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研究生:蔡佩衿
研究生(外文):Pei-Chin Tsai
論文名稱:蓖麻子毒蛋白A鏈與人類第三型白血球抗原B關聯性轉錄物作用位置之研究
論文名稱(外文):BAT3, a Faithful Partner of RTA: Identification of the Interactive Sites on Ricin A Chain and Human HLA-B Associated Transcript-3
指導教授:林榮耀林榮耀引用關係
指導教授(外文):Jung-Yaw Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:英文
論文頁數:68
中文關鍵詞:蓖麻子A 鏈第三型白血球抗原B轉錄物酵母菌突變相互作用
外文關鍵詞:ricinRTABAT3human HLA-B associated transcript-3yeast two-hybridmutantmutationinteraction
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蓖麻子毒蛋白(ricin)屬於第二類型核醣體去活性蛋白質的一種,它是由A鏈與B鏈共價組合而成。 蓖麻子毒蛋白的A鏈(RTA)是一個專一性高的氮-醣苷酶, 主控對細胞的毒殺作用,而B鏈是一個能主導此毒蛋白進入細胞的醣蛋白。 在之前的實驗中我們用yeast two-hybrid系統以RTA為餌來篩選人類Jurkat 細胞中的cDNA庫。 利用此法我們找到了一個能與RTA作用的蛋白,經過比對後發現它是一段人類第三型白血球抗原B關聯性轉錄物(human HLA-B-associated transcript 3),簡稱BAT3。 BAT3是一個含有高度脯胺酸(proline)的蛋白,它在細胞中的作用尚未被鑑定。 為了探討RTA在真核細胞中與BAT3作用的機制,本論文之目的旨在研究它們是利用哪些區域互相作用。
RTA的分子立體結構已經被解出,它是由三個構造及功能上不同的區域 (domains), 即RTA I、RTA II及RTA III所構成。 本研究中,把上述三個區域的RTA接入yeast two-hybrid系統之GAL4 DNA結合區域載體,pAS2-1中。 BAT3的二次結構是由電腦程式PCGENE模擬而來,由分析結果我們發現了兩個具有雙極性(amphipathic)的特殊α-螺旋可能為BAT3與RTA作用的位置位於BAT3中。 我們針對此兩個預測得到的雙極性α-螺旋加以定點突變以破壞α-螺旋的雙極性,但並不破壞α-螺旋的結構,來觀察這些差異對RTA與BAT3結合有何影響。 此外,針對BAT3還做了一些連續性的刪除性突變(deletion),這些構築均用於此系統來找出兩蛋白相互結合的位置。
由實驗結果發現RTA可以在細胞中專一性的與BAT3結合,結合強度相較於p53與SV40 large T antigen的結合強度高達2.5倍左右。 此外,RTA III本身即可與BAT3結合,但結合強度約只剩原來的2%左右,本實驗可推論出RTA III仍然需要其他區域的幫助來與BAT3相互結合。 研究中亦發現RTA III的C端對於RTA與BAT3的作用較為重要。 而BAT3上用來與RTA作用的位置位於740-1000胺基酸的區域之內,在此區域中,我們推測得到的雙極性α-螺旋B中的F846或N847扮演著重要的角色,將此兩個氨基酸同時突變成F846R與N847I,則完全失去與RTA作用的能力。 由這些實驗結果我們不但找到了RTA與BAT3相互作用的位置,還推測出蓖麻子毒蛋白毒殺細胞的機制可能不只單純來自於它對蛋白生合成的抑制。 蓖麻子毒蛋白本身可能是一個多功能的蛋白,利用不同部位的RTA來操控不同的生物功能。

Ricin, a member of type II ribosome-inactivating protein (RIP), is composed of an A and a B chains. The A chain of ricin is an N-glycosidase, which plays the main role in cytotoxicity, while B chain is a lectin and directs the whole protein into cells. We used ricin A-chain (RTA) as bait to screen the target proteins from a human Jurkat cDNA library by yeast two-hybrid system. One of the clones interacts with RTA, and it was identified as a fragment of human HLA-B-associated transcript 3 (BAT3), from amino acid residues 614-1044. BAT3 is a functional-unknown protein with high proline content. Present investigation is to study the function of RTA via interacting with BAT3 in mammalian cells, and to examine the interactive sites of RTA and BAT3.
In the experiment, RTA was found to interact specifically with BAT3 in vivo, and the interaction was about 2.5 fold higher than that of p53 to SV40 large T antigen. The 3D structure of RTA has already been solved, and it is composed of three structural and functional different domains, namely RTA I, RTA II, and RTA III. In this study, the three domains of RTA were constructed into a GAL4 DNA binding domain (DNA-BD) cloning vector, pAS2-1, in order to examine which domain interacts with BAT3. By using PCGENE software to simulate the secondary structure of BAT3 through computer analysis, two amphipathic α-helixes were identified which might be involved in its interaction with RTA. The two candidate amphipathicα-helixes were mutated by site directed mutagenesis to affect its amphipathic characteristics, and several systematic deletion clones were also constructed to study its specific binding sites with RTA.
Present investigation showed that RTA III can interact with BAT3. However, it is only 2% of the original intensity, which indicated that other domains of RTA also involved in the interaction. This investigation also suggests that RTA C-terminus seemed to be important for the interaction. The fragment, amino acid residues 740-1000 of BAT3, was shown to be sufficient for the interaction with RTA. Within this region, one or both of F846 and N847 in the simulatedα-helixes B are important sites for the interaction. Because when they are mutated to F846R and N847I, BAT3 lost the interaction ability. From this experiment, we not only identified the interactive sites on RTA and BAT3, but also speculated that the mechanism of ricin triggered cell death and protein synthesis inhibition are mediated by different portions of RTA.

1. Abbreviations ------------------------------------- 1
2. Chinese abstract ------------------------------------- 2
3. Abstract ------------------------------------- 4
4. Introduction ------------------------------------- 6
5. Materials ------------------------------------- 10
6. Methods ------------------------------------- 12
7. Results ------------------------------------- 17
8. Discussion ------------------------------------- 23
9. Figures ------------------------------------- 27
10. Tables ------------------------------------- 54
11. References ------------------------------------- 58

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