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研究生:林東屏
研究生(外文):Tung-Ping Lin
論文名稱:HDAC10及其結合蛋白之棎討
論文名稱(外文):Studies of HDAC10 and its associated factors
指導教授:楊文明楊文明引用關係
指導教授(外文):Wen-Ming Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:79
中文關鍵詞:HDAC10
外文關鍵詞:HDAC10
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組蛋白去乙醯基酵素 (HDACs,histone deacetylases) 藉由改變染色質的結構進而調控基因的轉錄。HDACs催化組蛋白末端上離胺酸 (lysine ε-amino groups) 以抑制轉錄進行。目前,已有11個HDAC成員被定義。HDAC10因去乙醯化酵素功能區域類似酵母菌Hdalp蛋白,且存在於細胞核及細胞質,故被歸為class II HDAC。
許多的證據說明,HDAC的酵素受質不僅限於組蛋白,還包括許多核內外乙醯化的非組蛋白。觀察HDAC10在細胞的位置,發現HDAC10可存在於細胞核與細胞質。由這些結果推測HDAC10可能在核內外扮演去乙醯化非組蛋白的角色。截至目前,HDAC10的功能仍不清楚。因此,透過本篇論文將探討HDAC10在細胞核內外的功能與壓力下HDAC10細胞位置的改變。
在細胞核內部份,利用專一性乙醯化抗體及轉錄活性試驗證明HDAC10去乙醯化轉錄輔助抑制因子KAP-1及轉錄因子Pax3。當細胞大量表現HDAC10會減弱其KAP-1及Pax3轉錄因子的抑制作用。加入HDAC 抑制劑 (TSA,trichotatin A) 後,KAP-1及Pax3的轉錄抑制活性因HDAC10受抑制而回復。藉由層次比重離心法證實這三者蛋白可存於類似的濃度梯度。綜合這些結果,歸納出HDAC10在核內轉錄調控層面上的角色。在研究HDAC10蛋白複合體時,發現HDAC10可結合乙醯態PRMT5 (protein arginine methyltransferse 5) 卻不執行去乙醯化作用。此外,非乙醯態熱休克蛋白70 (heat shock protein70) 與HDAC10穩定結合透過純化HDAC10蛋白複合體。給予細胞熱休克後似乎促使HDAC10與熱休克蛋白70聚集一起。最後,也觀察到在proteasome抑制劑 (proteasome inhibitor,MG132) 處理下HDAC10似乎也伴隨熱休克蛋白70進入細胞內。綜合以上,本論文發現HDAC10的酵素受質包括核內轉錄因子及核外特殊蛋白。透過HDAC10調控核內轉錄抑制因子及HDAC10在壓力下細胞位置的改變,來定義出HDAC10在細胞中可能的生物角色。
Histone deacetylase (HDAC) family functions as enzymes involving in gene transcription and chromatin remodeling. HDAC catalyzes the deacetylation of lysine residues in the histone N-terminal tails and plays a role in transcriptional repression. So far, at least 11 members have been identified. Because HDAC10 has been found in both the cytoplasm and the nucleus and its catalytic domain similar to yeast Hdalp, it belongs to class II HDAC.
The function of HDAC10 is unknow. It was found that HDAC10 was located in both nucleus and cytoplasm. Recently, many lines of evidence show that HDACs modify not only histones but also some non-histone proteins in the nucleus and cytoplasm. These evidences suggest the possible roles of nuclear and cytoplasmic HDAC10s deacetylate non-histone proteins. In this thesis, the nuclear and cytoplasmic functions of HDAC10 and the cellular localization of HDAC10 under stress will be studied.
Using a sepicific acetylated antibody and transcriptional assay, it was demonstrated that HDAC10 deacetylates co-repressor KAP-1 and transcrption factor Pax3 in the nucleus. The repressional activities of KAP-1 and Pax3 were decreased when HDAC10 was overexpressed. Under HDAC inhibitor trichostatin A (TSA) treatment, the depressingly transcriptional levels of KAP-1 and Pax3 have been raised. By glycerol gradient analysis, HDAC10, KAP-1 and Pax3 were detected in the same fractions. All these results suggest that HDAC10 plays important roles in transcriptional regulation. In studies of the HDAC10 stable complex, I also found that PRMT5 (protein arginine methyltransferase 5) and heat shock protein 70 (hsc70) were in the HDAC10 complex. HDAC10 interacts with acetylated PRMT5 but it doesn’t deacetylate PRMT5. By immunoaffinity assay, deacetylated hsc70 stably associates with HDAC10, but not the acetyalted hsc70. HDAC10 seems to aggregate with heat shock protein 70 after heat shock. Finally, HDAC10 was detected in the nucleus with heat shock protein 70 under proteasome inhibitor (MG132) treatment. In summary, our results suggest that HDAC10 possiblely catalyzes substrates include both nuclear transcriptional factor and cytoplasmic proteins. Taken together, our results demonstrated the possible roles of HDAC10 in the transcription and response for cellular stress.
壹、中文摘要 4
貳、英文摘要 5
參、緒論 6
一、前言 6
二、介紹 6
1. HDAC family於轉錄調控的角色 6
2. HDAC受質可為非組蛋白核心 (non-core histone protein) 8
肆、研究目的 8
伍、研究策略 8
陸、材料與方法 9
一、質體DNA之構築 9
二、細胞培養及基因轉移感染 11
三、蛋白交互作用 11
1. 免疫沈澱反應 (Co-immunoprecipitaion) 及西方免疫點墨反應 (Western immunoblot) 11
2. GST- pull down 12
四、轉錄活性之檢測 13
五、螢光顯微鏡 (Fluorescence microscopy) 13
六、層次比重離心法 (Density-gradient centrifugation) 13
柒、結果 15
一、HDAC10在細胞內可形成穩定的蛋白質複體 15
二、HDAC10結合HDAC10結合非乙醯化的熱休克蛋白70 15
三、HDAC10結合乙醯化態PRMT5 (protein arginine methyltransferase) 16
四、HDAC10可去乙醯化轉錄抑制輔助子KAP-1,並降低其轉錄抑制活性 18
五、HDAC10去乙醯化轉錄因子Pax3,降低Pax3轉錄抑制活性 20
六、HDAC10結合另一class Ⅱ成員HDAC6 21
七、HDAC10 在正常或壓力環境下細胞中分佈情形 22
八、其他與HDAC10結合的蛋白在細胞分佈情形 23
捌、討論 24
一、HDAC10在細胞質功能 24
二、細胞核內HDAC10的酵素功能 25
三、可能調控HDAC10細胞核內外shuttling的機制 26
玖、參考文獻 29
拾、圖 34
十一、附圖 58
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