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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
| dc.contributor.author | Po-Shiun Huang | en |
| dc.contributor.author | 黃柏勳 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:33:22Z | - |
| dc.date.available | 2014-08-18 | |
| dc.date.copyright | 2009-08-18 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43936 | - |
| dc.description.abstract | 異次三元體 Gα, Gβ, and Gγ 組成的 G 蛋白質為最常見的訊息傳遞分子，並且掌管許多重要的生理反應，是傳遞胞外訊息一個相當重要的媒介。其中以 Gα 次單元作為訊息啟動的樞紐，與 GTP 結合時轉為活化態並且引發下游訊息傳遞，直到 Gα 次單元本身的 GTPase 將 GTP 水解為 GDP 才終結反應。此外在G 蛋白質訊息途徑中，另一個關鍵性的蛋白質稱為 Regulator of G-protein signaling (RGS)，其在 G 蛋白質中扮演相當重要的角色，利用催化 Gα 次單元的 GTPase 活性以加速訊息傳遞的中止。過去在原生動物、真菌以及哺乳動物都相繼發現 RGS，直到最近才在阿拉伯芥中發現第一個植物 RGS (AtRGS1)，也是第一個被發現具有 N 端七個穿膜區結構的 RGS。本篇論文針對此 AtRGS1的 RGS domain 進行研究，利用動物的 Gα 次單元以及 RGS 進行物種間的交互作用，以螢光方法偵測 RGS 活性並佐以結構以及序列分析探討其物種間的差異。結果發現植物AtRGS1 也能催化動物 Gα 次單元 (Gαi1) 的 GTPase 活性，然而動物 RGS (RGS2, RGS4, and RGS9) 卻無法催化阿拉伯芥 Gα 次單元 (AtGPA1)。利用 Lucifer yellow 螢光標定的 RGS 即時偵測 AtRGS1 對於各種 Gα 次單元間的交互作用，結果顯示動物 Gαi1 及植物 AtGPA1 都能與 AtRGS1接觸，並且具有相似的親和性，因此進一步證實物種間的交互作用。綜合上述結果以及序列分析，植物 AtRGS1比較相似於動物 RGS4 所屬的R4 subfamily。雖然 AtRGS1 與動物 RGS 在保留性序列中大部分相似，但植物 AtRGS1 以及 AtGPA1 仍然具有其特殊性，因此可能是阻礙動物 RGS 與植物 AtGPA1交互作用的主要因素。 | zh_TW |
| dc.description.abstract | Heterotrimeric G-protein, composed of Gα subunit and Gβγ dimer, is one of the most prevalent cellular signaling molecules and relays outside stimuli to mediate a variety of physiological responses. The G-protein signaling cascades are switched on via the GTP bound Gα subunit and then off due to the GTP hydrolysis that is catalyzed by the intrinsic GTPase of Gα subunit. Furthermore, this signaling pathway can be modulated by a group of proteins, regulator of G-protein signaling (RGS), which function as GTPase activating proteins (GAPs) to accelerate the GTPase activity of Gα proteins and then shorten the duration of signal transduction. Accordingly, RGS has been served as a negative regulator in G-protein signaling and its functional characteristic also makes RGS as a potential therapeutic target.
