cost:\2490000 )

    タンパク質のパルミトイル化脂質修飾は、酵母から動・植物まで保存された普遍的な翻訳後修飾で、タンパク質の輸送や機能を制御する。近年の網羅的なプロテオミクス解析により、多種多様なパルミトイル化タンパク質が次々と報告されているが、そのシグナル伝達機構は殆ど不明である。本研究では、神経シナプスの中心的タンパク質であるPSD-95をモデルとして、パルミトイル化シグナル伝達機構の全容を明らかにする。
    パルミトイル化修飾は、可逆的な脂質修飾でタンパク質の局在や機能を動的に制御する。近年のプロテオミクス解析により、多種多様なパルミトイル化タンパク質が次々と報告されているが、そのシグナル伝達機構は殆ど不明である。本研究では、神経シナプスの中心的足場タンパク質であるPSD-95等をモデルとして、パルミトイル化シグナル伝達機構の全容を明らかにする。具体的には、(1)パルミトイル化反応の制御機構の解明、(2)パルミトイル化反応が可逆的である意義を解明する。
    2020年度は、PSD-95の脱パルミトイル化酵素ABHD17の相互作用分子群の解析を進めた。2019年度にショットガン法にて同定したABHD17相互作用分子(263種)の中で、生理的に重要かつ新規性の高いと考えられた10種類余りのタンパク質について、解析を進めた。これら相互作用分子の結合特異性は、タグ付きABHD17ノックインマウスと野生型マウスの精製タンパク質プロファイルを比較することで評価した。また、PSD-95のパルミトイル化酵素の一つであるZDHHC2の基質タンパク質をパルミトーム解析により探索した。野生型マウス脳とZDHHC2ノックアウトマウス脳におけるパルミトームの比較により、in vivoにおけるZDHHC2特異的な基質タンパク質の有無を検討、解析した。これらの特異性と定量性を重視した生化学的解析により、ABHD17とDHHC2の反応機構、活性制御機構の解明に資する重要な知見を得た。さらに、パルミトイル化反応が可逆的である意義を明らかにするために、パルミトイル化サイクルを受けないPSD-95変異体の性状解析を推し進めた。
    令和2年度が最終年度であるため、記入しない。
    令和2年度が最終年度であるため、記入しない。

  8. S-アシル化修飾ゾーンとシナプス機能

    Grant number:18H04873  2018.4 - 2020.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\7410000 ( Direct Cost: \5700000 、 Indirect Cost:\1710000 )

    S-アシル化脂質修飾は、酵母から動・植物まで保存された普遍的な翻訳後修飾で、タンパク質の輸送や機能を制御する。S-アシル化反応は可逆反応であり、アシル化、脱アシル化両反応のバランスが外界刺激により制御されている。多種多様なS-アシル化タンパク質は、様々なオルガネラ膜や細胞膜に特異性をもって局在化するが、興味深いことに、私共が単離したS-アシル化関連酵素群も、小胞体やゴルジ体のみならず、エンドソームや細胞膜といった多様な細胞内局在を示す。そこで、私共は“タンパク質合成直後の小胞体やゴルジ体における選別輸送に加えて、細胞膜のような最終到達地においても、S-アシル化酵素と脱S-アシル化酵素が協調して「S-アシル化修飾ゾーン」を形成し、タンパク質の動的な局在制御を担っている”という仮説を検証する。本研究では、神経細胞のシナプス膜をモデルとして、その形成と機能発現における「S-アシル化修飾ゾーン」の役割を解明する。2019年度は、特異性を重視した免疫沈降法により脱アシル化酵素ABHD17の結合タンパク質を多数同定した。また、ミトコンドリアに局在化させた脱S-アシル化プローブ(mitoDPP)と私共の脱S-アシル化酵素ライブラリーを活用して、ミトコンドリアにおける脱S-アシル化酵素としてABHD10を同定した(Bryan Dickinson 博士(シカゴ大)との国際共同研究、Nat Chem Biol誌に発表)。さらに、私共のS-アシル化酵素ライブラリーを活用した国際共同研究により、CCR5(HIV-1ウイルスの受容体)のS-アシル化酵素(ZDHHC3,7)を同定し、その輸送機構を解明した(Franck Perez博士(キュリー研究所)との国際共同研究、Science Advances誌に発表)。
    令和元年度が最終年度であるため、記入しない。
    令和元年度が最終年度であるため、記入しない。

