Updated on 2024/03/12

写真a

 
YOKOI Norihiko
 
Organization
Graduate School of Medicine Center for Neurological Diseases and Cance Division Assistant Professor
Graduate School
Graduate School of Medicine
Undergraduate School
School of Medicine Department of Medicine
Title
Assistant Professor
Contact information
メールアドレス
External link

Degree 1

  1. 博士(理学) ( 2009.3   名古屋大学 ) 

Research Interests 13

  1. ADAM22

  2. LGI1

  3. PSD-95

  4. シナプス伝達

  5. 人工金属酵素

  6. 自己免疫疾患

  7. 蛋白質パルミトイル化修飾

  8. 蛋白質結晶構造解析

  9. 蛋白質複合体

  10. てんかん

  11. 病原変異体

  12. 蛋白質電子伝達反応

  13. 金属蛋白質

Research Areas 2

  1. Life Science / Neuroscience-general

  2. Nanotechnology/Materials / Inorganic/coordination chemistry  / 生物無機化学

Research History 5

  1. 名古屋大学大学院医学系研究科   神経情報薬理学   助教

    2023.9

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  2. National Institute for Physiological Sciences   Assistant Professor

    2015.4 - 2023.8

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  3. National Institute for Physiological Sciences   Designated assistant professor

    2013.4 - 2015.3

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  4. National Institute for Physiological Sciences

    2010.4 - 2013.3

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  5. National Institute for Physiological Sciences

    2009.4 - 2010.3

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Education 1

  1. 名古屋大学大学院   理学研究科   物質理学専攻(化学系)

    - 2009.3

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Professional Memberships 1

  1. 日本神経科学会

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Awards 5

  1. 第5回 自然科学研究機構若手研究者賞

    2016.6  

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  2. 若手優秀発表者賞 包括脳ネットワーク 2015年度冬のシンポジウム

    2015.12  

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  3. New Journal of Chemistry Interface Poster Prize(最優秀ポスター発表賞) 『The 4th Asian biological inorganic chemistry conference (AsBIC-IV)』

    2008.11  

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  4. 学生講演賞 『第58回錯体化学討論会』

    2008.9  

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  5. 学生講演賞 『日本化学会第88春季年会』

    2008.3  

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Papers 27

  1. Regulatory mechanisms of the LGI1–ADAM22 protein levels to prevent epilepsy Reviewed

    Yokoi Norihiko, Fukata Yuko, Fukata Masaki

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

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  2. Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum. 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   2022.6

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    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

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  3. 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   Vol. 298 ( 6 ) page: 102048   2022

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    DOI: 10.1016/j.jbc.2022.102048

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  4. 14-3-3 proteins stabilize LGI1-ADAM22 levels to regulate seizure thresholds in mice International journal

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

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

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    Authorship:Lead 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

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  5. Trans-synaptic LGI1-ADAM22-MAGUK in AMPA and NMDA receptor regulation. Reviewed International journal

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

    Neuropharmacology   Vol. 194   page: 108628 - 108628   2021.8

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    Exquisitely-regulated synaptic transmission and plasticity underlie higher brain functions such as learning and memory. PSD-95, a member of the MAGUK family, scaffolds an array of postsynaptic proteins including AMPA and NMDA receptors, and plays essential roles in excitatory synaptic transmission and postsynaptic organization. Epilepsy-related secreted protein LGI1 and its receptor ADAM22 represent major constituent elements of the PSD-95-containing synaptic protein complex in the brain. Recent studies begin to reveal a trans-synaptic configuration of the LGI1-ADAM22 complex and its pivotal role in AMPA and NMDA receptor-mediated synaptic transmission through regulating MAGUKs. Especially interesting is that without the association with LGI1-ADAM22, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Here, we review roles of LGI1-ADAM22 in synaptic function, and discuss its modes of action on the MAGUK regulation: as (i) a trans-synaptic hub, (ii) an extracellular scaffold, and (iii) an allosteric activator. We also highlight patho-physiological roles of the LGI1-ADAM22-MAGUK linkage in synaptic disorders such as epilepsy and autoimmune limbic encephalitis.

