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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曾秀如(Shiou-Ru Tzeng) | |
| dc.contributor.author | Yi-Ting Kuo | en |
| dc.contributor.author | 郭怡廷 | zh_TW |
| dc.date.accessioned | 2021-06-08T00:45:24Z | - |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-03 | |
| dc.identifier.citation | 1. Yang, Q., L.S. Chen, and V. Gandhi, Mechanism-based combinations with Pim kinase inhibitors in cancer treatments. Curr Pharm Des, 2014. 20(42): p. 6670-81. 2. Le, B.T., et al., Targeting Pim kinases for cancer treatment: opportunities and challenges. Future Med Chem, 2015. 7(1): p. 35-53. 3. Arunesh, G.M., et al., Small molecule inhibitors of PIM1 kinase: July 2009 to February 2013 patent update. Expert Opin Ther Pat, 2014. 24(1): p. 5-17. 4. Chen, J., et al., Pim-1 plays a pivotal role in hypoxia-induced chemoresistance. Oncogene, 2009. 28(28): p. 2581-92. 5. Bachmann, M. and T. Moroy, The serine/threonine kinase Pim-1. Int J Biochem Cell Biol, 2005. 37(4): p. 726-30. 6. Blanco-Aparicio, C. and A. Carnero, Pim kinases in cancer: diagnostic, prognostic and treatment opportunities. Biochem Pharmacol, 2013. 85(5): p. 629-43. 7. Warfel, N.A. and A.S. Kraft, PIM kinase (and Akt) biology and signaling in tumors. Pharmacol Ther, 2015. 151: p. 41-49. 8. Merkel, A.L., E. Meggers, and M. Ocker, PIM1 kinase as a target for cancer therapy. Expert Opin Investig Drugs, 2012. 21(4): p. 425-36. 9. Zemskova, M., et al., The PIM1 kinase is a critical component of a survival pathway activated by docetaxel and promotes survival of docetaxel-treated prostate cancer cells. J Biol Chem, 2008. 283(30): p. 20635-44. 10. Lori, C., et al., Effect of single amino acid substitution observed in cancer on Pim-1 kinase thermodynamic stability and structure. PLoS One, 2013. 8(6): p. e64824. 11. Magnuson, N.S., et al., Why target PIM1 for cancer diagnosis and treatment? Future Oncol, 2010. 6(9): p. 1461-78. 12. Narlik-Grassow, M., C. Blanco-Aparicio, and A. Carnero, The PIM family of serine/threonine kinases in cancer. Med Res Rev, 2014. 34(1): p. 136-59. 13. Thomas, M., et al., The proto-oncogene Pim-1 is a target of miR-33a. Oncogene, 2012. 31(7): p. 918-28. 14. Bullock, A.N., et al., Structure and substrate specificity of the Pim-1 kinase. 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Kumar, A., et al., Crystal structures of proto-oncogene kinase Pim1: a target of aberrant somatic hypermutations in diffuse large cell lymphoma. J Mol Biol, 2005. 348(1): p. 183-93. 22. Pasqualucci, L., et al., Hypermutation of multiple proto-oncogenes in B-cell diffuse large-cell lymphomas. Nature, 2001. 412(6844): p. 341-6. 23. Campagnoli, M.F., et al., Molecular basis of Diamond-Blackfan anemia: new findings from the Italian registry and a review of the literature. Haematologica, 2004. 89(4): p. 480-9. 24. Raran-Kurussi, S. and D.S. Waugh, The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose-binding protein but their folding is either spontaneous or chaperone-mediated. PLoS One, 2012. 7(11): p. e49589. 25. Boehr, D.D., H.J. Dyson, and P.E. Wright, An NMR perspective on enzyme dynamics. Chem Rev, 2006. 106(8): p. 3055-79. 26. Rossi, P., et al., A microscale protein NMR sample screening pipeline. J Biomol NMR, 2010. 46(1): p. 11-22. 27. Goto, N.K., et al., A robust and cost-effective method for the production of Val, Leu, Ile (delta 1) methyl-protonated 15N-, 13C-, 2H-labeled proteins. J Biomol NMR, 1999. 13(4): p. 369-74. 28. Kay, L.E., NMR studies of protein structure and dynamics. J Magn Reson, 2005. 173(2): p. 193-207. 29. Tugarinov, V. and L.E. Kay, Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. J Am Chem Soc, 2003. 