{"created":"2023-06-20T13:20:37.836115+00:00","id":669,"links":{},"metadata":{"_buckets":{"deposit":"b147deed-67f9-4d10-ba82-15ff75e3c8e9"},"_deposit":{"created_by":1,"id":"669","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"669"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000669","sets":["2:428:15"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"CHAVAS, Leonard M. G."}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"シャバス, レオナルド ミッシェル ガブリエル"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2005-09-30"}]},"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":"Structure Determination of Human Cytoplasmic Neu2 <br /><br />　To date, l3 mammalian sialidases have been cloned including four from humans. So far, only some bac-terial and viral neuraminidase's structures are known, nothing from human or mammals. In this study, the crystal structure of the human cytosolic sialidase Neu2 was determined at the atomic level, either in an apo form or in complex with diverse inhibitors of substrate of reaction. <br /><br />　The core of the enzyme folds as a six-bladed β-propeller with 26 β-strands and five α-helices, typical of viral and bacterial sialidases. Neu2 adopts an irregular six-bladed structure caused by β-bulges of the third strand, as well as longer β-stands compared to the \"classical\" propeller foldings. <br /><br />　In the Neu2-DANA complex structure, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) lies in a half-chair conformation interacting with 10 amino acids of the active site. Interaction between Neu2 and the inhibitor DANA shows similarities with bacterial and viral counterparts but also exhibits some differences in the active site arrangement and dynamic nature of the loops containing residues respon-sible for catalysis and substrate recognition An acidic crevice at the center of the β-propeller forms an activating core for the enzymatic catalysis and the basic residues at the mouth of the crevice coordinate substrates. <br /><br />　Two loops are disordered on the top side of the apo form Neu2: one contains Glu<SUP>111</SUP> important for the substrate binding, and the other Asp<SUP>46</SUP> for the catalysis. Soaking the apo form crystals with monosaccharide such as galactose, glucose, or maltose orders the former loop into α2, illustrating a plausible two steps model for a dynamic in the substrate recognition.<br /><br />　In the Neu2 structures, two Asp-boxes STDHGRTW (residues 129-136) and SHDHGRTW (residues 199-206) are easily identified (where boldface letters indicate residues structurally important for Asp-boxes). A third Asp-box STNDGLDF (residues 248-255) was found to have similar folding property comparing with the two firsts. The sequence of the third Asp-box in Neu2 extends the consensus se-quence of Asp-boxes as Ser-X-(Asp/Gln)-X-Gly-X-(Thr/Asp)-〓, where 〓stands for aromatic residues. A new screening may therefore give further insights into the function of these structural elements. <br /><br />　Finally, different crystal forms of the sialidase have been refined in order to facilitate inhibitors screenings. The influenza virus neuraminidase plays a critical role in the life cycle of the virus and has been the focus of new drug developments. Drug design of new agents against the viral sialidase comes from the fact that whereas diverse kinds of influenza virus neuraminidases were identified, their catalytic site is completely conserved among all influenza subtypes in terms of amino acids organization. To date, no data exist on whether the human sialidases can interact as well with viral and/or bacterial targeting drugs. To answer this question, complex structures of Neu2 with overall six different influenza virus in-hibitors have been solved, illustrating the inhibitors lying in the human sialidase active site coordinated in a similar way. By comparing tle recognition mechanism of each inhibitor, in Neu2 or in influenza virus neuraminidase, new drug design studies might be started in order to preferentially recognize either human or viral sialidases. <br /><br /><br />　Biochemical and Crystallographic Studies on Rab27a/b and Their Effectors <br /><br />　The integrity of eukaryotic cells stongly depends on membrane traffic, which is tightly linked to the regulation by a number of protein families such as Ras, Rho, Rab and Ran. Those proteins bind to var-ious types of effector proteins, and execute versatile functions. More precisely, the Rab27 subfamily, consisting of Rab27a and Rab27b, is directly implicated in the hansport of lysosome-related organelles, such as melanosomes in melanocytes or lytic granules in cytotoxic T-lymphorytes. In order to clarify the regulation process of such transport phenomena, biochemical and crystallization experiments have been carried out on both Rab27a/b, alone and in complex with two effectors Slac2-a and Slp4-a. <br /><br />　The individual GTPases, Rab27a and Rab27b, were purified for suitable crystallization experiments, both in their active (GTP-bound) or inactive (GDP-bound)forms. Extensive crystallization screenings have been performed, resulting in the apparition of some protein diffracting crystals in diverse conditions for Rab27b in its inactive form. The crystals grew in a tetragonal space group but did not diffract at higher resolution than 4.2 Å. Seleno-methionine substituted Rab27b protein was then purified and crystallized, but the crystal's diffraction limit did not improve. Further refinements in terms of protein deletion mutants and crystallization are now under way in order to obtain highly diffracting crystals. <br /><br />　The complexes between the GTPases and their effectors, respectively Slac2-a and Slp4-a, could be generated and purified, and have been subjects of intensive crystallization trials with the help of a crystallization robot. Unfortunately, to date no protein crystals suitable for X-ray structure determination have been obtained. ","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899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":"2005"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"CHAVAS, LEONARD M. G.","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"0","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲899_要旨.pdf","filesize":[{"value":"274.2 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/669/files/甲899_要旨.pdf"},"version_id":"3629ede5-8290-437b-8dc8-ec69eae59089"}]},"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 Glycoconjugate Alteration and Vesicle Transport","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Structural and Biochemical Studies on Glycoconjugate Alteration and Vesicle Transport"},{"subitem_title":"Structural and Biochemical Studies on Glycoconjugate Alteration and Vesicle Transport","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["15"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"669","relation_version_is_last":true,"title":["Structural and Biochemical Studies on Glycoconjugate Alteration and Vesicle Transport"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:12:11.904252+00:00"}