    DOI: 10.1016/j.neuropharm.2021.108628

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  6. Acyl-PEGyl exchange gel-shift (APEGS) assay for palmitoylation quantification

    Fukata Masaki, Yokoi Norihiko, Fukata Yuko

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

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    Language:Japanese   Publisher:Japanese Electrophoresis Society  

    <p>Protein palmitoylation is the most common, dynamic lipid modification and has become topical, as recent proteomic analyses using acyl-biotinyl exchange chemistry have identified numerous palmitoylated proteins in diverse cells, tissues and species. However, their physiological importance remains poorly understood, as there have been no methods to assess the palmitoylation stoichiometries of proteins. The fields have been long awaiting a quantitative method such as Phos-tag SDS-PAGE to monitor the phosphorylation stoichiometry. Here, we developed a simple, sensitive, and specific method to quantify the palmitoylation states of endogenous proteins, <i>i.e.</i>, stoichiometries and site-occupancies. The method was named the acyl-PEGyl exchange gel shift (APEGS) method. The APEGS assay consists of four chemical steps: (1) cleavage of disulfide bonds with tris-(2-carboxyethyl) phosphine (TCEP), (2) blockade of free cysteine thiols with <i>N</i>-ethyl maleimide (NEM), (3) specific cleavage of palmitoylation thioester linkages with hydroxylamine (NH<sub>2</sub>OH), and (4) labeling of newly exposed cysteinyl thiols with maleimide-conjugated PEG (mPEG), which causes the mobility shift of palmitoylated proteins on SDS-PAGE. Western blotting (WB) with antibodies against proteins of interest would separate palmitoylated bands from non-palmitoylated. Quantifying the relative intensities of bands would provide information about the palmitoylation stoichiometry and palmitoylation states. Advantageous features of the APEGS assay include: (1) versatile application to any biological samples, (2) no need for protein purification, (3) reliably examining whether proteins of interest are palmitoylated if antibodies are available, and (4) investigating the dynamic changes in palmitoylation states. Thus, the APEGS assay contributes to understanding regulatory mechanisms for protein palmitoylation.</p>

    DOI: 10.2198/electroph.65.41

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

    Hasegawa D, Ohnishi Y, Koyama E, Matsunaga S, Ohtani S, Nakanishi A, Shiga T, Chambers JK, Uchida K, Yokoi N, Fukata Y, Fukata M

    J Vet Intern Med   Vol. 33 ( 3 ) page: 1440 - 1445   2019.4

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

    DOI: 10.1038/s41467-018-03947-w

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

  9. 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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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier B.V.  

    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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  10. Neurobiology of autoimmune encephalitis 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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  11. The LGI1-ADAM22 protein complex in synaptic transmission and synaptic disorders 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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  12. 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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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:SOC NEUROSCIENCE  

    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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  13. Local Palmitoylation Cycles and Specialized Membrane Domain Organization 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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  14. Postsynaptic nanodomains generated by local palmitoylation cycles

    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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  15. 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.

    DOI: 10.1038/nm.3759

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  16. 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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  17. 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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  18. SYNAPTIC PLASTICITY REGULATED BY PROTEIN-PROTEIN INTERACTIONS AND POSTTRANSLATIONAL MODIFICATIONS 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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    Language:English   Publishing type:Part of collection (book)   Publisher:ELSEVIER ACADEMIC PRESS INC  

    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.

    DOI: 10.1016/B978-0-12-394308-8.00001-7

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  19. Dual modification of a triple-stranded beta-helix nanotube with Ru and Re metal complexes to promote photocatalytic reduction of CO2 Reviewed

    Norihiko Yokoi, Yuki Miura, Chen-Yuang Huang, Nobuyuki Takatani, Hiroshi Inaba, Tomomi Koshiyama, Shuji Kanamaru, Fumio Arisaka, Yoshihito Watanabe, Susumu Kitagawa, Takafumi Ueno

    CHEMICAL COMMUNICATIONS   Vol. 47 ( 7 ) page: 2074 - 2076   2011

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ROYAL SOC CHEMISTRY  

    We have constructed a robust beta-helical nanotube from the component proteins of bacteriophage T4 and modified this nanotube with Ru-II(bpy)(3) and Re-I(bpy)(CO)(3)Cl complexes. The photocatalytic system arranged on the tube catalyzes the reduction of CO2 with higher reactivity than that of the mixture of the monomeric forms.