In mammalian, diverse RGS proteins form a RGS superfamily and are widely distributed in various tissues. However, in Arabidopsis, there is only one RGS protein (AtRGS1), the first RGS found to contain N-terminal seven-transmembrane domain. This study was designed to compare the similarity between the RGS domain of AtRGS1 and mammalian RGS proteins via fluorescent assay. The GAP activity for RGS proteins were conducted by the fluorescent GTP analogue, BODIPY TR-GTP, and the results exhibited that plant AtRGS1 can accelerate the GTPase activity of both cognate plant Gα (AtGPA1) and mammalian Gαi1, but this phenomenon was not consistent with mammalian RGS4, which only had GAP activity toward mammalian Gαi1 but not plant AtGPA1. These results were further confirmed by monitoring the interaction between Lucifer yellow-modified RGS and Gα proteins in real time. Moreover, the affinity assay also reported plant AtRGS1 had the similar affinity toward plant AtGPA1 and mammalian Gαi1, which further supported the existence of inter-species interaction. Finally, according to above results and coupling with structural and sequence analysis, it can be concluded that the plant AtRGS1 is more similar to the mammalian R4 subfamily RGS; however, AtRGS1 and AtGPA1 still contain some plant-specific residues to cause the barrier for inter-species interaction, thus blocking the interaction between mammalian RGS proteins and plant AtGPA1. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:33:22Z (GMT). No. of bitstreams: 1 ntu-98-R96b47415-1.pdf: 4188895 bytes, checksum: b3bd681d19348e38ecac649fb39ab2fc (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | Contents
謝誌 i Contents ii List of Figures iv List of Tables v 中文摘要 vi Abstract vii Introduction 1 1.1 G-protein signaling pathway 1 1.2 Mammalian G-protein system 2 1.2.1 G-protein coupled receptor (GPCR) 2 1.2.2 Heterotrimeric G-protein and downstream effectors 3 1.2.3 RGS superfamily 6 1.3 Arabidopsis thaliana G-protein system 9 1.3.1 Heterotrimeric G-protein 9 1.3.2 Putative GPCR 11 1.3.3 RGS protein 12 1.3.4 Potential downstream effectors 13 1.3.5 Biochemical characteristic of plant Gα subunit 14 1.4 The motivation of this study 14 1.5 The purpose of this study 16 Materials and Methods 20 2.1 Materials 20 2.2 Plasmids construction 21 2.3 Proteins expression and purification 23 2.3.1 Mammalian and Arabidopsis thaliana G-alpha subunits 23 2.3.2 Mammalian and Arabidopsis thaliana RGS proteins 24 2.3.3 Full length AtRGS1 25 2.4 Functional assay of Gα proteins: AlF4- dependent activation and GDP/GTP auto-exchange rate measurement 26 2.5 Intrinsic GTPase activity of Gα proteins and GTPase accelerating activity of RGS proteins 27 2.6 Lucifer yellow labeling 28 2.6 Real-time monitoring the interaction between LY-labeled RGS and Gα subunits 29 Results 31 3.1 Sequence analysis of G-protein related subunits between human and Arabidopsis 31 3.1.1 Sequence alignment of RGS proteins 31 3.1.2 Sequence alignment of Gα proteins 33 3.2 Purification of mammalian and Arabidopsis thaliana Gα proteins 35 3.3 Purification of mammalian and Arabidopsis thaliana RGS proteins 36 3.4 Functional assay of purified Gα proteins 37 3.4.1 AlF4- and GTPγS dependent activation assays 37 3.4.2 GDP/GTP auto-exchange rate and intrinsic GTPase activity 40 3.5 GTPase accelerating activity of RGS proteins 43 3.6 Lucifer yellow labeling 45 3.6.1 Lucifer yellow labeling sites 45 3.6.2 Purification of LY-modified RGS proteins 47 3.6.3 GAP activity of LY-labeled RGS proteins 50 3.7 Lucifer yellow dependent interaction assay 52 3.7.1 Interaction between LY-labeled RGS and Gα proteins 52 3.7.2 Affinity assay 