  9. Molecular mechanism for synapse-nanocolumn formation and synapse disorders

    Grant number:17H03678  2017.4 - 2020.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Fukata Masaki

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    Authorship:Principal investigator 

    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

    Regulated trans-synaptic communication plays a pivotal role in synaptic transmission. Although recent studies have identified a large number of synaptic adhesion molecules, the molecular mechanism for trans-synaptic communication remains incompletely understood. In this study, we showed the crystal structure of human LGI1-ADAM22 complex, revealing a 2:2 heterotetrameric assembly through the intermolecular LGI1-LGI1 interaction. A pathogenic R474Q mutation of LGI1 is located in the LGI1-LGI1 interface and disrupts the higher-order assembly of the LGI1-ADAM22 complex in vitro and in a mouse model for familial epilepsy. These studies support the notion that the LGI1-ADAM22 complex functions as the trans-synaptic machinery for precise synaptic transmission and disruption of this linkage causes the epileptic disorder.

  10. LGI1を中心とするシナプス蛋白質ネットワークの老化と認知症の分子病態

    Grant number:17H05709  2017.4 - 2019.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\10660000 ( Direct Cost: \8200000 、 Indirect Cost:\2460000 )

    神経シナプスの機能障害は認知症発症の重要な初期病態の一つと考えられている。これまでに、私共は脳の速い興奮性シナプス伝達の大部分を司るAMPA型グルタミン酸受容体の制御機構の解明に取り組み、AMPA受容体制御タンパク質としてLGI1-ADAM22(リガンド-受容体)を見出した。本研究では、これまでの研究をさらに発展させ、LGI1-ADAM22経路の全容とその破綻による病態機構を解明することを目指した。平成30年度は、LGI1-ADAM22のX線結晶構造解析の知見に基づき(東京大学、深井周也博士との共同研究)、LGI1-ADAM22がヘテロ4量体としてシナプス間を架橋することを発表した(Yamagata, Miyazaki et al, Nature Commun, 2018)。また、認知機能障害やけいれん発作、記憶障害を呈する自己免疫性辺縁系脳炎患者の血清や髄液より得たLGI1自己抗体を用いて、神経細胞の過興奮やシナプス伝達異常の原因となる病態機構の一端を明らかにした。これら自己抗体は、nativeのLGI1分子と高い親和性で結合することから、LGI1の詳細な局在解析に使用できると共に、LGI1に相互作用する分子群を網羅的に同定し、LGI1の作用機序を解明する上でも極めて有用であると考えられる。さらに、私共はLGI1およびADAM22の解析のみならず、これら分子が属するファミリー分子群(LGI2, LGI3, LGI4やADAM11、ADAM23)に関しても解析を進めた。このように本研究では、LGI1とADAM22の分子構造基盤を明らかし、LGI1-ADAM22の作用機序と病態機構の解明を推し進めた。
    平成30年度が最終年度であるため、記入しない。
    平成30年度が最終年度であるため、記入しない。

  11. 神経シナプス膜のナノドメイン構築と脂質環境

    Grant number:16H01371  2016.4 - 2018.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\9880000 ( Direct Cost: \7600000 、 Indirect Cost:\2280000 )

    神経シナプスは、シナプス伝達に関わる機能蛋白質が高度に集積する特殊な細胞膜領域からなる。蛋白質構成に加え固有の膜脂質構成がシナプスの微小膜環境を形成すると予測されるが、その脂質環境の本態や生理機能はいまだ不明である。これまでに私共は(1)シナプス後膜(PSD)の足場蛋白質PSD-95のパルミトイル化脂質修飾が、PSD構成単位である“PSDナノドメイン”の形成を担うこと、(2)シナプス膜に集積する蛋白質の多くが、高度かつ多重にパルミトイル化されていることを見出した。そこで本研究では、パルミトイル化脂質修飾のON・OFFを担う酵素群がナノドメイン脂質環境に与える影響、神経機能における生理機能を明らかにすることを目指す。平成28年度、私共はPSD-95脱パルミトイル化酵素としてα/β-hydrolase domain-containing protein(ABHD)17A、17B、17Cを同定した。しかし、ABHD17酵素の基質特異性、活性制御機構、細胞内局在、生理機能については未だ十分に明らかにされていない。そこで平成29年度は、オランダ・ユトレヒト大学のHoogenraad博士との共同研究を通じて、ABHD17の新規基質として微小管結合タンパク質MAP6同定した。MAP6はパルミトイル化修飾により輸送小胞膜に局在するが、ABHD17により脱パルミトイル化をうけると小胞膜から離脱し、微小管に結合することを見出した。そしてMAP6は微小管を安定化し、軸索形成や神経細胞の極性化を促進することを見出した(Tortosa E et al, Neuron 2017)。このように、パルミトイル化サイクルがシナプス機能のみならず、軸索形成や神経細胞の成熟・極性化にも重要な役割を担うことを明らかにした。
    29年度が最終年度であるため、記入しない。
    29年度が最終年度であるため、記入しない。