    DOI: 10.1039/c0cc03015e

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  20. Construction of Robust Bio-nanotubes using the Controlled Self-Assembly of Component Proteins of Bacteriophage T4 Reviewed

    Norihiko Yokoi, Hiroshi Inaba, Makoto Terauchi, Adam Z. Stieg, Nusrat J. M. Sanghamitra, Tomomi Koshiyama, Katsuhide Yutani, Shuji Kanamaru, Fumio Arisaka, Tatsuo Hikage, Atsuo Suzuki, Takashi Yamane, James K. Gimzewski, Yoshihito Watanabe, Susumu Kitagawa, Takafumi Ueno

    SMALL   Vol. 6 ( 17 ) page: 1873 - 1879   2010.9

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

    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.

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  22. Molecular design of heteroprotein assemblies providing a bionanocup as a chemical reactor Reviewed

    Tomomi Koshiyama, Norihiko Yokoi, Takafumi Ueno, Shuji Kanamaru, Shingo Nagano, Yoshitsugit Shiro, Fumio Arisaka, Yoshihito Watanabe

    SMALL   Vol. 4 ( 1 ) page: 50 - 54   2008.1

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    DOI: 10.1002/smll.200700855

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  23. Ligand design for the improvement of stability of metal complex.protein hybrids Reviewed

    Norihiko Yokoi, Takafumi Ueno, Masaki Unno, Toshitaka Matsui, Masao Ikeda-Saito, Yoshihito Watanabe

    CHEMICAL COMMUNICATIONS   ( 2 ) page: 229 - 231   2008

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    We have succeeded in improving the stability of Fe(Schiff-base).heme oxygenase (HO) hybrids by ligand design based on the crystal structure of Fe(N,N'-bis(salicylidene)-3.4-diaminobenzene propionic acid).HO.

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  24. Crystal structure based design of functional metal/protein hybrids Reviewed

    Takafumi Ueno, Norihiko Yokoi, Satoshi Abe, Yoshihito Watanabe

    JOURNAL OF INORGANIC BIOCHEMISTRY   Vol. 101 ( 11-12 ) page: 1667 - 1675   2007.11

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    Preparation of metal/protein hybrids is growing into important topics in the field of bioinorganic chemistry. X-ray crystal structure analyses of them provide direct information on unique interactions of metal cations or metal cofactors to understand and design enzymatic functions. In this mini review, the authors focus on the recent studies on the metal/protein hybrids concerning crystal structure analyses since 2002 and our related works. The precise structural determination promise us to deeply understand coordination chemistry in protein scaffold and shows intriguing suggestions on rational design and application use for biocatalysts, metal drugs and so on. (C) 2007 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.jinorgbio.2007.06.025

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  25. Coordination design of artificial metalloproteins utilizing protein vacant space Reviewed

    Ueno Takafumi, Abe Satoshi, Yokoi Norihiko, Watanabe Yoshihito

    COORDINATION CHEMISTRY REVIEWS   Vol. 251 ( 21-24 ) page: 2717 - 2731   2007.11

  26. Design of artificial metalloenzymes using non-covalent insertion of a metal complex into a protein scaffold Reviewed

    Takafumi Ueno, Tomomi Koshiyama, Satoshi Abe, Norihiko Yokoi, Masataka Ohashi, Hiroshi Nakajima, Yoshihito Watanabe

    JOURNAL OF ORGANOMETALLIC CHEMISTRY   Vol. 692 ( 1-3 ) page: 142 - 147   2007.1

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    Construction of artificial metalloenzymes is one of the most attractive targets in the field of inorganic and catalytic chemistry, since they show remarkable chemoselectivity and reactivity in aqueous media. For the purpose, covalent modification of protein and cofactors have usually been utilized to attach a metal complex(es) to a protein scaffold. This article focuses on non-covalent insertion of metal complexes into protein environments. The discussion includes the screening of stable metal complex/protein composites, crystal structures, molecular design for regulating enantio selectivity of the target catalytic reactions. Our recent results show that the non-covalent conjugation will provide us a new way in semi-synthesis of artificial metalloenzymes. (c) 2006 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jorganchem.2006.08.043

