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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳宏文(Hung-wen Chen) | |
dc.contributor.author | Chin-Chien Chou | en |
dc.contributor.author | 周志謙 | zh_TW |
dc.date.accessioned | 2021-06-13T03:26:13Z | - |
dc.date.available | 2008-08-04 | |
dc.date.copyright | 2006-08-04 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-28 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31966 | - |
dc.description.abstract | 人類胎盤轉錄因子hGCMa是屬於GCM轉錄因子基因家族的成員之一。其特徵為帶有一含有鋅離子的DNA結合區域,稱為GCM domain。 hGCMa能夠調控融合蛋白syncytin基因表現,使胎盤滋養層細胞彼此融合為融合滋養層細胞,促使胎盤絨毛構造發育完整,以利於胎兒與母體間氣體與養分的交換。
已知hGCMa蛋白的活性受到後轉譯修飾如ubiqutination及acetylation的調控。在本實驗計畫中,我們進一步探討另一後轉譯修飾sumoylation對hGCMa蛋白活性的影響。我們證明hGCMa蛋白與SUMO E2蛋白Ubc9存有專一性的交互作用,且此專一性的結合位置是位於hGCMa N端附近的GCM domain。而在細胞中與試管中Ubc9促進hGCMa與SUMO-1作共價性鍵結修飾,且主要修飾位置是在hGCMa的K156上。此外,在共同轉染實驗中Ubc9與SUMO E3蛋白PIAS2β,能夠增強hGCMa sumoylation的情況。而探討SUMO-1對hGCMa基因轉錄活性的研究中,證實了SUMO-1能夠抑制hGCMa的轉錄活性;反之,共同轉染SENP1此一參與desumoylation反應的酵素,的確增加hGCMa的轉錄活性。 綜合上述證據顯示,hGCMa確實能夠進行SUMO後轉譯修飾,且抑制hGCMa的轉錄活性。這也是GCM基因家族中第一個被鑑定出能夠受SUMO後轉譯修飾所調控的GCM蛋白。故本篇報告有助於釐清hGCMa的轉錄活性調控,以及了解滋養層細胞融合的機制。 | zh_TW |
dc.description.abstract | Human placental transcription factor GCMa (hGCMa) is a member of the GCM gene family with a characteristic zinc-containing DNA-binding domain called GCM domain. hGCMa regulates expression of the fusogenic protein, syncytin, in order to facilitate the fusion of cytotrophoblasts into syncytiotrophoblasts on placental villi. Syncytiotrophoblast mediates fetal-maternal exchange of gas and nutrients.
Previous studies indicated that hGCMa activity can be regulated by post-translational modifications such as ubiquitination and acetylation. In this study, we further investigated whether sumoylation, another post-translational modification, can regulate hGCMa activity. We demonstrated that hGCMa specifically interacts with the SUMO E2 protein, Ubc9, via the N-terminal GCM domain of hGCMa. In addition, Ubc9 promoted the conjugation between hGCMa and SUMO-1 in vivo and in vitro. The lysine156 residue in hGCMa was identified as the major sumoylation site. Moreover, coexpression of the SUMO E3 protein, PIAS2β, and Ubc9 further promoted the degree of hGCMa sumoylation. Examining the role of SUMO-1 in the regulation of hGCMa activity revealed that SUMO-1 modification decreases the transcriptional activity of hGCMa. Conversely, coexpression of SENP1, a desumolyation enzyme, indeed increased hGCMa-mediated transcriptional activation. Overall, our results support that hGCMa can undergo sumoylation, which further suppresses hGCMa transcriptional activity. To our knowledge, hGCMa is the first identified GCM member that can be post-translationally modified by SUMO-1. Our results further our understanding on how hGCMa activity is regulated, which may also help to understand the regulation of trophoblastic fusion. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:26:13Z (GMT). No. of bitstreams: 1 ntu-95-R93b46034-1.pdf: 5098954 bytes, checksum: 5284dcd2bb2f7c17c730956216c7258f (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 目 錄……………………………………………………………….. I
圖表目錄…………………………………………………………….. III 縮寫表……………………………………………………………….. IV 中文摘要…………………………………………………………….. VI 英文摘要 Abstract…………………………………………………... VII 第一章 緒論 1.1 胎盤………………………………………………... 1 1.2 gcm轉錄因子基因家族........................................... 3 1.3 後轉譯修飾………………………………………... 7 1.4 SUMOylation……………………………………… 11 1.5 研究動機及目標....................................................... 16 第二章 材料及方法 2.1 重組質體構築……………………………………... 18 2.2 細胞培養…………………………………………... 24 2.3 活體外SUMO反應分析………………………….. 26 2.4 西方墨點法 (Western blotting)…………………… 28 2.5 共同免疫沉澱法 (Co-immunoprecipitation)…….. 28 2.6 蛋白表現…………………………………………... 29 2.7 GST沉澱試驗 (GST pull-down assay)…………... 30 2.8 螢光報告基因Lucifrease活性檢測……………… 30 第三章 實驗結果 3.1 hGCMa與Ubc9在活體內外的交互作用。…….. 32 3.2 定位Ubc9與hGCMa的結合區域。……………. 33 3.3 hGCMa在活體內的SUMOylation修飾。……… 34 3.4 hGCMa在活體外的SUMOylation修飾。……… 37 3.5 hGCMa蛋白與SUMO-1結合位置分析。……… 38 3.6 SUMO調控hGCMa的轉錄活性。……………... 39 第四章 討論與總結……………………………………………….. 41 第五章 圖表……………………………………………………….. 47 第六章 參考文獻………………………………………………….. 57 圖表目錄 圖一 GCMa轉錄因子生物功能與作用途徑…………………….. 47 圖二 hGCMa與Ubc9在活體內的交互作用…………………….. 48 圖三 hGCMa與Ubc9在活體外的交互作用…………………….. 49 圖四 定位Ubc9與hGCMa的結合區域…………………………. 50 圖五 hGCMa在活體內的SUMOylation、Ubc9與PIAS2β 促進hGCMa SUMOylation以及顯性抑制型Ubc9抑制 hGCMa SUMOylation………………………………………. 51 圖六 hGCMa不同片段在活體內的SUMOylation………………. 52 圖七 hGCMa在表現HA-hGCMa的BeWo stable cell line的SUMOylation………………………………………... 53 圖八 hGCMa在活體外的SUMOylation修飾…………………… 54 圖九 hGCMa蛋白與SUMO-1結合位置分析…………………... 55 圖十 SUMO-1抑制hGCMa的轉錄活性………………………... 56 | |
dc.language.iso | zh-TW | |
dc.title | SUMO修飾對hGCMa活性的功能探討 | zh_TW |
dc.title | Regulation of hGCMa activity by SUMOylation | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李明亭(Ming-Ting Lee),黃銓珍(Chang-Jen Huang),繆希椿(Jesse Shi-Chuen Miaw),張功耀(Kung-Yao Chang) | |
dc.subject.keyword | 胎盤,轉錄因子, | zh_TW |
dc.subject.keyword | hGCMa,SUMO,Ubc9 PIAS,SENP1, | en |
dc.relation.page | 63 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-29 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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