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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43464完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊啟伸(Chii-Shen Yang) | |
| dc.contributor.author | Chung-Hung Hsieh | en |
| dc.contributor.author | 謝昌泓 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:22:02Z | - |
| dc.date.available | 2012-08-21 | |
| dc.date.copyright | 2009-08-21 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-19 | |
| dc.identifier.citation | 1. Neubig, R.R. & Siderovski, D.P. Regulators of G-protein signalling as new central nervous system drug targets. Nat Rev Drug Discov 1, 187-97 (2002).
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Differences in regional and subcellular localization of G(q/11) and RGS4 protein levels in Alzheimer's disease: correlation with muscarinic M1 receptor binding parameters. Synapse 47, 58-65 (2003). 25. Huuhka, K. et al. RGS4 polymorphism and response to electroconvulsive therapy in major depressive disorder. Neurosci Lett 437, 25-8 (2008). 26. Ding, J. et al. RGS4-dependent attenuation of M4 autoreceptor function in striatal cholinergic interneurons following dopamine depletion. Nat Neurosci 9, 832-42 (2006). 27. Hooks, S.B., Martemyanov, K. & Zachariou, V. A role of RGS proteins in drug addiction. Biochem Pharmacol 75, 76-84 (2008). 28. Mirnics, K., Middleton, F.A., Stanwood, G.D., Lewis, D.A. & Levitt, P. Disease-specific changes in regulator of G-protein signaling 4 (RGS4) expression in schizophrenia. Mol Psychiatry 6, 293-301 (2001). 29. Riddle, E.L., Schwartzman, R.A., Bond, M. & Insel, P.A. Multi-tasking RGS proteins in the heart: the next therapeutic target? Circ Res 96, 401-11 (2005). 30. Wieland, T., Lutz, S. & Chidiac, P. Regulators of G protein signalling: a spotlight on emerging functions in the cardiovascular system. Curr Opin Pharmacol 7, 201-7 (2007). 31. Manzur, M. & Ganss, R. Regulator of G protein signaling 5: a new player in vascular remodeling. Trends Cardiovasc Med 19, 26-30 (2009). 32. Gu, S., Cifelli, C., Wang, S. & Heximer, S.P. RGS proteins: identifying new GAPs in the understanding of blood pressure regulation and cardiovascular function. Clin Sci (Lond) 116, 391-9 (2009). 33. Gudermann, T., Nurnberg, B. & Schultz, G. Receptors and G proteins as primary components of transmembrane signal transduction. Part 1. G-protein-coupled receptors: structure and function. J Mol Med 73, 51-63 (1995). 34. Johnson, J.A. & Lima, J.J. Drug receptor/effector polymorphisms and pharmacogenetics: current status and challenges. Pharmacogenetics 13, 525-34 (2003). 35. Gonzalez-Maeso, J. & Sealfon, S.C. Agonist-trafficking and hallucinogens. Curr Med Chem 16, 1017-27 (2009). 36. Bosier, B. & Hermans, E. Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci 28, 438-46 (2007). 37. Abrams, P. et al. Muscarinic receptors: their distribution and function in body systems, and the implications for treating overactive bladder. Br J Pharmacol 148, 565-78 (2006). 38. Michal, P., El-Fakahany, E.E. & Dolezal, V. Muscarinic M2 receptors directly activate Gq/11 and Gs G-proteins. J Pharmacol Exp Ther 320, 607-14 (2007). 39. Zhong, H. & Neubig, R.R. Regulator of G protein signaling proteins: novel multifunctional drug targets. J Pharmacol Exp Ther 297, 837-45 (2001). 40. Hague, C. et al. Selective inhibition of alpha1A-adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop. J Biol Chem 280, 27289-95 (2005). 41. Bernstein, L.S. et al. RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. J Biol Chem 279, 21248-56 (2004). 42. Gold, S.J., Ni, Y.G., Dohlman, H.G. & Nestler, E.J. Regulators of G-protein signaling (RGS) proteins: region-specific expression of nine subtypes in rat brain. J Neurosci 17, 8024-37 (1997). 43. Snow, B.E. et al. A G protein gamma subunit-like domain shared between RGS11 and other RGS proteins specifies binding to Gbeta5 subunits. Proc Natl Acad Sci U S A 95, 13307-12 (1998). 