{"created":"2023-06-20T13:23:16.955660+00:00","id":4063,"links":{},"metadata":{"_buckets":{"deposit":"81881dd9-391e-44a6-9ac8-ff9f92333e97"},"_deposit":{"created_by":21,"id":"4063","owners":[21],"pid":{"revision_id":0,"type":"depid","value":"4063"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00004063","sets":["2:428:15"]},"author_link":["2240","2238","2239"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"中村, 健介"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"ナカムラ, ケンスケ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2013-03-22"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"In order to gain insights into regulations of various membrane structures in cell, structural \nand functional studies were carried out on two target proteins: Arfaptin, a protein \ninvolved in tubule formation at trans-Golgi network; and Atg16L, a protein involved \nin formation of autophagosomes.\nThe BAR (Bin/Amphiphysin/Rvs) domain forms a curved helical homodimer that can\nsense or induce the curvature of the membrane it associate with. BAR domain binds to acidic\nmembranes with its basic concaved face in a rather nonspecific manner. This is in contrast to\nother membrane-binding domains, many of which recognize the head groups of specific\nphospholipids. BAR domains are often found in conjunction with other membrane-binding\nmodules, such as a PH (pleckstrin homology) domain and a PX (phox homology) domain,\nwhich define spatial and functional specificities in membrane association. For instance, many\nmembers of the Sorting Nexin (SNX) family, including SNX9, have two membrane-binding\nproperties attributable to its curvature sensing BAR domain and phosphoinositide-binding PX\ndomain; this allows for the selective targeting to high curvature sub-domains of endosomal\ncompartments enriched in specific phosphopnositides. Upon associating with target\nmembranes, the SNX PX-BAR domain drives membrane tubulation for tubule-based sorting.\nBAR domain-containing protein Arfaptin functions in both tubule formation and\nstabilization; Arfaptin-1 stabilizes the fission sites of secretory granules at the trans-Golgi\nnetwork; Arfaptin-2 induces the membrane tubules in Golgi-membranes. The two isoforms\nhave amino acid sequence similarity of 68% and share similar domain structures. Both\nisoforms localize in the Golgi region, when exogenously expressed in cells, through the\nassociation with small GTPase Arf-like 1 (Arl1). Arfaptin-2 colocalizes with Arl1 on dynamic\nvesicular and tubular structures emanating from the Golgi, suggesting that Arl1 regulates\nArfaptin-mediated membrane deformation at the trans-Golgi.\nThe two isoforms of Arfaptin are similarly affected by Arl1, but they associate with\ndifferent regulators. Arfaptin-2, but not Arfaptin-1, associates with another small GTPase\nRac1. The crystal structures of the Rac1–Arfaptin-2 BAR domain complex, reported by\nTarricone, et al, show how Rac1 binds to the concaved face of the Arfaptin-2 BAR\nhomodimer, in the way that would interfere with the membrane-association of the BAR\ndomain. In addition, Rac1 also interferes with the Arf-association of Arfaptin-2. Hence, the\nfunction of Arfaptin-2 is intricately regulated by the crosstalk between the small GTPases,\nArf/Arl1 and Rac1. However, the molecular basis of this crosstalk remained unrevealed, for\nthe complex structure of Arl1–Arfaptin-2 was not known.\nThe crystal structures were determined for Arfaptin-1 in free form and Arfaptin-1 or\nArfaptin-2 in complex with Arl1. In the complex structures, two molecules of Arl1 were\nsymmetrically bound on each side of the crescent-shaped homodimer of Arfaptin BAR\ndomain, leaving the concave face open for membrane association. This conformation provided\nthe structural basis for recruitment of Arfaptins onto Golgi membranes by Arl1. Arl1 and\nRac1, another binding partner of Arfaptin-2, bound to Arfaptin-2 with the mM-order\ndissociation constants in Surface Plasmon Resonance (SPR) experiments. Structural\ncomparison between Rac1–Arfaptin-2 and Arl1–Arfaptin-2, combined with SPR experiments,\nindicated that Rac1 interferes with the one of two molecules of Arl1 on Arfaptin-2. These\nresults provided structural basis for the recruitment of Arfaptins onto Golgi membranes by\nArl1 and the crosstalk between Arf/Arl1 and Rac1.\nAutophagy is the basic catabolic process in Eukaryotic cells. The process involves unique\ntrafficking event in which cytoplasmic constituents are isolated and delivered from the\ncytoplasm to lysosome for degradation. In addition to the basic roles in catabolism, the\npathway is also utilized in the elimination of intracellular pathogen and presentation of antigen\nin Mammal. There are three different types of autophagy: chaperone-mediated autophagy,\nmicroautophagy, and macroautophagy. In the chaperone-mediated autophagy, degradation\ntargets that contain specific motif are recognized and translocated into lysosomes by\nchaperone heat shock cognate 70 (Hsc70) and the receptor lysosome-associated membrane\nprotein 2A (LAMP2A). In the microautophagy, lysosome directly engulfs the cytoplasmic\ncomponents. In the macroautophagy, cellular constituents are isolated by the transient\ndouble-membrane–bound structure called autophagosome and delivered to lysosome by the\nfusion of the two membrane-bound structures.