{"_buckets": {"deposit": "d178b5c7-53b4-4a23-8fb1-2727ea5a3a74"}, "_deposit": {"created_by": 1, "id": "990", "owners": [1], "pid": {"revision_id": 0, "type": "depid", "value": "990"}, "status": "published"}, "_oai": {"id": "oai:ir.soken.ac.jp:00000990", "sets": ["20"]}, "author_link": ["10107", "10109", "10108"], "item_1_biblio_info_21": {"attribute_name": "書誌情報（ソート用）", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2003-09-30", "bibliographicIssueDateType": "Issued"}, "bibliographic_titles": [{}]}]}, "item_1_creator_2": {"attribute_name": "著者名", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "庄司, 志咲子"}], "nameIdentifiers": [{"nameIdentifier": "10107", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_creator_3": {"attribute_name": "フリガナ", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "ショウジ, シサコ"}], "nameIdentifiers": [{"nameIdentifier": "10108", "nameIdentifierScheme": "WEKO"}]}]}, "item_1_date_granted_11": {"attribute_name": "学位授与年月日", "attribute_value_mlt": [{"subitem_dategranted": "2003-09-30"}]}, "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_1": {"attribute_name": "ID", "attribute_value_mlt": [{"subitem_description": "2003512", "subitem_description_type": "Other"}]}, "item_1_description_12": {"attribute_name": "要旨", "attribute_value_mlt": [{"subitem_description": "This study provides a first direct evidence that autophagy suppresses intracellular abnormal protein accumulation related with disease.\u003cbr /\u003e     Misfolded proteins expressed in cells, which have escaped chaperon mediated refolding or proteasome-dependent degradation, accumulate and aggregate. Aggregates of misfolded and damaged proteins might have several detrimental effects on cellular function by interfering with cellular processes for physical or mechanical reasons. Now, a large number of diseases are recognized as \u0027conformational diseases\u0027, caused by adoption of non-native protein conformations that lead to aggregation.\u003cbr /\u003e     Current studies focus on the degradation pathway by which cells protect themselves from toxicity associated with protein misfolding and aggregation. The major cellular degradation systems for protein and organelle turn over are ubiquitin-proteasome system (UPS) and autophagy. Autophagy is an intracellular degradation system in which cytoplasmic components, including organelles, are directed to the lysosome/vacuole by a membrane-mediated process. A linkage between UPS dysfunction and pathogens of conformational diseases has been well studied. On the other hand, the relationship between autophagy dysfunction and conformational diseases was not clear in previous studies.\u003cbr /\u003e     In this study, I examined whether autophagy suppresses accumulation of two-type abnormal proteins. One is mutant α1-antitrypsin Z (ATZ), which accumulates in endoplasmic reticulum (ER). It has known that accumulation of the mutant ATZ in the ER causes liver injury. Other is expanded-polyglutamine (polyQ), which form aggregate in cytoplasm or in nucleus. The aggregates of proteins containing abnormal long length polyQ-tracts induce neurodegeneration and cell death in cultured cells. To determine whether autophagy is involved in the degradation of these abnormal proteins, I used two specific autophagy marker molecules: Apg5 that is an essential for autophagy and LC3 that is associated with autophagosome membrane.\u003cbr /\u003e     Part I focused on the relationship between ATZ accumulation and autophagic degadation. First, I examined the effects of Apg5 gene disruption on ATZ accumulation. Western blot analysis using anti α\u003cSUB\u003e1\u003c/SUB\u003e-antitrypsin antibody showed the steady-state intracellular level of ATZ was increased in Apg5- deficient (APG5\u003cSUP\u003e-/-\u003c/SUP\u003e) mouse ES cells. Intracellular distribution of ATZ showed ER-pattern in both wild type and APG5\u003cSUP\u003e-/-\u003c/SUP\u003e cells by fluorescence microscopy using CFP-tagged ATZ (ATZ-CFP). ATZ-CFP fluorescence intensity in APG5\u003cSUP\u003e-/-\u003c/SUP\u003e cells was higher than that in wild type. These results indicate that ATZ accumulation in ER is accelerated in autophagy-deficient cells. Next, I examined the fate of ATZ in APG5\u003cSUP\u003e-/-\u003c/SUP\u003ecells by pulse-chase radiolabeling experiments. In APG5\u003cSUP\u003e-/-\u003c/SUP\u003ecells, the degradation rate of ATZ was slowed down. This result shows that ATZ is degraded by autophagy. In addition, the result of pulse-chase experiment using various proteasome inhibitors showed that the contribution of autophagy to degradation of ATZ is coequal to that of proteasome. Further, I found that viability of APG5\u003cSUP\u003e-/-\u003c/SUP\u003e cells expressing ATZ in presence of proteasome inhibitor was decreased. This result suggests that ATZ is more toxic in cells deficient in both autophagy and ubiquitin-proteasome system.