44. Hart, M.J. et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science 280, 2112-4 (1998). 45. Kozasa, T. et al. p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13. Science 280, 2109-11 (1998). 46. Burchett, S.A. Psychostimulants, madness, memory... and RGS proteins? Neuromolecular Med 7, 101-27 (2005). 47. Blazer, L.L. & Neubig, R.R. Small molecule protein-protein interaction inhibitors as CNS therapeutic agents: current progress and future hurdles. Neuropsychopharmacology 34, 126-41 (2009). 48. Wang, Y., Lee, Y., Zhang, J. & Young, K.H. Identification of peptides that inhibit regulator of G protein signaling 4 function. Pharmacology 82, 97-104 (2008). 49. Roman, D.L. et al. Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay. Mol Pharmacol 71, 169-75 (2007). 50. Kimple, A.J. et al. The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face. Biochim Biophys Acta 1774, 1213-20 (2007). 51. Chen, Y.C. in Institute of Microbiology and Biochemistry (National Taiwan University, Tiipei, 2008). 52. Taymans, J.M. et al. Detailed localization of regulator of G protein signaling 2 messenger ribonucleic acid and protein in the rat brain. Neuroscience 114, 39-53 (2002). 53. Chen, Y.C. (2008). Development of a new drug screening strategy using fluorescent probe modified regulator of G protein signaling (RGS) and Galpha proteins. National Taiwan University, Taipei. 54. Yang, C.S., Skiba, N.P., Mazzoni, M.R. & Hamm, H.E. Conformational changes at the carboxyl terminus of Galpha occur during G protein activation. J Biol Chem 274, 2379-85 (1999). 55. Johnston, C.A. et al. GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling. Proc Natl Acad Sci U S A 104, 17317-22 (2007). 56. Huang, P.S. (2009). A study on the inter-species interaction among G=protein related signaling subunits from Arabidopsis thaliana and mammals. National Taiwan University, Taipei. 57. Huang, C.S. (2008). Molecular mechanism of Arabidopsis thaliana G-protein system and its signaling regulatory protein, AtRGS1 National Taiwan University, Taipei. 58. Mittmann, C. et al. Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure. Cardiovasc Res 55, 778-86 (2002). 59. Owen, V.J. et al. Expression of RGS3, RGS4 and Gi alpha 2 in acutely failing donor hearts and end-stage heart failure. Eur Heart J 22, 1015-20 (2001). 60. Neitzel, K.L. & Hepler, J.R. Cellular mechanisms that determine selective RGS protein regulation of G protein-coupled receptor signaling. Semin Cell Dev Biol 17, 383-9 (2006). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43464 | - |
| dc.description.abstract | 異三元體 G 蛋白質系統 (heterotrimeric G-protein system)，在人體中調控超過一半以上的賀爾蒙以及多數的生理反應。接受多樣的外界訊息分子須倚賴種類繁複的 G 蛋白質耦合接受器 (G-protein coupled receptor，GPCR)，並且經由觸發細胞內部各種訊息路徑，進而產生相對應的生理反應去面對外界的改變。雖然 G 蛋白質系統是由 GPCR 啟動，但是整體系統的專一性及作用時間的長短卻是由 G 蛋白質調節蛋白質 (regulator of G-protein signaling，RGS) 負責。RGS 在整個 G 蛋白質系統中，透過加速 Gα 次單元體的 GTP 水解以達到控制訊息傳遞的結束。除了在 G 蛋白質系統中的重要性之外，RGS 在人體中的分布，主要集中在中樞神經及心血管系統，此一特點大大的增加了藥物的組織專一性。綜合以上幾點，針對 RGS 與 Gα 次單元體交互作用為標靶的藥物，十分具有開發的潛力。
為了加速 RGS 藥物的發展，本實驗的目的是希望發展出一套以螢光為基礎進行 RGS-Gα 蛋白質交互作用的觀測系統。前人的研究初步證明此系統的可行性，但是螢光訊號變化卻仍相當微弱。本研究透過以結構為基礎的一系列最佳化過程，找出適當的螢光標定觀測點。更進一步發現，α 螺旋及環狀區塊的交界處，是交互作用觀測點的特性。並且成功的由阿拉伯芥的 RGS 蛋白質證明，此概念更可推廣至具有與 RGS4 具有結構同源性的 R4 家族。綜合以上實驗結果，除了證明挑選螢光標定點策略的可行，也證明此策略的廣泛應用性。 | zh_TW |
| dc.description.abstract | Heterotrimeric G-protein system mediates more than 60% of hormones in human and senses extracellular signals via G-protein coupled receptor (GPCR) which triggers cytosolic signaling cascades to respond to the environment. This signal is highly specific and regulated by a group of proteins, called regulator of G-protein signaling (RGS). RGS serves as GTPase accelerating protein (GAP) which acts specifically at corresponding Gα protein and terminates signaling pathway. Besides, RGS can distribute specifically at central nervous system, cardiovascular system or other tissues, and since it shows high specificity toward Gα protein, it becomes a promising drug target for therapeutic usage.