\nThe autophagosomes originate from a small single-layer membrane structure termed\nPrecursor Membrane Structure (PAS). Through hemi-fusions with additional membrane\nstructures, PAS develops into a flat double-membrane structure, termed the isolation\nmembrane, which expands and enwraps portions of the cytoplasm. As their ends meet, it\nresults in a large double-membrane vesicle, or an autophagosome, that isolates the degradation\ntargets. The maturation of autophagosome involves an ubiquitin-like conjugation system in\nAutophagy-related protein (Atg) family; in which LC3, a mammalian homologue of yeast\nAtg8, is conjugated to phosphatidylethanolamine (PE) by E3-like ligase Atg16(L) complex.\nEthanolamine-conjugated LC3 and its orthologue GATE-16, as well as the yeast Atg8,\npromote tethering and hemi-fusion of membranes in vitro. Thus the conjugation system is\nconsidered essential for the autophagosome maturation. Atg16L complex is suggested to\nfunction during the elongation step, as the complex dissociates from the membrane upon the\ncompletion of autophagosome.\nThe structure and function of those Atg proteins are extensively studied in yeast. Multiple\ncrystal structures are determined for the fragments of the yeast Atg16 complex, which consists\nof Atg5-Atg12 conjugate and Atg16. The model of the overall structure has been proposed by\nFujioka, et al; Atg5-Atg12 conjugate binds to N-terminal helix of Atg16, which dimerizes\nthrough its C-terminal coiled-coil domain to form a complex that contains two copies of each\nof the three Atg proteins. The complex associates with membranes through Atg5, and induce\nhomotypic tethering of vesicles in vitro even in the absence of Atg8. It has confirmed in vivo\nand in vitro that Atg16 is required for the efficient conjugation of LC3 and PE. Moreover, the\ndimerization of Atg16 at the coiled-coil domain is reported to be essential for autophagy in\nvivo.\nThe corresponding protein complex in Mammal consists of Atg5-Atg12 conjugate and\nAtg16L; thus named Atg16L complex. The mammalian Atg16L complex is similar to the\nyeast Atg16 complex: It associates with membranes through Atg5; dimerizes through the\ncoiled-coil domain of Atg16L; and has E3-ligase activity toward LC3, the homologue of Atg8.\nHowever, Atg16L is significantly different from the yeast Atg16. It binds to small GTPase\nRab33 at the region following the coiled-coil domain, and also has an additional WD40\ndomain the C-terminus. Recent reports identified a single nucleotide polymorphism in\nN-terminus of the WD40 domain that is associated with inflammatory Crohn's disease. Thus,\nthose additional regions of Atg16L may be involved in the distinct mammalian-autophagic\nfunctions that are absent in yeast.\nThe mammalian Atg16L directly interacts with Rab33b, the Golgi-resident small GTPase\ninvolved in Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking. Rab33b\ninteracts with Atg16L, at the region following the coiled-coil region (residues 80-200), and\nrecruits not only Atg16L but also Atg12, which does not directly bind Rab33b. This indicates\nthat Rab33b is able to recruit the entire Atg16L complex. Indeed, the expression of\nRab33b-binding domain of Atg16L strongly inhibited autophagosome formation. However,\nthe molecular mechanism of this recruitment and subsequent function of Atg16L complex\nremained unrevealed, for the structure of the mammalian Atg16L and its complex with\nAtg5-Atg12 is not yet known.\nThe X-ray crystal structure was determined for the coiled-coil domain of mouse Atg16L;\nwhich had an antiparallel coiled-coil dimer in contrast to the yeast parallel Atg16. The\ndimerization interface had hydrophobic interactions and hydrogen bonds at the residues that\nwere not conserved between mammal and yeast. These findings suggested that the overall\nstructure of mammalian Atg16L complex is quite distinct form that of the yeast Atg16\ncomplex.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第1586号 ","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"高エネルギー加速器科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"13 物質構造科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2012"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"NAKAMURA, Kensuke","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-26"}],"displaytype":"simple","filename":"甲1586_要旨.pdf","filesize":[{"value":"325.0 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/4063/files/甲1586_要旨.pdf"},"version_id":"e943d56a-a621-43ed-b42f-b1e39bc7e444"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Structural and biochemical studies on protein complexes regulating membrane traffic","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Structural and biochemical studies on protein complexes regulating membrane traffic"},{"subitem_title":"Structural and biochemical studies on protein complexes regulating membrane traffic","subitem_title_language":"en"}]},"item_type_id":"1","owner":"21","path":["15"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-11-15"},"publish_date":"2013-11-15","publish_status":"0","recid":"4063","relation_version_is_last":true,"title":["Structural and biochemical studies on protein complexes regulating membrane traffic"],"weko_creator_id":"21","weko_shared_id":21},"updated":"2023-06-20T15:16:01.221719+00:00"}