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標題: AIF核內運輸與hHR23A之研究
Expression of hHR23A as a potential nuclear transporter of AIF
作者: 黃智洋
Huang, Chih-Yang
Contributors: 邱繡河;陳志毅
周寬基
中興大學
關鍵字: 促細胞凋亡因子;核內運輸蛋白;細胞凋亡
AIF;hHR23A;Apoptosis
日期: 2008
Issue Date: 2012-09-04 10:49:21 (UTC+8)
Publisher: 生物醫學研究所
摘要: AIF(apoptosis-inducing factor)為位在粒線體內膜膜上之蛋白質,在正常細胞中,能夠藉由維持電子傳遞鏈複合體I的穩定性,進而影響細胞能量的產生及自由基的清除,並且也會維持粒線體的構造;反之,當細胞遭受到不當刺激時,粒線體膜通透性改變,使得原本存在於粒腺體間質中之AIF,被釋放出來,進入細胞核內造成DNA片段化,進一步促進細胞凋亡。AIF蛋白質序列上具有兩段特殊之訊號序列,分別為粒線體定位訊號序列(mitochondria localization signal, MLS)及細胞核定位訊號序列(nuclear localization signal, NLS),當其蛋白質合成後會藉由MLS運送至粒線體之中,而在細胞凋亡早期時,會藉由其NLS運送至細胞核之中。但近年研究發現,許多未具有NLS之蛋白質亦能進入細胞核內,如Smad、β-catenin、ERK2及PTEN,表示細胞內可能有其他未知之機制能輔助這些蛋白質進入細胞核內。
我們利用酵母菌雙雜合系統找出能與AIF交互作用之蛋白質,發現AIF在細胞核及細胞質中均能與hHR23A(human homologue Rad23 A)有交互作用。hHR23A為酵母菌Rad23之相似蛋白質,Rad23被認為參與在核甘酸修復與蛋白質降解,而其在哺乳類細胞中有兩個相似蛋白質,分別為hHR23A及hHR23B。目前研究指出,hHR23B的功能較相似於酵母菌Rad23,也參與在核甘酸修復。而hHR23A的重要性較不如hHR23B,但hHR23A會與其他許多蛋白質作用,如p53、EF2、BRCA2及Hsp70等,因此對於hHR23A之功能仍未清楚。
此外,經由免疫沉澱法也證實hHR23A確實能與AIF有交互作用,並且根據實驗結果發現,利用干擾性RNA抑制hHR23A蛋白質表現後,再誘發細胞凋亡,AIF無法進入細胞核之內;而缺乏hHR23A之細胞對於抗癌藥亦有較高之抗藥性,表示缺乏hHR23A,進而影響AIF進入細胞核,使得細胞有較高之存活率。此外,根據實驗結果也發現,當細胞處以粒線體ATP合成抑制劑後,再誘發細胞凋亡,AIF也無法進入細胞核之內,同時hHR23A在細胞內之分布也受到影響,表示AIF進出細胞核並非藉由擴散運輸,而是藉由主動運輸,因此當缺乏ATP時,即使進入細胞凋亡,AIF仍無法進入到細胞核之內。由我們之實驗結果可知,AIF進入細胞核,可能是藉由與hHR23A結合後,經由hHR23A運輸進入細胞核內,而其過程是需要ATP的幫助。
Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidoreductase activity, which is encoded by the nuclear gene. Following induction of apoptosis, AIF is released from mitochondria and translocated to cytosol, and then to the nucleus. The mechanism of AIF translocation from mitochondria to nucleus is still unclear. It has been suggested that translocation is due to the presence of nuclear localization signal (NLS). However, proteins without NLS, such as Smad, PTEN and ERK, could also be translocated into nucleus. There could be a protein that transports protein into nucleus.
Therefore, we used yeast two-hybrid system to find the nuclear transporter and we identified a protein, hHR23A, which could bind to AIF both in cytosol and in nucleus. hHR23A is a mammalian protein that is homologous to yeast Rad23 protein. Although hHR23B has been shown to be correlated with DNA repair, the function of hHR23A is yet to be determined. We propose that hHR23A could be a protein transporter to bring AIF into nucleus following cell stress.
We then raised antibodies to AIF and hHR23A respectively, and by immunoprecipitation we used these antibodies to pull down AIF and hHR23A proteins, of which the protein identities were further determined by MALDI-TOF. We then constructed shRNA(small-hairpin RNA) of hHR23A to study whether down-regulation of hHR23A could interfere nuclear translocation of AIF during apoptosis. Moreover, we constructed deletion mutants of AIF and hHR23A to map out the interaction domains on AIF and hHR23A. Our preliminary data showed that hHR23A bound to AIF directly. Knockdown of hHR23A expression could reduce nuclear translocation of AIF after treating with apoptosis inducer, staurosporine. Furthermore, cell survival rate of hHR23A knockdown cell increased markedly when cells were treated with cisplatin. Addition of DNP, an inhibitor of ATP synthesis, could also diminish nuclear translocation of AIF and hHR23A, and increase cisplatin resistance. Our results suggest that hHR23A is involved in a novel nuclear transportation, which facilitates nuclear translocation of AIF during apoptosis.
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