錯誤褶疊蛋白質誘導之擬熱休克反應機制之探討

Understanding the mechnism of misfolded protein induced- heat-shock-like response

陳銘澤(Ming-Tse Chen)

指導教授 ： 葉靖輝

國立中央大學/理學院/生命科學系/碩士(2008年)

摘要

本研究旨在探討水稻小分子量熱休克蛋白質基因Oshsp18.0受脯胺酸(Proline)類似物Azetidine-2-carboxylate (AZC)之誘導模式及其相關調控機制。生物藉由誘導熱休克基因來抵抗外界環境溫度的改變。除溫度改變外，環境中存在之重金屬離子、化學物質或胺基酸類似物，甚至僅酸鹼度的改變皆可誘導熱休克基因之表現。熱休克基因的表現導致細胞內熱休克蛋白質大量累積，有助於增加細胞對嚴苛環境之耐受性，因此植物體於逆境時常累積大量小分子量熱休克蛋白質。將水稻白化幼苗處理致死高溫(48℃)或AZC後可見嚴重離子滲漏之現象。離子滲漏現象可因預先處理非致死性高溫(41℃)而緩和。水稻染色體中存在九個第一族小分子量熱休克蛋白質基因成員。我們針對Oshsp18.0進行詳細研究；發現此基因受高溫、銅、鎘、砷、AZC、酸鹼變化誘導。AZC會取代Proline之位置結合至寡胜肽並使寡胜肽之正常褶疊受到抑制。細胞誘導unfolded protein response (UPR) 以消除AZC引起之不正常蛋白質累積。將水稻白化苗預先處理蛋白質生合成抑制劑(cycloheximide)後，Oshsp18.0對AZC有反應延遲之現象；相對地，Oshsp18.0於高溫逆境並未有反應延遲現象。基因之表現受到轉錄因子及轉錄元素之間交互作用調控；且已有研究顯示，Oshsp18.0及Oshsp17.3二者重疊之啟動子區域存在一段包含九個核苷酸之序列(AZRE)與AZC具有專一性之關聯。綜合以上結果，推論存在特定轉錄因子會與AZRE進行專一性反應。
為獲得此轉錄因子之序列，我們利用yeast one-hybrid之技術釣取此轉錄因子之基因。由篩選所得之基因中，確有參與在UPR相關之基因(包括促進胜肽褶疊、運送、降解及終止蛋白質的合成)。我們選取一具高度重覆性之基因－10-kDa chaperonin－進行gel retardation實驗。結果顯示，此蛋白質並未能在in vitro情況下與AZRE進行鍵結；因此有必要繼續由所篩選所得之基因中挑選其他候選者進行分析。

關鍵字

脯胺酸類似物 ； 錯誤褶疊蛋白質 ； 熱休克反應 ； 小分子量熱休克蛋白質 ； 錯誤褶疊蛋白質反應 ； 內質網逆境

並列摘要

We focused on studying the mechanism of gene induction of Oshsp18.0 under treatment of L-azetidine-2-carboxylate (AZC), a proline analog, in this study. Organisms respond to elevated temperature by up-regulating heat shock genes. Not only in temperature stress, but also in various kinds of chemical irritations and pH change induces expression of heat shock genes. Induction of heat shock genes results in accumulation of heat shock proteins. Accumulated heat shock proteins enhances thermotolerance or resistance to following severe stress. AZC induces expression of small heat shock protein genes, however, it causes sever ion-leakage to a comparable level of lethal heat stress treatment. By pre-treatment of nonlethal heat stress, the ion-leakage dropped. Hence, acclimation enhances thermotolerance while AZC causes severe ion-leakage. We chose one of the nine class I small heat shock protein genes in rice, Oshsp18.0, for detailed analysis via RT-PCR. Oshsp18.0 is expressed under heat stress, AZC, Cu2+, Cd2+ or As3+. Oshsp18.0 is expressed at a higher level under heat stress, AZC, As3+ and lower level under Cu2+ and Cd2+. Even change of pH slightly induces expression of Oshsp18.0. As a prline analog, AZC is likely to incorporate into polypeptides which causes misfolded protein and unfolded protein response. Pre-treatment of inhibitor of protein de novo synthesis, cycloheximide, gene expression of Oshsp18.0 delayed under AZC treatment which was not observed in other treatments. This implies different gene regulation mechanism is involved under AZC treatment. Induction of heat shock genes depends on the interaction of transcription factor and regulatory element located on the promoter region of heat shock genes. A 9-base-pair element, AZRE (5’ -CGTCCAGGAC- 3’), was previously identified in the promoters of Oshsp18.0 and Oshsp17.3. AZRE responded to AZC treatment specifically. To identify the transcription factor(s) that specifically interact with AZRE, we used yeast one-hybrid technology to screen cDNA library which was prepared from AZC treatment. Of the candidates, genes involved in the unfolded protein response were obtained. For its repetition, 10-kDa chaperonin (10Cpn), was selected for further analysis. 10Cpn is a constitutively expressed gene as shown via RT-PCR. In vitro binding assay showed no interaction with AZRE. Other candidate genes must be tested in the future.

並列關鍵字

Oshsp18.0 ； misfolded protein ； heat shock response ； small heat shock protein ； proline analog ； azetidine-2-carboxylic acid ； AZC ； ER stress ； unfolded protein response

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