  12. Optogenetic manipulation of protein palmitoylation for synapse organization

    Grant number:16K14560  2016.4 - 2018.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    Masaki Fukata, FUKATA Yuko, YOKOI Norihiko, HIRATA Tetsuya, INAHASHI Hiroki, KANADOME Takashi, SUZUKI Yumi, WATANABE Mie

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    Authorship:Principal investigator 

    Grant amount:\3640000 ( Direct Cost: \2800000 、 Indirect Cost:\840000 )

    The neuronal synapse comprises specialized membrane domains and their dynamic remodeling plays an important role in synaptic plasticity. So far, we proposed that the dynamic change in (de)palmitoylating enzyme activity at local synapses is a critical factor for reorganization of synaptic membrane domains. In this study, we aimed at creating the optogenetic probes that spatio-temporally manipulate the (de)palmitoylating enzyme activity. Taking advantage of LOV2, CRY2-CIB1, LOVTRAP and Zdk1-iLID systems, we got a promising clue.

  13. 記憶と脳の安定性を保持するLGI1リガンドの老化と認知症における役割

    Grant number:15H01570  2015.4 - 2017.3

    科学研究費助成事業  新学術領域研究(研究領域提案型)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\9620000 ( Direct Cost: \7400000 、 Indirect Cost:\2220000 )

    認知症の制御には認知症関連蛋白質の発掘は欠かせない。私共は認知症発症の最初期にはシナプス伝達機構の破綻が関与すると考え、AMPA型グルタミン酸受容体を介したシナプス伝達の制御機構に焦点を当てて研究を進めてきた。昨年度は、てんかん関連蛋白質LGI1の欠損が、足場蛋白質PSD-95を介したシナプス伝達制御に必須であることを明らかにした(Lovero、深田ら PNAS 2015)。今年度は、このPSD-95の機能制御を担う新たな分子を探索、同定した(横井、深田ら J Neurosci 2016)。PSD-95は、興奮性シナプスのシナプス後肥厚部(PSD)に濃縮し、AMPA受容体やNMDA受容体、Neuroliginなどを裏打ちし、シナプス伝達やシナプス形成を制御する。PSD-95がPSDに局在するためには、パルミトイル化脂質修飾が必要である。パルミトイル化修飾は可逆反応であり外界刺激に依存して制御される。これまでに私共はPSD-95パルミトイル化酵素としてDHHC2、DHHC3を同定してきたが、脱パルミトイル化酵素に関しては長らく不明であった。今回、私共はPSD-95脱パルミトイル化酵素としてABHD17A、17B、17Cを同定した。そして、ABHD17を海馬神経細胞に過剰発現させると、PSD-95とAMPA受容体のシナプス局在が激減し、樹状突起スパインの数が減少することを見出した。また、これら酵素群をノックダウンするとPSD-95の脱パルミトイル化が有意に阻害されることを見出した。さらに、私共は蛋白質のパルミトイル化状態(ストイキオメトリー)を検出する生化学的手法(APEGS法)を開発した。すなわち、シナプス蛋白質のパルミトイル化状態の変容が脳蛋白質老化の一因になっているかを検討することが可能となる基盤的技術の開発にも成功した。
    28年度が最終年度であるため、記入しない。
    28年度が最終年度であるため、記入しない。

  14. Identification of depalmitoylating enzymes

    Grant number:26291045  2014.4 - 2017.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    FUKATA Masaki, FUKATA Yuko, YOKOI Norihiko, SEKIYA Atsushi, MURAKAMI Tatsuro, TAKAHASHI Naoki, SUZUKI Yumi, WATANABE Mie