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  27. Design of metal cofactors activated by a protein-protein electron transfer system

    T Ueno, N Yokoi, M Unno, T Matsui, Y Tokita, M Yamada, M Ikeda-Saito, H Nakajima, Y Watanabe

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 103 ( 25 ) page: 9416 - 9421   2006.6

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    Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. investigations of protein-protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein-protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/ cytochrome P450 reductase (CPR) through protein-protein complex formation during the enzymatic reaction. We report that a Fe-III(Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH2CH2COOH-Schiff-base)(HO)-H-. composite indicates the presence of a hydrogen bond between the propionic acid carboxyl group and Arg-177 of HO. Furthermore, the ET rate from NADPH/ CPR to the composite is 3.5-fold faster than that of Fe(Schiff-base)(HO)-H-., although the redox potential of Fe(10-CH2CH2COOH-Schiff-base)(HO)-H-. (-79 mV vs. NHE) is lower than that of Fe(Schiff-base)(HO)-H-. (+15 mV vs. NHE), where NHE is normal hydrogen electrode. This work describes a synthetic metal complex activated by means of a protein-protein ET system, which has not previously been reported. Moreover, the result suggests the importance of the hydrogen bond for the ET reaction of HO. Our Fe(Schiff-base)(HO)-H-. composite model system may provide insights with regard to design of ET biosystems for sensors, catalysts, and electronics devices.

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MISC 8

  1. zDHHCパルミトイル化酵素とABHD17脱パルミトイル化酵素

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

    Kanadome T, Yokoi N, Fukata Y, Fukata M

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

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  3. 抗DCC抗体関連性辺縁系脳炎と診断した猫の1例

    大谷彰平, 大西ゆみ, 小山英志, 松永悟, 中西章男, 志賀崇徳, チェンバーズ ジェームズ, 内田和幸, 横井紀彦, 深田優子, 深田正紀, 長谷川大輔

    獣医神経病学会   Vol. 44th   2018

  4. 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

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  5. ケミカルシャペロンによるタンパク質の構造異常の修復はてんかんモデルマウスにおいてけいれん感受性を軽減する

    横井紀彦, 深田優子, 深田正紀

    ライフサイエンス新着論文レビュー First Author’s     2015.1

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  6. 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

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  7. 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

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

  8. Structure-Function Relationship of the Heme Oxygenase

    Masaki Unno, Takafumi Ueno, Norihiko Yokoi, Toshitaka, Matsui, Tomoe Kawanami, Mari Iwasaki, Shusuke Kusama, Yoshihito Watanabe, Masao Ikeda-Saito

    Photon Factory Activity Report   Vol. 24 ( B ) page: 230   2006

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Presentations 6

  1. 神経修飾リガンドLGI1の変異を原因とするヒト家族性てんかんの分子病態機構 Invited

    第89回日本薬理学会年会 シンポジウム「神経機能と疾患発症に関わる新たなシグナリング機構解明への挑戦」  2016.3.10 

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  2. 神経分泌蛋白質LGI1の変異を原因とする“てんかん”の分子病態機構の解明と治療法の開拓 Invited

    横井紀彦, 深田正紀, 深田優子

    生理学研究所研究会「シナプス恒常性維持の分子基盤とその破綻」  2013.6.6 

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  3. 脳内蛋白質複合体の研究を通して、生物無機化学の未来について考えること Invited

    横井紀彦

    分子研研究会「錯体化学から始まる学術展開の可能性」  2021.3.11 

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  4. 生理的なシナプス蛋白質複合体の変異体マウスを用いた機能解析 Invited

    横井紀彦

    研究会「生物無機化学の新潮流と展望」  2013.5.25 

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  5. 水素結合による蛋白質電子伝達システムと人工金属錯体の複合機能化 Invited