\u003cbr /\u003e     In Part II, I examined whether autophagy is induced by expanded-polyQ aggregation, and whether the poly-Qaggregation is enhanced by autophagy dysfunction. First, I performed western blotting of rat PC12 cells expressing proteins containing polyQ-tracts using anti LC3-antibody to know induction of autophgy, The amount of LC3-II that is the autophagosome membrane binding form of LC3 marked increased in cells expressing abnormal long length polyQ (Q79), which form aggregate. In contrast, such increase of LC3-II was not detected by expression of proteins containing short length po1yQs (Q22), which do not form aggregate. To confirm autophagosome formation, I observed GFP- and YFP-LC3 stable transformant expressing long length polyQs (HA-Q79 and Q81-CFP) or short length polyQ tracts (HA-Q22 and Q19-CFP) in fluorescence microscopy. Punctate signals of GFP- and YFP-LC3 indicating autophagy induction were increased in the cells expressing abnormal length polyQs but not normal length polyQs. These results indicate that the formation of polyQ-aggregate induced autophagy. To know that autophagosome encloses polyQ-aggregate, I observed the higher magnified images of cells having polyQ-aggregates in laser scanning confocal microscopy and electron microscopy. Although I could not observe autophagosome enclosing large aggregates, small polyQ-aggregate and polyQ-proteins were detected in ring-structure of autophagosome. This result suggested that autophagosome sequesters small polyQ-aggregates and polyQ-proteins but not its larger aggregates. Next, I examined the effect of autophagy dysfunction on polyQ-aggregation by counting of number of aggregates and western blotting. The number of poly-Q aggregate and the amount of detergent-insoluble polyQ-proteins (aggregated polyQ proteins) were marked increased in APG5\u003cSUP\u003e-/-\u003c/SUP\u003e cells. In addition, I found that polyQ-inducing cell death is enhanced in autophagy-deficient cells. These results show that autophagy suppresses polyQ-aggregate formation and reduces its cellular damage.\u003cbr /\u003e     Taken all together, this study revealed that autophagy is involved in degradation of two-type abnormal proteins, ATZ and polyQ, even though the distribution of these proteins is different. This finding suggests that autophagy is general anti-conformational disease system.", "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724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": "生命科学研究科"}]}, "item_1_select_9": {"attribute_name": "専攻", "attribute_value_mlt": [{"subitem_select_item": "18 遺伝学専攻"}]}, "item_1_text_10": {"attribute_name": "学位授与年度", "attribute_value_mlt": [{"subitem_text_value": "2003"}]}, "item_1_text_20": {"attribute_name": "業務メモ", "attribute_value_mlt": [{"subitem_text_value": "（2018年2月20日）本籍など個人情報の記載がある旧要旨・審査要旨を個人情報のない新しいものに差し替えた。承諾書等未確認。要確認該当項目修正のこと。"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "SHOJI, Shisako", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "10109", "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", "download_preview_message": "", "file_order": 0, "filename": "甲724_要旨.pdf", "filesize": [{"value": "339.1 kB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_11", "mimetype": "application/pdf", "size": 339100.0, "url": {"label": "要旨・審査要旨 / Abstract, Screening Result", "url": "https://ir.soken.ac.jp/record/990/files/甲724_要旨.pdf"}, "version_id": "22e7ea94-66f3-495a-a16a-4b19b6789a41"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "jpn"}]}, "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": "AUTOPHAGY IS INVOLVED IN THE  DEGRADATION OF MISFOLDED PROTEINS  IN MAMMALIAN CELLS", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "AUTOPHAGY IS INVOLVED IN THE  DEGRADATION OF MISFOLDED PROTEINS  IN MAMMALIAN CELLS"}, {"subitem_title": "AUTOPHAGY IS INVOLVED IN THE DEGRADATION OF MISFOLDED PROTEINS IN MAMMALIAN CELLS", "subitem_title_language": "en"}]}, "item_type_id": "1", "owner": "1", "path": ["20"], "permalink_uri": "https://ir.soken.ac.jp/records/990", "pubdate": {"attribute_name": "公開日", "attribute_value": "2010-02-22"}, "publish_date": "2010-02-22", "publish_status": "0", "recid": "990", "relation": {}, "relation_version_is_last": true, "title": ["AUTOPHAGY IS INVOLVED IN THE  DEGRADATION OF MISFOLDED PROTEINS  IN MAMMALIAN CELLS"], "weko_shared_id": 1}