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    Authorship:Principal investigator 

    Grant amount:\16640000 ( Direct Cost: \12800000 、 Indirect Cost:\3840000 )

    Protein palmitoylation, the most common lipid modification, dynamically regulates neuronal protein localization and function. Its unique reversibility is conferred by DHHC-type palmitoyl acyl-transferases (palmitoylating enzymes) and still controversial palmitoyl-protein thioesterases (depalmitoylating enzymes). Here, we identified the membrane-anchored serine hydrolases, ABHD17A, 17B, and 17C, as the physiological PSD-95 depalmitoylating enzyme that regulates PSD-95 palmitoylation cycles in neurons. This study describes the first direct evidence for the neuronal depalmitoylating enzyme and provides a new aspect of the dynamic regulatory mechanisms of synaptic development and synaptic plasticity. In addition, our established APEGS assay, which provides unbiased and quantitative information about the palmitoylation state and dynamics, revealed the distinct regulatory mechanisms for synaptic palmitoylation.

  15. 新規AMPA受容体制御因子群によるシナプス機能制御の解明

    Grant number:20670005  2008 - 2012

    科学研究費助成事業  若手研究(S)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\80730000 ( Direct Cost: \62100000 、 Indirect Cost:\18630000 )

    シナプス間の情報伝達効率は使用状況によって柔軟に変化し、記憶や学習など脳高次機能の分子基盤を成す。この機構の破綻はてんかん等の神経疾患の重要な一因と考えられている。AMPA型グルタミン酸受容体(AMPA受容体)は脳内の興奮性神経伝達の大部分を司るので、AMPA受容体の輸送機構は現在の神経科学における極めて重要な命題である。本研究ではこの命題に対し、独自の生化学的手法により同定した1)パルミトイル化脂質修飾酵素および2)てんかん関連リガンドLGI1に着目して、AMPA受容体制御機構の全容を明らかにすることを目的としている。
    1)AMPA受容体の発現量はシナプスに存在する足場蛋白質PSD-95(パルミトイル化PSD-95)の量に依存する。今年度はパルミトイル化PSD-95を特異的に認識するプローブを開発し、内在性パルミトイル化PSD-95の動態を可視化することに成功した(Fukata Y et al,投稿中)。これは脂質修飾を受けた蛋白質を特異的に認識する抗体を得たという点でも画期的だと言える。
    2)一方、私どもは前年度に引き続きLGI1のノックアウトマウスの性状解析を行い、LGI1ノックアウトマウスにおけるAMPA受容体機能異常および、海馬組織における局在異常を見出した(Yokoi N et al,投稿準備中)。さらに、てんかんの焦点を明らかにするために脳の領域特異的な機能回復実験を進めている。このように今年度の研究計画は達成できたと考えている。

  16. PSD-95パルミトイル化酵素によるシナプス機能制御

    Grant number:20022043  2008 - 2009

    科学研究費助成事業  特定領域研究

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\8000000 ( Direct Cost: \8000000 )

    これまでに私どもはPSD-95パルミトイル化脂質修飾酵素P-PATファミリー(DHHC2, 3, 7, 15)がAMPA受容体の機能を制御することを見出した。本研究ではこれまでの結果をさらに発展させ、1)神経活動依存的にP-PAT機能を制御する情報伝達機構、および2)P-PATの生理機能を明らかにし、P-PATによるAMPA受容体の動態制御機構の解明を目指す。本年度の研究実績は以下のとおりである。
    4種類のP-PATのうち海馬ではDHHC2およびDHHC3の発現が高く主要な働きを果たしていることを見出した。興味深いことに、DHHC3は細胞体のゴルジ装置に限局しているが、DHHC2は樹状突起内にも存在し、神経活動感受的にシナプス近くに素早く移動することが分かった。さらに、神経活動抑制下では、AMPA受容体のシナプス発現量が上昇することが知られているが、DHHC2のRNA干渉実験の結果、DHHC2によるPSD-95のパルミトイル化が、神経活動抑制によって誘導されるAMPA受容体のシナプス後膜への集積(AMPA受容体恒常性維持)に必須であった(Noritake et al. J. Cell Biol. 2009)。以上の知見は、パルミトイル化酵素ファミリー分子の局在多様性が、極性化した神経細胞における基質蛋白質の局在を巧妙に制御していることを示唆する。これらの成果は世界的にも高く評価され、総説(深田らNature Rev. Neuroscience, 2010)にまとめられた。さらに、私どもは、生細胞で内在性PSD-95のパルミトイル化動態を可視化することを目的として、パルミトイル化PSD-95を特異的に検出するprobeの作成に成功した。このように今年度の研究計画は達成できたと考えている。