    横井紀彦

    分子研研究会「金属と分子集合」  2007.6.1 

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  6. LGI1–ADAM22抗てんかん蛋白質複合体の量的制御機構の解明 Invited

    横井紀彦, 深田正紀, 深田優子

    生理研研究会「機能的神経回路の構築と動作を支える分子細胞基盤」  2022.2.4 

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KAKENHI (Grants-in-Aid for Scientific Research) 6

  1. 抗てんかん作用の発揮を目指したLGI1-ADAM22複合体の形成制御機構の解明

    Grant number:22K06451  2022.4 - 2025.3

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    横井 紀彦

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    Grant amount:\4160000 ( Direct Cost: \3200000 、 Indirect Cost:\960000 )

    30%近くのてんかんは難治性のてんかんと言われており、新たな治療戦略の創出が求められている。そのためには脳の異常興奮がどのように抑制されているのかを解明することが重要になる。我々は神経分泌蛋白質LGI1と膜蛋白質ADAM22の複合体の量が脳の異常興奮の抑制の鍵であることを見出してきた。本研究では、LGI1-ADAM22複合体形成を制御する分子機構を見出し、そして、それら分子機構を利用してLGI1-ADAM22複合体の量を増やすことで、てんかん治療効果に繋げることを目指す。
    現在、およそ30%のてんかんは難治性のてんかんと言われており、新たな治療戦略の創出が求められている。脳の異常興奮がてんかんの原因の一つと考えられていることから、てんかん治療戦略の開発には脳の興奮性の制御機構の解明が重要になる。我々は、てんかん原因遺伝子産物である神経分泌蛋白質LGI1と膜蛋白質ADAM22がシナプスにおいて複合体を形成すること、そして、それら蛋白質の変異によって複合体の量が減少し、てんかんが誘引されることを報告してきた。このことから、LGI1-ADAM22複合体の量が脳の異常興奮を抑制する鍵と着想し、複合体の量を制御する分子機構を明らかにすることで、新たなてんかん治療戦略に繋がると考えた。そして、LGI1-ADAM22複合体の量を制御する分子機構として、ADAM22のリン酸化による14-3-3との複合体形成や、ADAM22を含む1.2 MDaの巨大蛋白質複合体の形成を明らかにしてきた(Yokoi et al. Cell Rep. 2021)。本研究課題ではADAM22のリン酸化機構を利用することで、マウス脳内のLGI1-ADAM22複合体の量を制御し、てんかん治療戦略につなげることを目指す。我々はこれまでに、14-3-3に対して、ADAM22のリン酸化S832とS855が結合することを見出してきた。2022年度はS855に対するリン酸化特異的抗体を作製し、マウス脳内のS855リン酸化を検出する手段を得た。さらに、ADAM22 S832とS855に対するリン酸化酵素の活性誘導化合物を培養神経細胞やマウスへ作用させて、ADAM22のリン酸化と14-3-3との結合、そして、マウスの痙攣感受性への影響について検討を進めた。
    本研究の進展は、ADAM22のリン酸化によるLGI1-ADAM22複合体の安定化機構とてんかん発症の関係を明らかにし、新たなてんかん治療戦略につながる可能性がある。本年度作製したADAM22 Ser855特異的抗体は、これまでに作製したSer832特異的抗体と合わせて、今後のてんかん研究に対して、重要な研究ツールとなり得る。
    2023年度はこれまでに見出したADAM22のリン酸化機構に着目し、この機構を利用することで、生体(マウス)内でのADAM22、およびLGI1-ADAM22複合体の安定化を試みる。また、ADAM22と14-3-3の複合体形成によるシナプス蛋白質複合体の形成制御機構についても検討を進める。

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  2. Regulatory mechanisms of the antiepileptic ligand-receptor level to control excitation-inhibition balance in the brain

    Grant number:19K06893  2019.4 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Yokoi Norihiko

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    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    Decreased transsynaptic LGI1-ADAM22 protein complexes, because of their mutations, cause epilepsy. However, it remains unclear how LGI1-ADAM22 levels are regulated and how much LGI1-ADAM22 function is required. Here, we found that ADAM22 is stoichiometrically phosphorylated in the mouse brain. Without the phosphorylation, the amounts of the ADAM22 and LGI1 proteins were significantly decreased in the mouse brain. We demonstrated that quantitative phosphorylation of ADAM22 by protein kinase A (PKA) mediates high-affinity binding of ADAM22 to 14-3-3 and the binding prevents AP2-mediated endocytosis of ADAM22. Leveraging a series of ADAM22 and LGI1 hypomorphic mice, we found the LGI1 and ADAM22 levels 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.

