担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Brain Communications  

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 > 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 > 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

Open Access

Web of Science

Scopus

PubMed

その他リンク： https://academic.oup.com/braincomms/article-pdf/5/6/fcad295/53898834/fcad295.pdf

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbc.2022.102048

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

出版者・発行元：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

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.neuropharm.2021.108628

PubMed

掲載種別：研究論文（学術雑誌）   出版者・発行元：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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：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, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. <italic>Adam22</italic>^{<italic>ΔC5/ΔC5</italic>} 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

その他リンク： https://syndication.highwire.org/content/doi/10.1073/pnas.2022580118

掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-020-16883-5

その他リンク： http://www.nature.com/articles/s41467-020-16883-5

掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA119.010491

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/ana.25666

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

PubMed

DOI： 10.1038/s41589-019-0399-y

掲載種別：研究論文（学術雑誌）  

DOI： 10.1126/sciadv.aax0821

PubMed

担当区分：最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/978-1-4939-9532-5_7

PubMed

担当区分：責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media LLC  

DOI： 10.1038/s41467-018-03947-w

PubMed

その他リンク： http://www.nature.com/articles/s41467-018-03947-w

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Scopus

PubMed

担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：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

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

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

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CURRENT BIOLOGY LTD  

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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroscience  

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

Web of Science

Scopus

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

DOI： 10.1212/NXG.0000000000000046

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/bs.ctm.2015.10.003

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Molecular Biology of the Cell  

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

Web of Science

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC CLINICAL INVESTIGATION INC  

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

Web of Science

PubMed

担当区分：筆頭著者,　最終著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pone.0114905

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroscience  

Autoimmune forms of encephalitis have been associated with autoantibodies against synaptic cell surface antigens such as NMDA- and AMPA-type glutamate receptors, GABAB receptor, and LGI1. However, it remains unclear how many synaptic autoantigens are yet to be defined. Using immunoproteomics, we identified autoantibodies against theGABAA receptor inhumansera 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 β3 subunit of the GABAA receptor. The β3-subunit-containing GABAA 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 theβ3 subunit was knocked down. Our developed multiplex ELISA testing showed that both patients had similar levels of GABAA 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 ofsymptompresentation of encephalitis to rat hippocampal neuron cultures specifically decreased both synaptic and surface GABAA 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' GABAA receptor antibodies play a central role in the patients' symptoms. Therefore, this study establishes anti-GABAA receptor encephalitis and expands the pathogenic roles of GABAA receptor autoantibodies. © 2014 the authors.

DOI： 10.1523/JNEUROSCI.4415-13.2014

Web of Science

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

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.

DOI： 10.1242/dev.107995

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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.

DOI： 10.1113/jphysiol.2013.252122

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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.

DOI： 10.1074/jbc.M112.431676

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.201302071

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

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.

DOI： 10.1021/ja311416v

Web of Science

PubMed

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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.

DOI： 10.1111/j.1460-9568.2012.08129.x

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M112.348094

Web of Science

PubMed

記述言語：英語   掲載種別：論文集(書籍)内論文   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pgen.1002194

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC CELL BIOLOGY  

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.

DOI： 10.1091/mbc.E10-10-0824

Web of Science

PubMed

担当区分：責任著者   記述言語：英語   出版者・発行元：NATURE PUBLISHING GROUP  

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.

DOI： 10.1038/nrn2788

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

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

DOI： 10.1038/emboj.2009.325

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC NEUROSCIENCE  

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.

DOI： 10.1523/JNEUROSCI.1863-09.2009

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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.

DOI： 10.1371/journal.pgen.1000748

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.200903101

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   出版者・発行元：PERGAMON-ELSEVIER SCIENCE LTD  

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.

DOI： 10.1016/j.plipres.2009.02.001

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC CELL BIOLOGY  

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.

DOI： 10.1091/mbc.E08-09-0944

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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.

DOI： 10.1074/jbc.M808920200

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC MICROBIOLOGY  

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.

DOI： 10.1128/MCB.01144-08

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

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.

DOI： 10.1007/s00424-008-0465-x

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC NEUROSCIENCE  

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.

DOI： 10.1523/JNEUROSCI.2171-08.2008

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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.

DOI： 10.1074/jbc.M802140200

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.200702054

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America  

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 α-tubulin. Notably, the p150Glued CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the α-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 α-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 p150Glued and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end. © 2007 by The National Academy of Sciences of the USA.

DOI： 10.1073/pnas.0703876104

Web of Science

Scopus

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

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.

DOI： 10.1016/j.bbamem.2007.01.009

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1242/jcs.03356

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC NEUROSCIENCE  

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.

DOI： 10.1523/JNEUROSCI.4214-06.2006

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.ymeth.2006.05.015

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE  

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.

DOI： 10.1126/science.1129947

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.200601051

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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.

DOI： 10.1016/j.immuni.2006.03.011

Web of Science

PubMed

J-GLOBAL

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.200503011

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROCKEFELLER UNIV PRESS  

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.

DOI： 10.1083/jcb.200501121

Web of Science

PubMed

J-GLOBAL

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

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.

DOI： 10.1016/j.neuron.2004.12.005

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Developmental Cell  

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. Copyright © 2004 Cell Press.

DOI： 10.1016/j.devcel.2004.10.017

Web of Science

Scopus

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE  

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.