  17. パルミトイルサイクルによるG蛋白質の動態制御機構

    Grant number:20054022  2008 - 2009

    科学研究費助成事業  特定領域研究

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\5000000 ( Direct Cost: \5000000 )

    GTP結合蛋白質の多くはパルミトイル化脂質修飾を受けることにより、特定の膜マイクロドメインに局在し分子スイッチとして機能する。例えば三量体G蛋白質Gαや低分子量G蛋白質H-Rasなどがパルミトイル化修飾を受ける。しかし、パルミトイル化酵素が不明であったためパルミトイル化修飾によるG蛋白質の動態制御機構に関しては不明であった。昨年度までに、私どもは23種類のパルミトイル化酵素の中から、Gαをパルミトイル化する酵素としてDHHC3/7を同定した。今年度は1)DHHC3/7によるG蛋白質動態制御機構の解明、および2)パルミトイル化酵素ファミリーの多様な活性制御機構の解明を目指した。今年度の研究実績は以下のとおりである。
    GFP融合型Gαqを用いたFRAP/FLIP法、および光変換蛍光蛋白質Dendra2との融合型Gαqを用いたphotoconversion法を用いて細胞内Gαqの動態を解析した。その結果、細胞膜に存在するパルミトイル化Gαqは脱パルミトイル化を受けた後速やかにDHHC3/7が存在するゴルジ装置に移行し、DHHC3/7により再パルミトイル化され再び細胞膜に輸送されることを見出した(堤らMol. Cell. Biol. 2009)。また、神経細胞のような極性化した細胞においては、細胞体のゴルジ装置においてDHHC3がGαqやPSD-95など多くの基質を恒常的にパルミトイル化するが、樹状突起上のポストシナプスにおいては別のサブファミリーに属するDHHC2が、神経活動感受的にPSD-95をパルミトイル化することが分かった(則竹らJ. Cell BioL. 2009)。これらの成果は世界的にも高く評価され、総説(深田らNature Rev. Neuroscience, 2010)にまとめられた。このように今年度の研究計画は達成できたと考えている。

  18. 新規パルミトイル化酵素群の活性制御機構の解明

    Grant number:18687008  2006 - 2008

    科学研究費助成事業  若手研究(A)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\30030000 ( Direct Cost: \23100000 、 Indirect Cost:\6930000 )

    パルミトイル化は他の脂質修飾とは異なり可逆的であり、外界刺激依存性にサイクリングしている。これまでに、我々は全23種類からなる新規パルミトイル化酵素群をゲノムワイドに単離し、その機能解析を進めてきた。今年度はパルミトイル化酵素ファミリーの活性制御機構を明らかにするために、DHHCパルミトイル化酵素に結合する蛋白質を脳組織から特異性高く精製し、高感度質量分析装置を駆使してその構成蛋白質を明らかにした。具体的にはDHHC2結合蛋白質としてDHHC20を含む複数の蛋白質を見出した。また、DHHC3結合蛋白質としてintegrinなど複数の膜蛋白質を同定した。さらに、DHHC8,DHHC21に結合する蛋白質を同様の手法を用いて次々に同定しつつある。これらの解析を通じて、パルミトイル化酵素群のサブファミリー特異的な活性制御機構の一端を明らかにした。
    一方、我々は前年度までに神経活動の下流でPSD-95パルミトイル化酵素DHHC2の活性がダイナミックに変化することを見出している。今年度はDendra2融合蛋白質を用いたphotoconversion法を駆使して、PSD-95やDHHC2酵素の動態を神経細胞で可視化し、これら蛋白質の動態制御機構を検討した。興味深いことに、神経活動を遮断することによりPSD-95はより膜近傍に集積し、AMPA受容体の恒常性維持に重要な役割を果たしていることを明らかにした。本研究課題は平成20年度科学研究費補助金(若手研究(S))に係る交付内定通知の受理(5月)に伴い、若手研究(S)に専念するため廃止した。

