Title: Synaptic scaffolding molecule (S-SCAM) membrane-associated guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion molecules at inhibitory synapses in rat hippocampal neurons Sumita K, Sato Y, Iida J, Kawata A, Hamano M, Hirabayashi S, Ohno K, Peles E, Hata Y Ref: Journal of Neurochemistry, 100:154, 2007 : PubMed
Synaptic scaffolding molecule (S-SCAM) is a synaptic protein, which harbors five or six PSD-95/Discs large/ZO-1 (PDZ), a guanylate kinase and two WW domains. It interacts with NMDA receptor subunits, neuroligin and beta-catenin, and is involved in the accumulation of neuroligin at excitatory synapses. In this study, we have demonstrated S-SCAM is localized at inhibitory synapses in rat primary cultured hippocampal neurons. We have identified beta-dystroglycan (beta-DG) as a binding partner for S-SCAM at inhibitory synapses. WW domains of S-SCAM bind to three sequences of beta-DG. We have also revealed that S-SCAM can interact with neuroligin 2, which is known to be exclusively localized at inhibitory synapses. The WW domains and the second PDZ domain of S-SCAM are involved in the interaction with neuroligin 2. Beta-DG, neuroligin 2 and S-SCAM form a tripartite complex in vitro. Neuroligin 2 is detected in the immunoprecipitates by anti-beta-DG antibody from rat brain. S-SCAM, beta-DG and neuroligin 2 are partially co-localized in rat hippocampal neurons. These data suggest that S-SCAM is associated with beta-DG and neuroligin 2 at inhibitory synapses, and functions as a linker between the dystrophin glycoprotein complex and the neurexin-neuroligin complex.
The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.
Title: Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction Mima J, Hayashida M, Fujii T, Narita Y, Hayashi R, Ueda M, Hata Y Ref: Journal of Molecular Biology, 346:1323, 2005 : PubMed
Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.
Synaptic scaffolding molecule (S-SCAM) is a synaptic protein that consists of PDZ domains, a guanylate kinase domain, and WW domains. It interacts with N-methyl-d-aspartate receptor subunits, neuroligin, and beta-catenin. Here, we identified Axin as a novel binding partner of S-SCAM. Axin was co-immunoprecipitated with S-SCAM from rat brain, detected in the post-synaptic density fraction in rat brain subcellular fractionation, and partially co-localized with S-SCAM in neurons. The guanylate kinase domain of S-SCAM directly bound to the GSK3beta-binding region of Axin. S-SCAM formed a complex with beta-catenin and Axin, but competed with GSK3beta for Axin-binding. Thereby, S-SCAM inhibited the Axin-mediated phosphorylation of beta-catenin by GSK3beta.
Title: Synaptic scaffolding molecule is involved in the synaptic clustering of neuroligin Iida J, Hirabayashi S, Sato Y, Hata Y Ref: Molecular & Cellular Neurosciences, 27:497, 2004 : PubMed
S-SCAM has a similar molecular organization to PSD-95. Both of them interact with a cell adhesion molecule, neuroligin. We previously reported that beta-catenin binds S-SCAM and recruits it to synapses. We have here examined using rat primary cultured neurons whether neuroligin recruits S-SCAM to synapses or S-SCAM determines the localization of neuroligin. Overexpressed neuroligin formed larger clusters under co-expression of S-SCAM but not of PSD-95. Overexpressed neuroligin blocked synaptic accumulation of PSD-95 but not of S-SCAM. S-SCAM mutant containing the neuroligin-binding region interfered with synaptic accumulation of neuroligin and PSD-95, whereas the similar mutant of PSD-95 had no effect. Biochemical studies revealed that neuroligin forms a ternary complex with S-SCAM and PSD-95 through manifold interactions. These findings imply that S-SCAM is tethered by beta-catenin to synapses and induces synaptic accumulation of neuroligin, which subsequently recruits PSD-95 to synapses.
Title: Crystallization and preliminary X-ray analysis of carboxypeptidase Y inhibitor IC complexed with the cognate proteinase Mima J, Hayashida M, Fujii T, Hata Y, Hayashi R, Ueda M Ref: Acta Crystallographica D Biol Crystallogr, 60:1622, 2004 : PubMed
Carboxypeptidase Y (CPY) inhibitor I(C) is a naturally occurring serine carboxypeptidase inhibitor from Saccharomyces cerevisiae, the sequence of which is not homologous with any other known proteinase inhibitor and is classified as the phosphatidylethanolamine-binding protein (PEBP). I(C) has been crystallized in complex with the deglycosylated form of CPY by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant. The crystals of the complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 81.13, b = 186.6, c = 65.14 A. Diffraction data were collected to 2.7 A resolution. Structure determination of the complex is in progress by the molecular-replacement method using the structure of CPY as a search model.