DOI： 10.1126/science.1090262

Web of Science

PubMed

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Molecular Biology of the Cell  

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 β-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 β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II 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 β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of "E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion".

DOI： 10.1091/mbc.E03-08-0582

Web of Science

Scopus

PubMed

J-GLOBAL

担当区分：筆頭著者   記述言語：英語   出版者・発行元：Current Opinion in Cell Biology  

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.

DOI： 10.1016/S0955-0674(03)00097-8

Web of Science

Scopus

PubMed

J-GLOBAL

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cell  

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 IQGAP1, 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.

DOI： 10.1016/S0092-8674(02)00800-0

Web of Science

Scopus

PubMed

J-GLOBAL

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Methods in molecular biology (Clifton, N.J.)  

DOI： 10.1385/1-59259-281-3:121

Scopus

PubMed

担当区分：筆頭著者   記述言語：英語   出版者・発行元：Nature Reviews Molecular Cell Biology  

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.

DOI： 10.1038/35103068

Web of Science

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Web of Science

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Molecular and Cellular Biology  

We have previously proposed that IQGAP1, an effector of Rac1 and Cdc42, negatively regulates cadherin-mediated cell-cell adhesion by interacting with β-catenin and by causing the dissociation of α-catenin from cadherin-β-catenin-α-catenin complexes and that activated Rac1 and Cdc42 positively regulate cadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with β-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 α-catenin, we found that EGFP-α-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-β-catenin complexes increased during action of TPA. Constitutively active Rac1 and IQGAP1 carboxyl terminus, a putative dominant-negative mutant of IQGAP1, inhibited the disappearance of α-catenin from sites of cell-cell contact induced by TPA. Taken together, these results indicate that α-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 α-catenin relocalization.

DOI： 10.1128/MCB.21.6.2165-2183.2001

Web of Science

Scopus

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

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.

Web of Science

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biochemical and Biophysical Research Communications  

Cadherin is a well-known cell-cell adhesion molecule, and it binds to β-catenin, which in turn binds to α-catenin. However, little is known about the regulatory mechanism underlying the cadherin-mediated cell-cell adhesion. Here we purified two novel β-catenin-interacting proteins with molecular masses of 180 kDa (p180) and 150 kDa (p150) from bovine brain cytosol by using glutathione S-transferase (GST)-β-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 β-catenin and KIAA0313 in the yeast two-hybrid system and overlay assay, respectively. β-Catenin was coimmunoprecipitated with KIAA0313 in Madin-Darby canine kidney II (MDCKII) cells, bovine brain cytosol, and EL cells. KIAA0313 and β-catenin were partly colocalized at sites of cell-cell contact in MDCKII cells. Taken together, our data suggest that KIAA0313 associates with β-catenin through KIAA0705 in vivo at sites of cell-cell contact. (C) 2000 Academic Press.

DOI： 10.1006/bbrc.2000.3002

Web of Science

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Genes to Cells  

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)/ROKα/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. and 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 (MACK) 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 MaCK inhibited both the formation of filopodia and accumulation of C-terminal threonine- phosphorylated ERM proteins at filopodin induced by Cdc42(V12). Conclusion: The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MaCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.

DOI： 10.1046/j.1365-2443.2000.00348.x

Web of Science

Scopus

担当区分：筆頭著者   記述言語：英語   出版者・発行元：COMPANY OF BIOLOGISTS LTD  

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.

Web of Science

記述言語：英語   出版者・発行元：CURRENT BIOLOGY LTD  

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.

DOI： 10.1016/S0955-0674(99)00014-9

Web of Science

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biological Chemistry  

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with β-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating α-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, L., Nagase, T., Nomura, N., Tani, H., Shoji, L., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832-835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of β-catenin, which contains the α-catenin-binding domain. IQGAP1 dissociated α-catenin from the β-catenin-α-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTPγS)·glutathione S-transferase (GST)Cdc42 and GTPγS·GST-Rac1 inhibited the binding of IQGAP1 to β-catenin in a dose-dependent manner in vitro, whereas neither GDP·GST-Cdc42, GDP·GST-Rac1, nor GTPγS·GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of α-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 Rac1 resulted in the reduction of E- cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with β-catenin, leading to stabilization of the cadherin-catenin complex.

DOI： 10.1074/jbc.274.37.26044

Web of Science

Scopus

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

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

記述言語：日本語   出版者・発行元：(公社)日本生化学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER ASSOC ADVANCEMENT SCIENCE  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

担当区分：責任著者   記述言語：日本語   出版者・発行元：オーム社  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2010195866

記述言語：日本語  

CiNii Books

担当区分：責任著者  

記述言語：日本語   出版者・発行元：日本生化学会  

CiNii Books

担当区分：最終著者,　責任著者   記述言語：日本語   出版者・発行元：共立出版  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2007209543

担当区分：最終著者,　責任著者   記述言語：日本語   出版者・発行元：共立出版  

CiNii Books

記述言語：日本語   出版者・発行元：日本組織細胞化学会  

CiNii Books

CiNii Research

記述言語：日本語   出版者・発行元：愛知県医師会  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2004099260

担当区分：筆頭著者  

担当区分：筆頭著者   記述言語：日本語   出版者・発行元：学研メディカル秀潤社  

CiNii Books

その他リンク： http://search.jamas.or.jp/link/ui/2001189016

担当区分：筆頭著者  

担当区分：筆頭著者   記述言語：日本語   出版者・発行元：秀潤社  

CiNii Books