  19. PSD-95パルミトイル化酵素によるAMPA受容体動態制御機構の解明

    Grant number:18022054  2006 - 2007

    科学研究費助成事業  特定領域研究

    深田 正紀, 深田 優子

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    Authorship:Principal investigator 

    Grant amount:\8000000 ( Direct Cost: \8000000 )

    シナプス伝達効率は神経活動依存性に柔軟に変化することから、記憶や学習の分子基盤を成すと考えられている。中でもグルタミン酸受容体の一つであるAMPA受容体は神経活動依存性にダイナミックにシナプスに輸送され、シナプス可塑性の中心的な役割を果たす。最近、私どもは足場蛋白質PSD-95の局在を制御するパルミトイル化脂質修飾酵素(P-PAT)を同定し、P-PATがPSD-95のパルミトイル化を介してAMPA受容体のシナプス膜での発現量を調節することを明らかにした。本研究では神経活動によるP-PATの活性制御機構の解明を通じて、AMPA受容体の動態制御機構を明らかにする。本年度の研究実績は以下のとおりである。
    P-PATはDHHC2,3,7,15という4種類の分子種から構成されているが、これら4種類のパルミトイル化酵素のどの分子種がグルタミン酸受容体の下流で調節されているかは不明であった。今年度、私どもはグルタミン酸受容体の下流で制御されうる酵素はDHHC2およびDHHC15のサブファミリーであることを見出した。また、興味深いことにDHHC3は細胞体のゴルジ装置に特異的に局在し、一方DHHC2は樹状突起内のシナプス近傍にも存在していることを見出した。さらに、私どもはP-PATがPSD-95のパルミトイル化を介してAMPA受容体の恒常性維持に関わっていることを見出した(グルタミン酸受容体の抑制→P-PATの活性化→PSD-95のパルミトイル化レベルの上昇→AMPA型グルタミン酸受容体のシナプスへの集積→神経活動の維持)(則竹ら投稿準備中)。この知見はシナプス活動がある一定範囲内で安定に維持されるという現象(例えばsynaptic scaling)を理解する上でも重要であると考えられる。このように今年度の研究計画は達成できたと考えている。

  20. パルミトイルサイクルによるG蛋白質の動態制御機構の解明

    Grant number:18057032  2006 - 2007

    科学研究費助成事業  特定領域研究

    深田 正紀, 深田 優子

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    Authorship:Principal investigator 

    Grant amount:\6100000 ( Direct Cost: \6100000 )

    細胞内情報伝達の要として機能するGTP結合蛋白質の多くは特定の膜マイクロドメインに局在することにより外界シグナルを細胞内に効率良く伝達することができる。パルミトイル化脂質修飾は三量体G蛋白質のGα(Gs,Gi,Gqなど)、低分子量G蛋白質(H/N-Rasなど)に見られ、これら分子の局在、機能を外界刺激依存性に制御していると考えられている。最近、私どもはゲノムワイドにパルミトイル化反応の責任酵素群を同定した。本研究では1)各G蛋白質をパルミトイル化する特異酵素の同定、2)パルミトイル化酵素の活性制御機構の解析、及び3)パルミトイル化酵素によるG蛋白質動態制御機構の解析を行う。
    昨年度までに、Gαs、Gαqのパルミトイル化酵素を独自のパルミトイル化酵素ライブラリーを用いてスクリーニングし、DHHC3、DHHC7にGαサブユニットに対して著しくパルミトイル化レベルを促進させる活性があることを見出した。今年度は新たにGαi2をパルミトイル化する酵素としてDHHC3、DHHC7およびDHHC21を見出した。さらに、DHHC3とDHHC7をノックダウンにより機能抑制したところ、内在性のGαqのパルミトイル化レベルが減少し、細胞膜から細胞質にGαqが移行することを見出した。さらに、DHHC3がゴルジ装置に局在し、Gαqが細胞膜とゴルジ装置間を常時サイクルしていることを見出した(堤ら投稿準備中)。現在、Gαqの細胞内動態をFRAP,photoconversion法および全反射顕微鏡を駆使して可視化し、パルミトイル化酵素によるGαqの動態制御機構を明らかにしようとしている。このように今年度の研究計画は達成できたと考えている。

