2011-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/685936摘要：人類 hPuf-A/KIAA0020蛋白在2001年被發現是一個新的次要組織相容性抗 (minor histocompatibility antigen)，在結構上具有6個類似Pumilio-RNA結合區域，胺基酸序列比對發現hPuf-A應該是斑馬魚puf-A同源基因，相似度可達63%。我們2010年12月被Cancer Research接受的論文中發現hPuf-A主要位於細胞核仁內，只有極少量存在細胞核質；利用Actinomycin D、DRB和camptothecin (分別是RNA pol I、pol II以及Topoisomerase I的抑制劑)處理細胞後，hPuf-A在核仁的分佈位置會轉移至核質，顯示hPuf-A的位置會受到RNA的存在和DNA的損傷而呈現動態的轉變；有趣的是當我們利用siRNA降解hPuf-A蛋白的表現，細胞週期雖無明顯變化，但缺少hPuf-A蛋白表現的細胞在處理camptothecin 和UV 後有明顯死亡，大量表現hPuf-A蛋白的細胞在處理camptothecin 和UV 後卻有明顯存活的情形；利用親和性層析與質譜分析以及免疫沉澱分析發現hPuf-A與PARP-1在同一個複合物中，in vitro實驗發現hPuf-A會抑制PARP-1的poly(ADPribosyl)ation活性，進而影響PARP-1在細胞凋亡時被caspase切割的數量(Chang et al, Cancer Res, 2011, in press)。 根據上述已被接受的實驗結果推論hPuf-A可能在DNA受到傷害後扮演了調控PARP-1的角色，同時在人類組織免疫染色的初步研究中發現hPuf-A的表現量會隨著癌化程度而增加，因此我們提出三年計畫，希望能進一步詮釋 hPuf-A的生理功能： 第一、 hPuf-A的哪一段區域負責與PARP-1作用 第二、 了解hPuf-A的表現量與癌化程度的因果關係 第三、 hPuf-A可以和哪一種RNA結合 <br> Abstract: Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish ortholog contains six Pumilio-homology RNA binding domains and has been shown to participate in the development of eyes and primordial germ-cells, but the cellular function of hPuf-A remains unclear. In our manuscript accepted by Cancer Research in 2010, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors (camptothecin and etoposide). Interestingly, knockdown of hPuf-A sensitized cells to camptothecin and UV treatment leading to apoptosis and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometry analysis identified poly(ADP-ribose) polymerase 1 (PARP-1) as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to camptothecin and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3 (Chang et al. Cancer Res 2011, in press). According to the above results, hPuf-A may regulate the poly(ADP-ribosyl)ation of PARP-1 in response to DNA damage. Additionally, preliminary data showed that the expression of hPuf-A increased in human carcinoma specimens compared with normal tissues. Therefore, we would like to launch a three-year proposal to further characterize the function of hPuf-A. Specific aim #1: To identify which region of hPuf-A is responsible for the interaction of PARP-1. Specific aim #2: To investigate the correlation of hPuf-A with tumorigenesis. Specific aim #3: To isolate hPuf-A associated RNAs.hPuf-APARP-1pADPrhPuf-APARP-1pADPr