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標題: | 利用大腸桿菌系統進行AtMAPRs的類泛素化修飾之研究 Study of sumoylation of AtMAPRs using a reconstituted sumoylation system in E. coli |
作者: | Yuan-Chan Liu 劉沅瓚 |
指導教授: | 楊健志(Chien-Chih Yang) |
關鍵字: | AtMAPR2,AtMAPR5,MSBP1,類泛素, AtMAPR2,AtMAPR5,MSBP1,SUMO, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | AtMAPRs的同源蛋白廣泛存在於動植物中,參與固醇類的生合成等生理功能及具與固醇類、血基質等結合的能力。然而他們在植物中的功能仍尚未被清楚了解。AtMAPRs能在缺少E3泛素連接酶的情況下進行泛素化。另外,AtMAPRs在哺乳動物中的同源蛋白PGRMC1能在體內進行類泛素化(sumoylation)。泛素和類泛素在大小、構形、反應機制等多方面都高度相似。AtMAPRs是否也會被類泛素化就成了一個有趣的問題。而了解AtMAPRs的轉譯後修飾則能對於其他的生理功能有進一步的認識。本研究利用在E. coli中重建AtMAPRs與類泛素化系統,分析其被類泛素化能力。在此系統中,重組AtMAPRs與類泛素化系統被共表現於大腸桿菌內並部分產生類泛素化。分析含重組AtMAPRs及類泛素化系統的E. coli粗抽物,並以抗體偵測,發現AtMAPR2產生了由14 kDa上移到30 kDa的條帶。AtMAPR5也有類似的現象。這顯示AtMAPR2和AtMAPR5都可能會被類泛素化。而後進一步以液相層析串聯式質譜儀來分析這個結果。AtMAPR2的九個離胺酸中有四個都顯示被類泛素化修飾。離胺酸217則是AtMAPR5中唯一出現類泛素化修飾的離胺酸。將AtMAPR2的四個被辨認為類泛素化潛在的離胺酸(K18、K35、K61和K76)突變為精胺酸之後,依然能夠看到條帶上移的現象,訊號強度為正常AtMAPR2的21%。而將AtMAPR5的離胺酸217突變為精胺酸之後,AtMAPR5就不再出現上移條帶。使用MG132處理的阿拉伯芥中可以偵測到AtMAPR2和它的一個上移條帶,其身份仍待鑑定。本研究發現AtMAPR2可被類泛素化,並有多個類泛素化位點。AtMAPR5也可被類泛素化,但僅有離胺酸217一個位點。轉譯後修飾時常與蛋白質的生理功能相關,這些結果將提供日後AtMAPR2和AtMAPR5的研究方向。 Arabidopsis thaliana membrane-associated progesterone binding proteins (AtMAPRs) and its homologs are extensively found in metazoans and plants. Members have been reported to function in steroid biosynthesis and binding to heme and steroid. However, their functions in plants are poorly understood. AtMAPRs can be E3-independently ubiquitinated in in vitro ubiquitination assays, while the mammalian homolog of AtMAPRs, PGRMC1, is found to be sumoylated in vivo. Ubiquitin and SUMO are similar in terms of size, structure, mechanism and more. It is interesting to study that if AtMAPRs can be sumoylated as well. This study utilized a reconstituted sumoylation system in E. coli to analyze the sumoylation activity of AtMAPRs. In the system, recombinant AtMAPRs were co-expressed with the sumoylation machinery and were partially sumoylated. An upper-shifted AtMAPR2 (30 kDa) was detected in oppose to its original 14 kDa band. A similar band-shift was also observed for AtMAPR5. These indicate that AtMAPR2 and AtMAPR5 can potentially be sumoylated. The identification of sumoylated AtMAPR2 and sumoylated AtMAPR5 were further studied using LC-MS/MS. Four of the nine lysines of AtMAPR2 were identified to be sumoylation target sites. K217 was the only identified sumoylation site in AtMAPR5. The band shift for AtMAPR2K18R/K35R/K61R/K76R was still present, only to be 79% lighter than that of the wild type, and AtMAPR5K217R did not have a shifted band. Upper-shifting bands of AtMAPR2 was observed in MG132 treated Arabidopsis, its identity is still to be analyzed. This study showed that AtMAPR2 can be sumoylated and had multiple sumoylation sites. AtMAPR5 can be sumoylated and had only one site, K217. Since post-translational modifications are often related to the functions of a protein, these results provide directions for further functional studies of AtMAPR2 and AtMAPR5. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21878 |
DOI: | 10.6342/NTU201803970 |
全文授權: | 未授權 |
顯示於系所單位: | 生化科技學系 |
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