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研究生:夏慧伶
研究生(外文):Hui-Ling Shia
論文名稱:人類轉譯後修飾蛋白(SUMO)與染色體中心節蛋白(CENP-C)之分子交互作用研究
論文名稱(外文):Molecular studies on the interaction between human post-translational modifier protein SUMO and centromere protein CENP-C
指導教授:李水龍李水龍引用關係
指導教授(外文):Steven. S.-L. Li
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物醫學科學研究所
學門:生命科學學門
學類:生物化學學類
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:152
中文關鍵詞:人類轉譯後修飾蛋白染色體中心節蛋白
外文關鍵詞:SUMOCENP-C
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人類轉譯後修飾蛋白質SUMO-1/2/3與酵母菌SMT3蛋白質具同源性,而且酵母菌SMT3是從酵母菌MIF2中心節結合蛋白突變中分離出來的。研究顯示酵母菌MIF2蛋白至少有兩個區域與哺乳類CENP-C蛋白質具有相似性,因此提高興趣研究於人類CENP-C蛋白質與人類SUMO-1/2/3蛋白質可能的相互作用。從子宮頸癌上皮細胞 (HeLa cell) 中全部的RNA反轉錄作用成互補DNA及聚合酶連鎖反應放大一段CENP-C基因 (簡稱C38 Ж,C38可表現出CENP-C全長943個胺基酸中的333個胺基酸) 。將此片段載入pEGFP質體中,表現出具有綠色螢光蛋白質的EGFP-C38融合蛋白質 (fusion protein)。在子宮頸癌上皮細胞中做有關EGFP-C38與Flag-SUMO-1/2/3GG蛋白的co-localization 及in vivo sumoylation 的研究,結果證明EGFP-C38與Flag-SUMO-1/2/3GG蛋白 co-localization在細胞核中。共同免疫沉殿法中先用抗Flag抗體結合Flag蛋白質後再用抗Flag抗體或抗GFP 抗體去辨認Flag-SUMO-1/2/3GG和EGFP-C38-Flag的結合物 (conjugate) 。西方墨點法分析偵測到EGFP-C38-Flag--SUMO-1/2/3GG的修飾複合物(sumoylated complexes) ,但其分子量比預測的還要大。
His-C38蛋白片段在in vitro sumoylation中,經由蛋白質電泳及西方墨點法分析、抗SUMO-1或抗SUMO-2抗體辨認修飾過的His-C38蛋白片段,可辨認His-SUMO-1/2GG--His-C38的修飾複合物(sumoylated complexes)。然而經由MALDI-TOF分析 SUMO-1/2GG 的C-terminal 之glycine 與 C38-His蛋白片段中的lysine所鍵結形成的isopeptide bonds已分析出。乃是將C38基因片段分成兩段 C28、C10,分別搬到pET21b表現蛋白進而純化。實驗證明S-tagged SUMO-1/2GG會修飾C28-His、C10-His,已證明經由MALDI-TOF-TOF可分析到S-tagged SUMO-1/2GG與C10-His所形成的isopeptide bonds。而SUMO-1/2GG與C28-His所形成的isopeptides bond已經分析出來了。
Human post-translational protein modifier protein SUMO-1/2/3 genes code for proteins homologous to yeast SMT3 protein, which is encoded by a suppressor 3 of MIF2 mutation in centromere protein gene. The yeast MIF2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C. It would be of interest to investigate the possible interaction(s) between human CENP-C and SUMO-1/2/3 proteins. A CENP-C cDNA fragment was cloned using RT-PCR with total RNAs form Hela cells. This cDNA fragment encoding CENP-C amino acids 342-764 (MW 38 kDa designated C38) was tagged with EGFP. The sub-cellular localization and in vivo sumoylation in HeLa cells were carried out. The EGFP-C38 protein was shown to co-localize with active forms of Flag-SUMO-1/2/3GG proteins in nucleus of Hela cells. The EGFP -C38 protein was also shown co-immunoprecipitated with antibodies against SUMO proteins. The protein conjugates were analyzed on SDS-PAGE and their western blots were probed with either anti-GFP or anti- Flag antibodies. The molecular weight of EGFP-C38 protein was found to be higher than the expected MW, indicating that EGFP-C38 protein was sumoylized. This part ( 333 amino acids) of CENP-C protein (943 amino acids) was expressed and purified. The in vitro sumoylized His-C38 protein fragment was analyzed on SDS-PAGE, and the western blot was probed with either anti-SUMO-1 or anti-SUMO-2 antibodies. The C38-His protein fragment appeared to be sumoylized, and the isopeptide bond between the C-terminal glycine of SUMO and lysine of His-C38 was analyzed by MALDI-TOF-TOF. C38 cDNA was sub-fragmented into C28 and C10 fragments transformed to BL21 strain for expression protein and purified protein. S-tagged SUMO-1/2GG modify C28-His and C10-His fragments, the isopeptide bond between the C-terminal glycine of S-tagged SUMO-2GG and lysine of C10-His was identified analyzed by MALDI-TOF-TOF. The isopeptide bonds between either S-tagged SUMO-1GG and C28-His or S-tagged SUMO-1/2GG and C28-His /C10-His are being analyzed.
中文摘要…………………………………………………………………I
英文摘要………………………………………………………………...II
英文縮小表……………………………………………………………..III
壹、序言………………………………………………………………..1
一、人類染色體中心節(centromere) 結合蛋白(CENPs)…….………1
二、CENPs與MIF2的相關性……….………………………………..6
三、CENP-C蛋白質與其它蛋白質的關係……………………………7
四、SUMO的發現與酵母菌的中心節蛋白關係………………………9
五、不同SUMO在演化上的相關性及本身特性………………………9
六、SUMO與Ubiquitin的相關性……………………………………..12
七、SUMO的功能…….…………………………………………….…14貳、研究目的……………...……………….………………………….17
參、材料與方法……………………………………..….……………19
一、建構不同型式CENP-C質體……………………………………19
二、細胞處理……………………………………………….…………21
三、細胞的核酸萃取………………………………………………….23
四、反轉錄聚合酶連鎖反應 (RT-PCR)………………………..…….24
五、質體構築 (plasmid construction)…………………..………….…26
六、基因選殖 (cDNA cloning)…………………………………..…..30
七、轉殖HeLa cell (Transfection)………………………………….….31
八、共同免疫螢光反應………………………………………….….…31
九、共同免疫沈澱法……………………….……….…………………32
十、西方墨點法………………………………………………………..33
十一、蛋白質表現……………………..………………………………35
十二、體外SUMO的修飾作用………………………………………38
十三、SDS-PAGE 凝膠及胰蛋白酶切割…………………….………39
肆、結果………………………………………………………………42
一、 CENP-C38蛋白與SUMO蛋白質之間的關係…………………42
二、 CENP-C38蛋白與SUMO蛋白質共同表現在細胞的位置……43
三、在細胞中CENP-C38蛋白與活化SUMO-1/2/3的相互關係….46
四、在細胞外活化的SUMO-1/2GG蛋白質修飾C38蛋白…………..48
五、活化的SUMO-2GG蛋白修飾C10-His蛋白的異共價鍵鍵結…51
伍、討論……………………………………………………………….54
陸、參考文獻………………………………………………………….62
表………………………………………………………………………..67
圖………………………………………………………………………..72
附錄………………………………………………………………...…104
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