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研究生(外文):Wen-Yuan Chung
論文名稱(外文):Characterization og the function of RING domain of Nap2 gene
指導教授(外文):Chin-Hsiang Chien
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因為惡性腫瘤細胞表面的醣蛋白多會有sialylation程度增加的現象,因此有許多研究者想藉由唾液酸酶(sialidase)處理癌細胞,以降低癌細胞轉移以及入侵其他組織的能力。 在近年的研究中發現,大量表現唾液酸酶的細胞不單只會影響細胞表面醣蛋白唾液酸化的程度也會影響細胞內部的生理現象。
small nanH蛋白是梭狀芽胞桿菌的一種神經胺酸苷酶(neuraminidase),本實驗室以yeast two-hybrid篩選,發現了一個會與small nanH蛋白作用的未知功能基因,將它命名為Nap2(neuraminidase associated protein 2)。 經由北方墨點法分析(Northern blot)發現Nap2基因的轉錄產物全長應該是大於4kb。 我們在此研究的蛋白僅是NAP2蛋白中含有RING finger domain的部分氨基酸片段。 經過蛋白質序列比對後發現,R-NAP2蛋白的氨基酸序列包含了兩個可能的NLS序列和一個 RING finger domain。 共軛焦顯微鏡觀察發現EGFP-R-NAP2蛋白會分佈在細胞核外圍,且位在R-NAP2蛋白N端的NLS序列對R-NAP2分佈的影響較大,C端的NLS序列以及RING finger domain則較無顯著的影響。 以MTT assay分析發現,大量表現R-NAP2會抑制OVCAR-3、YM-1、HeLa和HEK293細胞株的細胞生長速率,但對HepG2/A2和Huh-7卻無顯著的影響。 根據西方墨點法(Western blot)和流式細胞儀的分析結果指出大量表現R-NAP2蛋白並不會誘導細胞走向細胞凋亡。 流式細胞儀(FACS)分析細胞週期的結果發現,大量表現R-NAP2 蛋白可能會造成細胞週期停頓(cell cycle arrest)在G2/M phase的現象。 由上述之實驗結果顯示,R-NAP2蛋白可能具有抑制腫瘤生長(tumor suppressor)的功能。
由於我們在此探討的都只是NAP2蛋白的部分氨基酸序列之功能,另外還帶有SPRY domain的NAP2蛋白全長是否也具有類似的功能或分佈在細胞的狀況還是值得我們進一步探討。
Because the cell surface of malignant cancer cells play a feature of hyper-sialylation of the glycoprotein, sialidase was thought to be a way to inhibit the metastasis and invasive ability of cancer cells. In recent years, it was found that sialidase may not only influence of the glycoproteins and glycolipids on the cell surface but also influence intra-cellular function.
Small nanH is a neuraminidase of Clostridium perfringens. After yeast two-hybrid screen, we found a novel gene which would interact with small nanH protein and it was named Nap2 (neuraminidase associated protein 2). The Northern blot analysis showed that the major full length transcript of Nap2 gene should be longer than 4kb. The domain we studied here was just part of the NAP2 protein sequence folded into RING finger domain, hence it was called RING domain of Nap2 (R-NAP2). There are two candidate NLS sequence and one RING finger domain in the protein sequence of R-NAP2. Confocal microscopy observation showed EGFP-R-NAP2 protein was localizated around nucleus. The NLS sequence near N-terminal but not the one near C-terminal, neither RING finger domain of R-NAP2, would affect the localization of R-NAP2 protein. In MTT assay analysis, transiently overexpression R-NAP2 protein would inhibit cell growth rate in OVCAR-3, YM-1, HeLa, and HEK 293 cell lines, but not in HepG2/A2 and Huh-7. Overexpression of R-NAP2 protein may not induce cell apoptosis according to the results of Western blot and FACS. On the other hand, the FACS analysis showed that overexpression of R-NAP2 may cause cell cycle arrest at G2/M phase. It showed that R-NAP2 protein may play a role as tumor suppressor.
But we still don’t know whether full length of NAP2 protein which contains another domain- SPRY domain would show similar function and localization as R-NAP2 protein dose.
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