  21. 低分子量G蛋白質Cdc42,Racと標的蛋白質IQGAPによる細胞極性の制御機構

    Grant number:14780488  2002 - 2003

    科学研究費助成事業  若手研究(B)

    深田 正紀

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    Authorship:Principal investigator 

    Grant amount:\3900000 ( Direct Cost: \3900000 )

    創傷の治癒過程や発生過程において細胞は極性をもって運動する。この極性形成や細胞運動には微小管とアクチン細胞骨格の協調した再編成が重要である。一方、低分子量GTP結合蛋白質Rhoファミリー(Cdc42,Rac1,RhoA)は細胞極性を制御していることが報告されているがその分子メカニズムはやはり不明であった。
    本年度はCdc42とRac1の標的蛋白質IQGAP1の新規結合蛋白質としてCLIP-170を同定した(Fukata et al. Cell 2002)。CLIP-170は伸長する微小管のプラス端に濃縮し、特定の細胞表層を認識し、微小管を配向、アンカーさせ、細胞極性形成に重要な役割を果たしていると考えられていた。私共はin vitroとin vivoのどちらの系においても活性型Rac1/Cdc42、IQGAP1及びCLIP-170が3者複合体を形成し、IQGAP1とCLIP-170の結合が活性型Rac1と活性型Cdc42により促進されることを明らかにした。さらに、ドミナントネガティブ型IQGFAP1を発現させることにより、CLIP-170の動態が阻害されることも見出した。
    微小管の細胞表層での捕捉機構は長らく細胞生物学上大きな謎であったが、本研究によりRhoファミリー(Rac1/Cdc42)と標的蛋白質IQGAP1による分子機構が初めて明らかになった。本研究成果は細胞極性形成機構を理解する上でも極めて重要であると考えられる。
    したがって、本年度の研究計画は達成できたと考えている。

  22. 細胞間接着制御系の網羅的解析

    Grant number:14014217  2002

    科学研究費助成事業  特定領域研究

    深田 正紀, 天野 睦紀

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    Authorship:Principal investigator 

    Grant amount:\6000000 ( Direct Cost: \6000000 )

    個体発生過程や組織の再構築時にはcadherinを介した細胞間接着のダイナミックな再構築が重要な役割を果たしている。このような細胞間接着の再構築には、空間的位置情報と時間情報が正確に細胞内に伝達、統合され、接着装置が再構築されることが必須である。しかし、その分子メカニズムは長らく不明であった。本研究では細胞間接着の分子基盤をより網羅的に明らかにすることを目的とした。
    本年度は細胞間接着を自由に再構築することのできるassay系(Ca2+スイッチ法、およびHepatocyte Growth Factor (HGF)によるcell scattering:細胞分散)を用いて、細胞間接着の各ステージ(接着初期、接着維持期、接着破壊時)に特異的なcadherin, catenins, IQGAP1および低分子量G蛋白質Rhoファミリー結合蛋白質をアフィニティーカラムや免疫沈降により同定した。とりわけIQGAP1結合蛋白質として同定したCLIP-170は微小管のプラス端に濃縮する蛋白質で、IQGAP1はCLIP-170との結合を介して微小管を細胞表層で捕捉することが明らかとなった(Fukata et al., Cell 2002)。IQGAP1はCLIP-170に誘導された微小管を細胞間接着部位にリクルートし、細胞間接着を形成するのに必要な蛋白質やvesicleを輸送する足場を提供している可能性が考えられる。さらに、β-catenin結合蛋白質としてPDZドメインを5個有するスキャフォールディング蛋白質KIAA0705とKIAA0705を介してβ-cateninに結合する蛋白質として低分子量G蛋白質Rap1の活性制御蛋白質PDZ-GEFを同定した。
    したがって、本年度の研究計画はほぼ達成することができたと考えている。

  23. 低分子量G蛋白質Cdc42/Rac1の標的蛋白質IQGAP1の生理機能の解析

    Grant number:00J06523  2000

    日本学術振興会  科学研究費助成事業  特別研究員奨励費

    深田 正紀

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Teaching Experience (On-campus) 3

  1. First Year Seminar B

    2023

  2. 特徴あるプログラム(ニューロサイエンス)

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    2023