Our goal is to develop a fluorescence-based screening system to monitor the interactions between RGS and Gα, and our data showed that the helix-loop junction at RGS proteins are the best positions for fluorescent probe modifications to serve such a purpose. This strategy was then successfully shown by using an AtRGS1domain protein with fluorescent probe modified at helix-loop junction area and a sensitive fluorescence change can be observed upon activation. A general principle to select proper residues for monitoring protein-protein interaction (PPI) was thus proposed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:22:02Z (GMT). No. of bitstreams: 1 ntu-98-R96b47408-1.pdf: 2832973 bytes, checksum: b3535856ec007ee864763ef75f557e5f (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 謝誌 .................................................................................................................................. i
Content ............................................................................................................................ ii List of Figures ................................................................................................................. v List of Tables .................................................................................................................. vi List of Abbreviation ...................................................................................................... vii 中文摘要 ....................................................................................................................... viii Abstract ........................................................................................................................... x Introduction .................................................................................................................... 1 Heterotrimeric G-Protein system .......................................................................... 1 The activation of G protein signaling pathway .................................................... 1 Back to ground state ....................................................................................... 2 RGS proteins: diversity and structures ................................................................ 3 The classification of diverse RGS .................................................................. 4 Structures ........................................................................................................ 6 The RGS drug ........................................................................................................ 11 Targets for RGS drug ........................................................................................... 13 RGS drug screening system ................................................................................. 15 The purpose of this study ..................................................................................... 15 Flow chart .............................................................................................................. 16 Materials and methods ................................................................................................. 18 Materials ................................................................................................................ 18 Plasmid Construction and Site-directed Mutagenesis ...................................... 18 Expression and purification of RGS and its mutants ........................................ 20 Expression and purification of Gαi ..................................................................... 21 Gαi activity assay .................................................................................................. 22 GTPase activity assay and GAP assay ................................................................ 23 LY Labeling and Purification .............................................................................. 24 Labeling efficiency assay ...................................................................................... 24 Interaction assay ................................................................................................... 24 Real time monitoring .................................................................................... 24 High-throughput detecting .......................................................................... 25 Results ............................................................................................................................ 26 Constructs of different labeling residue on RGS4 ............................................. 26 Expression and purification of Gαi ..................................................................... 28 Expression and purification of RGS4 and RGS4 mutants ............................... 29 Expression and purification of AtRGS1 domain and AtRGS1 domain(SC2) . 30 Gαi activity assay .................................................................................................. 31 AlF4- dependent conformational change of switch regions ....................... 31 GTPase activity assay ................................................................................... 33 GAP activity .......................................................................................................... 33 Labeling efficiency assay ...................................................................................... 36 Purification of LY labeled RGS ........................................................................... 39 GAP activity assay of LY labeled RGS ............................................................... 40 Interaction assay ................................................................................................... 42 Interaction assay through 96-well plate .............................................................. 44 Discussion ...................................................................................................................... 46 Selection strategy for sensitive fluorescent reporter ......................................... 46 Comparison of different RGS drug screening system ....................................... 47 Conclusion ..................................................................................................................... 50 Future works ................................................................................................................. 50 碩士論文口試問與答摘要 ............................................................................................ 51 References ...................................................................................................................... 53 | |
| dc.language.iso | en | |
| dc.subject | RGS 藥物 | zh_TW |
| dc.subject | 藥物篩選系統螢光標定 | zh_TW |
| dc.subject | 蛋白質蛋白質交互作用 | zh_TW |
| dc.subject | α 螺旋及環狀區塊交界處 | zh_TW |
| dc.subject | drug screening system | en |
| dc.subject | RGS drug | en |
| dc.subject | flurescence probe | en |
| dc.title | 以結構為基礎建構RGS 藥物之篩選平台 | zh_TW |
| dc.title | Structure based development of fluorescently modified regulator of G protein signaling (RGS) as a strategy for RGS drug screening | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 梁博煌(Po-Huang Liang),張麗冠(Li-Kwan Chang),黃慶璨(Ching-Tsan Huang),陳俊任(Chun-Jen Chen) | |
| dc.subject.keyword | 藥物篩選系統螢光標定,RGS 藥物,蛋白質蛋白質交互作用,α 螺旋及環狀區塊交界處, | zh_TW |
| dc.subject.keyword | drug screening system,flurescence probe,RGS drug, | en |
| dc.relation.page | 57 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-19 | |
| 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 | |
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