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  3. Regulatory mechanisms of synaptic transmission by an epilepsy-causative ligand-receptor complex

    Grant number:17K14969  2017.4 - 2019.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Young Scientists (B)

    Norihiko Yokoi

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    Grant amount:\4420000 ( Direct Cost: \3400000 、 Indirect Cost:\1020000 )

    Balance between excitation and inhibition in neural circuits is finely tuned, and upsetting this delicate balance can cause epilepsy. Mutations in the human gene LGI1, encoding a neuronal secreted protein, cause a familial epilepsy. Previously we reported that a secretion-defective LGI1 mutant does not fold correctly and prematurely degrade, and a secretable mutant is selectively defective in binding to one of its receptors, ADAM22 (Yokoi et al. Nat Med 2015). Here we found that another secretable LGI1 R474Q mutant binds to ADAM22, but the mutation disrupts the LGI1-LGI1 homomeric dimerization in the mouse brain. This result revealed the novel pathogenic molecular mechanisms to cause epilepsy (Yamagata, Miyazaki, Yokoi et al, Nat Commun 2018).

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  4. Studies on excitatory synaptic transmission regulated by neuronal ligand/receptor interactions

    Grant number:25890021  2013.8 - 2015.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Research Activity Start-up

    NORIHIKO Yokoi

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    Grant amount:\2730000 ( Direct Cost: \2100000 、 Indirect Cost:\630000 )

    LGI1 is a neuronal secreted protein, and its mutations cause an inherited form of human epilepsy, autosomal dominant lateral temporal lobe epilepsy (ADLTE). However, the patho-physiological functions of LGI1 still remain unclear. Here, we classified 22 reported LGI1 missense ADLTE mutations as either secretion defective or secretion competent, and we generated and analyzed two mouse models of ADLTE expressing the mutant protein representative of the two groups. Both mutations reduced synaptic LGI1-ADAM22 interaction by protein conformational defects in the mouse brain. A chemical chaperone, 4-phenylbutyrate, restored the folding of the secretion-deficient LGI1 mutant and its binding to ADAM22 and ameliorated the increased seizure susceptibility of the LGI1 mutant mice. This study establishes an essential role of the LGI1-ADAM22 interaction to regulate the brain excitability (Yokoi et al. Nat. Med. 2015, 21,19-26).

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  5. 新規シナプス伝達修飾分子LGI1/ADAM22/23の構造基盤の解明

    Grant number:10J02876  2010 - 2012

    科学研究費助成事業  特別研究員奨励費

    横井 紀彦

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    LGI1は、ヒトでてんかんを引き起こす30以上の変異が報告され、また、辺縁系脳炎を引き起こす自己抗体の主要な標的であることが知られている。つまり、LGI1がヒトの神経活動に重要な役割を担うことは明白である。これまでに我々は、LGI1ノックアウト(KO)マウスが生後三週間以内にてんかん発作により全て死亡すること、LGI1がシナプス膜貫通蛋白質ADAM22、ADAM23と複合体を形成すること、そして、AMPA型グルタミン酸受容体の機能制御を行うことを見出してきた。しかしながら、LGI1の機能阻害とてんかん発症の分子病態メカニズムは未だ明らかではなかった。申請者はヒトでてんかんを引き起こすLGI1変異体の分泌活性を網羅的に検討し、分泌型と分泌阻害型に分類した。次に、生理機能への変異の影響を評価するため、分泌阻害型、分泌型変異体LGI1を発現するトランスジェニックマウスを作成した。LGI1変異体のヘテロ接合型マウスは、ペンチレンテトラゾールに対する痙攣感受性が野生型に比べ高かったことから、ヒトのてんかんモデルマウスとなることが示された。次に、マウス脳内でのLGI1変異体の機能欠損を生化学的、組織化学的に調査した結果、野生型LGI1がシナプス間隙へ分泌され、ADAM22、ADAM23と結合するのに対し、分泌不全型変異体は構造異常のために小胞体に留まり、最終的には分解されること、分泌型変異体はADAM22との結合が選択的に阻害されていることを見出した。以上の結果はLGI1の変異によって引き起こされるてんかんが、分泌不全、タンパク間相互作用不全を原因とする構造病(conformational disease)であることを示し、LGI1/ADAM22による脳の興奮性シナプス伝達制御機構の重要性が示された。さらに分泌不全型変異体の培養細胞からの分泌が、ある小分子により促されることも見出した。これは、LGI1変異によって引き起こされるてんかんの治療法に知見を与えるものである。