Synaptic scaffolding molecule (S-SCAM) is a synaptic membrane-associated guanylate kinase with inverted domain organization (MAGI) that interacts with NMDA receptor subunits and neuroligin. In epithelial cells, the non-neuronal isoform of S-SCAM (MAGI-1) is localized at tight or adherens junctions. Recent studies have revealed that the polarized targeting of MAGI-1 to the lateral membrane is mediated by its C-terminal region and that MAGI-1 interacts with beta-catenin in epithelial cells. In this article, we report that S-SCAM interacts with beta-catenin in neurons. beta-Catenin is coimmunoprecipitated with S-SCAM from rat brain. Both S-SCAM and beta-catenin are localized at synapses and are partially colocalized. The C-terminal region of S-SCAM binds to the C-terminal region of beta-catenin. We have tested how the interaction between S-SCAM and beta-catenin plays a role in the synaptic targeting of S-SCAM and beta-catenin. S-SCAM is targeted to synapses via the C-terminal postsynaptic density-95/Dlg-A/ZO-1 (PDZ) domain. beta-Catenin is targeted to synapses with armadillo repeats. The overexpressed C-terminal region of beta-catenin blocks the synaptic targeting of S-SCAM. The overexpressed C-terminal region of S-SCAM is partially targeted to synapses and forms a small number of clusters. In the presence of overexpressed beta-catenin, the C-terminal region of S-SCAM forms more clusters at synapses. These data suggest that the synaptic targeting of S-SCAM is mediated by the interaction with beta-catenin.
Title: Interaction of S-SCAM with neural plakophilin-related Armadillo-repeat protein/delta-catenin Ide N, Hata Y, Deguchi M, Hirao K, Yao I, Takai Y Ref: Biochemical & Biophysical Research Communications, 256:456, 1999 : PubMed
Synaptic scaffolding molecule (S-SCAM) is a multiple PDZ domain-containing protein, which interacts with neuroligin, a cell adhesion molecule, and the NMDA receptor. In this study, we searched for S-SCAM-interacting proteins and obtained a neuralplakophilin-related armadillo-repeat protein (NPRAP)/delta-catenin. NPRAP/delta-catenin bound to the last PDZ domain of S-SCAM via its carboxyl-terminus in three different cell-free assay systems, was coimmunoprecipitated with S-SCAM from rat crude synaptosomes, and was localized at the excitatory synapses in rat hippocampal neurons. NPRAP/delta-catenin may be implicated in the molecular organization of synaptic junctions through the interaction with S-SCAM.
Title: nRap GEP: a novel neural GDP/GTP exchange protein for rap1 small G protein that interacts with synaptic scaffolding molecule (S-SCAM) Ohtsuka T, Hata Y, Ide N, Yasuda T, Inoue E, Inoue T, Mizoguchi A, Takai Y Ref: Biochemical & Biophysical Research Communications, 265:38, 1999 : PubMed
Synaptic scaffolding molecule (S-SCAM) has six PDZ domains through which it interacts with N-methyl-d-aspartate receptors and neuroligin at synaptic junctions. We isolated here a novel S-SCAM-binding protein. This protein has one PDZ, one Ras association, one Ras GDP/GTP exchange protein (Ras GEP) domain, and one C-terminal consensus motif for binding to PDZ domains. We named it nRap GEP (neural Rap GEP). nRap GEP moreover has an incomplete cyclic AMP (cAMP)-binding (CAB) domain. The domain organization of nRap GEP is similar to that of Epac/cAMP-guanine nucleotide exchange factor (GEF) I, except that Epac/cAMP-GEFI has complete CAB and Ras GEP domains but lacks the other two domains and the C-terminal motif. nRap GEP showed GEP activity for Rap1 but did not bind cAMP. nRap GEP was specifically expressed in rat brain. Immunohistochemical analysis revealed that nRap GEP and S-SCAM were localized at synaptic areas of the cerebellum. These results suggest that nRap GEP is a novel neural Rap1-specific GEP which is associated with S-SCAM.
At synaptic junctions, pre- and postsynaptic membranes are connected by cell adhesion and have distinct structures for specialized functions. The presynaptic membranes have a machinery for fast neurotransmitter release, and the postsynaptic membranes have clusters of neurotransmitter receptors. The molecular mechanism of the assembly of synaptic junctions is not yet clear. Pioneering studies identified postsynaptic density (PSD)-95/SAP90 as a prototypic synaptic scaffolding protein to maintain the structure of synaptic junctions. PSD-95/SAP90 belongs to a family of membrane-associated guanylate kinases and binds N-methyl-D-aspartate receptors, potassium channels, and neuroligins through the PDZ domains and GKAP/SAPAP/DAP through the guanylate kinase (GK) domain. We performed here a yeast two-hybrid screening for SAPAP-interacting molecules and identified a novel protein that has an inverse structure of membrane-associated guanylate kinases with an NH2-terminal GK-like domain followed by two WW and five PDZ domains. It binds SAPAP through the GK-like domain and NMDA receptors and neuroligins through the PDZ domains. We named this protein S-SCAM (synaptic scaffolding molecule) because S-SCAM may assemble receptors and cell adhesion proteins at synaptic junctions.
PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.
Title: Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. An alpha/beta hydrolase structure that is different from the alpha/beta hydrolase fold Hisano T, Hata Y, Fujii T, Liu JQ, Kurihara T, Esaki N, Soda K Ref: Journal of Biological Chemistry, 271:20322, 1996 : PubMed
L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.
Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.