54 3.8 Functional assay of truncated AtGPA1 55 3.9 Purification and GAP activity of Full Length AtRGS1 58 3.10 Purification of AtTHF1 and truncated AtTHF1(dt) 60 Discussions 63 4.1 The biochemical properties of purified Gα proteins and the truncated AtGPA1 63 4.2 Inter-species interaction between mammalian and A. thaliana G-protein related subunits 65 4.3 Expression and purification of full length AtRGS1 72 4.4 The physiological role of AtTHF1 73 Conclusions 76 Perspectives 77 References 79 碩士論文口試問與答 85 List of Figures Figure 1. G-protein signaling cycle. 2 Figure 2. Protein structures of mammalian G-protein system. 8 Figure 3. Gα and RGS proteins used in this study and their known interactions. 18 Figure 4. Schematic framework of this study. 19 Figure 5. Sequence alignment of RGS domain of human RGS4, RGS9, RGS2, and RGS12, and Arabidopsis AtRGS1. 32 Figure 6. Sequence alignment of the human Gαi1, Gαt, Gαq, Gαs and Arabidopsis AtGPA1. 34 Figure 7. SDS-PAGE and Western blot analysis of purified Gα proteins. 35 Figure 8. SDS-PAGE and Western blot analysis of purified RGS proteins. 37 Figure 9. Functionalities examination for purified Gα proteins by monitoring the intrinsic Trp fluorescence. 39 Figure 10. Functionalities examination for purified Gα proteins by using the fluorescent GTP analogue. 42 Figure 11. BODIPY TR-GTP as a fluorescent probe for the RGS-catalyzed GTPase activity measurement. 45 Figure 12. Lucifer yellow labeling sites. 46 Figure 13. Purification, SDS-PAGE analysis and UV illumination of LY-modified RGS proteins. 49 Figure 14. GAP activity measurement of LY-modified RGS proteins. 51 Figure 15. Real-time interaction measurement between LY-modified RGS and Gα protein. 53 Figure 16. Affinity measurement for the interaction between LY-AtRGS1 and AtGPA1 (or Gαi1). 55 Figure 17. Protein purification and functional assays of truncated AtGPA1. 57 Figure 18. Protein purification and GAP activity measurement of full length AtRGS1. 60 Figure 19. Protein purification of AtTHF1 and the functionality tests of the AtTHF1-coupled AtGPA1. 62 Figure 20. The interface between RGS4 and Gαi1. 71 List of Tables Table 1. Primers for plasmid construction 22 Table 2. Expression vectors 22 | |
| dc.language.iso | en | |
| dc.subject | 物種間交互作用 | zh_TW |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | G 蛋白質 | zh_TW |
| dc.subject | 訊息傳遞 | zh_TW |
| dc.subject | G 蛋白質訊息調節蛋白質 | zh_TW |
| dc.subject | 螢光 | zh_TW |
| dc.subject | 螢光標定 | zh_TW |
| dc.subject | RGS | en |
| dc.subject | fluorescence | en |
| dc.subject | Arabidopsis thaliana | en |
| dc.subject | G-protein | en |
| dc.subject | signal transduction | en |
| dc.subject | AtRGS1 | en |
| dc.subject | AtGPA1 | en |
| dc.subject | AtTHF1 | en |
| dc.subject | Gα | en |
| dc.subject | inter-species interaction | en |
| dc.subject | fluorescent labeling | en |
| dc.title | 阿拉伯芥與哺乳類動物之 G-protein 訊息系統相關蛋白質具有物種間交互作用 | zh_TW |
| dc.title | A Study on the Inter-species Interaction among G-protein Related Signaling Subunits from Arabidopsis thaliana and Mammals | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 梁博煌(Po-Huang Liang),黃慶璨(Ching-Tsan Huang),張麗冠(Li-Kwan Chang),陳俊任(Chun-Jen Chen) | |
| dc.subject.keyword | 阿拉伯芥,G 蛋白質,訊息傳遞,G 蛋白質訊息調節蛋白質,螢光,螢光標定,物種間交互作用, | zh_TW |
| dc.subject.keyword | Arabidopsis thaliana,G-protein,signal transduction,AtRGS1,AtGPA1,AtTHF1,RGS,Gα,fluorescence,fluorescent labeling,inter-species interaction, | en |
| dc.relation.page | 88 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-14 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-98-1.pdf 未授權公開取用 | 4.09 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。