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 (NH₂OH), 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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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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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

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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Language：English   Publisher：Elsevier Ltd  

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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Language：English   Publisher：ELSEVIER IRELAND LTD  

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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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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

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

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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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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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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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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/smll.201000772

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/smll.200700855

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

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.

DOI： 10.1039/b713468a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

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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DOI： 10.1016/j.ccr.2007.04.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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.

DOI： 10.1073/pnas.0510968103

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Language：Japanese   Publisher：オーム社  

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

Language：English   Publishing type：Article, review, commentary, editorial, etc. (bulletin of university, research institution)  

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Presentation type：Oral presentation (invited, special)  

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