  6. 蛋白質三次元構造を利用した電子伝達経路の構築

    Grant number:07J03483  2007 - 2008

    科学研究費助成事業  特別研究員奨励費

    横井 紀彦

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    Authorship:Other 

    本研究では、酸化還元分子を、蛋白質複合体を基盤として配列させることで、分子間の電子伝達反応を利用した新規化学反応システム構築を試みた。天然の酵素は複数の補因子の配列により、補因子間の電子移動反応を促進させ、高活性、高効率な化学反応システムを構築している。一方、蛋白質は、分子量数万といった巨大な分子にも関わらず、単一の構造を形成し、アミノ酸化学修飾を利用して人工化合物を蛋白質の望みの位置へ固定することが可能である。従って、複数の人工酸化還元分子を化学修飾で蛋白質表面へ導入し、配列させることで、新規の化学反応システムの構築が期待できる。本研究では、チューブ構造が人工的な分子を一次元的に配列させるのに有用な土台の一つと考え、我々が作成したベータヘリックス蛋白質[(gp5βf)_3]_2の繰り返しチューブ型構造を元にした触媒分子の配列化を試みた。
    酸化還元触媒であるフラビンのサクシニミジルエステル誘導体を[(gp5βf)_3]_2へ修飾したところ、そのフラビン集積体は、フラビンの光化学反応をスイッチとするクリック反応を、遊離のフラビンや、ポリリジンへ同様に修飾されたフラビンよりも約11倍速く進行させることを発見した。これは、強固なベータヘリックス構造を鋳型としたフラビンの配列の固定化によってフラビンの反応性を促進させることができたと考えられる。また、[(gp5βf)_3]_2のK41システイン変異体を作成し、そのシステインに対してRe(bpy)(CO)_3Cl錯体を、リシンにはRu(bpy)_3をそれぞれ修飾した。この複合体K41C_Re_<Cys>Ru_<NH>は、錯体の混合溶液と比べ、可視光照射下でのCO生成触媒反応を2.9倍多く進行させ、[(gp5βf)_3]_2が錯体間電子移動反応の鋳型分子として有用と示された。以上のように本研究では、ベータヘリックス構造を基盤とした機能分子の配列が、その分子の機能の制御に繋がる可能性を示すことに成功した。

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Teaching Experience (On-campus) 4

  1. 生物と薬物 実習

    2023

  2. 基礎セミナーB

    2023

  3. 基礎医学セミナー

    2023

  4. 生物と薬物 演習・学生発表

    2023

Teaching Experience (Off-campus) 3

  1. 分子細胞生理学I

    2023.6 The Graduate University for Advanced Studies)

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    Level:Postgraduate  Country:Japan

  2. 分子細胞生理学I

    2019.5 The Graduate University for Advanced Studies)

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    Level:Postgraduate  Country:Japan

  3. 細胞神経生物学

    2014.11 The Graduate University for Advanced Studies)

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    Level:Postgraduate  Country:Japan

 

Social Contribution 1

  1. 記憶をつくるタンパク質

    Role(s):Lecturer

    岡崎市教育委員会  出前授業、岡崎市立額田中学校  愛知県岡崎市額田中学校  2017.10

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    Audience: Junior students

    Type:Visiting lecture