125(45): p. 13868-78. 30. Tong, K.I., M. Yamamoto, and T. Tanaka, A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli. J Biomol NMR, 2008. 42(1): p. 59-67. 31. Krishnarjuna, B., et al., Amino acid selective unlabeling for sequence specific resonance assignments in proteins. J Biomol NMR, 2011. 49(1): p. 39-51. 32. Takeuchi, K., et al., 1-13C amino acid selective labeling in a 2H15N background for NMR studies of large proteins. J Biomol NMR, 2007. 38(1): p. 89-98. 33. Olsson, U. and M. Wolf-Watz, Overlap between folding and functional energy landscapes for adenylate kinase conformational change. Nat Commun, 2010. 1: p. 111. 34. Leavitt, S. and E. Freire, Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol, 2001. 11(5): p. 560-6. 35. Johnson, C.M., Differential scanning calorimetry as a tool for protein folding and stability. Arch Biochem Biophys, 2013. 531(1-2): p. 100-9. 36. Rosenzweig, R. and L.E. Kay, Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu Rev Biochem, 2014. 83: p. 291-315. 37. Waugh, D.S., An overview of enzymatic reagents for the removal of affinity tags. Protein Expr Purif, 2011. 80(2): p. 283-93. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17884 | - |
| dc.description.abstract | Pim1 激酶在細胞的週期調控、增生以及生存等方面都扮演著非常關鍵性的角色。在許多的惡性腫瘤，例如前列腺癌以及多種類型的白血病當中，都有Pim1激酶過度表現的現象，因此Pim1激酶被認為是現今藥物開發中非常重要的標靶蛋白。目前已透過X光結晶繞射的技術來得到許多和抑制物結合的蛋白在活化和不活化狀態下的結構，並利用這些結構來研究抑制物對於蛋白的調控機制。到目前為止，大部分激酶結構的研究是透過X光結晶繞射的技術，只有極少部分是探究激酶在溶液當中的結構和動力學的情形。蛋白質在溶液當中的運動在蛋白活性和功能上一直都扮演相當重要的角色，目前可以透過核磁共振光譜以及其他生物物理的技術來探討。因此，我的實驗目的主要是利用液相核磁共振技術來分析Pim1激酶在脫輔基和與抑制物結合狀態下的特性。為了能夠將核磁共振運用在大分子蛋白的分析，我們將大腸桿菌培養在重水環境當中來表現重組蛋白MBP Pim1，並藉由親和層析法和凝膠層析法純化得到Pim1。完成常用的三共振實驗後，需要透過後續的分析判斷完成Pim1 激酶在序列骨鏈上的判定。另外，我們採用了選擇性同位素標註的方式來提升核磁共振光譜判定的正確性。在核磁共振實驗以外，為了研究Pim1和抑制物結合後的能量變化，我們使用了等量滴定微量熱儀以及式差掃描量熱儀來分析。在等量滴定微量熱儀的結果顯示，Pim1激酶和抑制物的結合均為放熱反應。在式差掃描量熱儀的結果顯示，Pim1 和SGI-1776結合下的熔點溫度比Pim1脫輔基熔點溫度高了10oC，這顯示了在有藥物結合之下，Pim1是比較穩定的。在比較藥物對於蛋白穩定性的影響之外，我們也去比較了野生型Pim1以及其他會致病的突變型的穩定性，從核磁共振的光譜中發現，突變型N82K和L193F相較於野生型來說是較為不穩定的。總結，透過核磁共振和蛋白質動力學的分析結果，對於藥物設計是十分重要的。 | zh_TW |
| dc.description.abstract | Pim1 kinase plays pivotal roles in cell cycle control, proliferation and cell survival. Overexpression of Pim1 has been observed in many malignancies such as prostate cancer and several types of human leukemia [1]. For the reason above, Pim1 is emerging as an important target in drug discovery. X-ray crystallography has revealed various active and inactive conformational states of kinases, which are implicated in their regulation and modulation by inhibitors. However, current structural understanding of kinases is largely based on X-ray crystallographic studies, whereas very little data exist on the conformations and dynamics that kinases adopt in the solution state. Moreover, the important role of protein motions in regulating enzymatic activity and protein function has been demonstrated by NMR spectroscopy along with other biophysical techniques. Thus, the goal of my research project is to characterize Pim1 kinase in apo and inhibitor-bound states by modern solution NMR techniques. To extend the use of NMR to larger systems, the recombinant protein, MBP Pim1, has been produced in deuterated media and then purified with affinity column and gel-filtration. With the standard triple-resonance experiments, the assignment of backbone NMR resonances for Pim1 kinase is still in progress. To confirm spectra assignments, we also adopt several specific isotope labeling strategies. Moreover, in order to investigate the energetic basis of inhibitor binding to Pim1 variants, we have used isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The results show the interactions of Pim1 and inhibitors are exothermic reactions and the Tm value of Pim1 with SGI-1776 is higher than apo form. It means Pim1 in drug-bound state is more stable than apo form. Besides, we also explore the stability of mutant types N82K, L193F, P311T and WT by NMR. According to the spectra, WT Pim1 is more stable than N82K and L193F. In conclusion, the information from NMR and thermodynamic studies will provide enormous value in Pim1 kinase drug design. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T00:45:24Z (GMT). No. of bitstreams: 1 ntu-104-R02442030-1.pdf: 5823501 bytes, checksum: bc0184360ab7d4e305f1e491e201c1a6 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員審定書………………………..…………………………………………….# 摘要………..……………………….…………………………………………….…..Ⅰ Abstract………………………………………………………………………………Ⅱ Abbreviation list……………………………………………………………….……..Ⅳ Catalog………………………………………………………………………..……....Ⅵ 1. Introduction……………………………………………………………………….. 1 1-1. The introduction of Pim kinase…………………………………………………... 1 1-2.The characteristics of Pim1 and its function……………………………………… 1 1-3.Expression and regulation of Pim1. ……………………………………………… 2 1-4.The structure of Pim1……………………………………………………………... 3 1-5.Pim1 kinase in cancer…………………………………………………………….. 5 1-6.Pim kinase inhibitors……………………………………………………………… 6 1-7.Pim kinase as targets for cancer therapy………………………………………….. 7 2. The specific aim…………………………………………………………………… 7 3. Materials and methods……………………………………………………………. 8 3-1. Materials…………………………………………………………………………. 8 3-1-1 Biomaterials, reagents and consumables……………………………………….. 8 3-1-2 Instruments and equipment……………………………………………………. 11 3-1-3. The table list of the characteristic of kinase inhibitors……………………….. 12 3-2. Methods…………………………………………………………………………. 13 3-2-1. Inhibitors decision for Pim1 in structural biology analysis…………………... 13 3-2-2. Cloning of the Escherichia coli BL21 (DE3) strain MBP-Pim1……………... 14 3-2-3. Preparation of competent cells………………………………………………... 14 3-2-4. Plasmid transformation……………………………………………………….. 15 3-2-5. Primer design…………………………………………………………………. 16 3-2-6. Site Directed Mutagenesis……………………………………………………. 16 3-2-7. Recombinant protein expression and purification……………………………. 18 3-2-8. Nuclear magnetic Resonance (NMR) spectroscopy…………………………. 24 4. Results…………………………………………………………………………….. 32 4-1. Protein expression and purification…………………………………………….. 32 4-1-1. Expression test for target protein decision……………………………………. 32 4-1-2. 15N-13C-2H labeled MBP-Pim1 expression and purification in perdeuterated M9 medium…………..…………………………………………………………………... 34 4-1-3. TEV protease expression and purification……………………………………. 34 4-2. Protein NMR analysis…………………………………………………………... 35 4-2-1. NMR buffer optimization…………………………………………………….. 35 4-2-2. The 1H-15N HSQC spectra of Pim1 with drugs bound compared with the 1H-15N HSQC spectra of apo-Pim1………………………………………………….. 36 4-2-3. 1H-15N HSQC spectra observation for WT Pim1 with SGI-1776 and SD-169 at different temperature.………………………………………………………………... 36 4-2-4. Pim1 NMR assignment………………………………………………………. 37 4-2-5. Selective amino acid labeled Pim1…………………………………………… 37 4-2-5-1. Selective amide group labeled……………………………………………… 37 4-2-5-1-1. 15N-lysine selectively labeled WT Pim1…………………………………. 38 4-2-5-1-2. 14N-arginine selectively reverse labeled WT Pim1………………………. 38 4-2-5-2. 1-13C selective amino acid labeled strategy………………………………… 38 4-2-5-2-1. 1-13C-leucine selectively labeled WT Pim1……………………………… 39 4-2-5-2-2. 1-13C-Valine selectively labeled WT Pim1………………………………. 39 4-2-5-3. 13C-1H-methyl-TROSY spectra of 13C-IVL-labelled Pim1………………… 40 4-2-6. Summary of backbone assignment of Pim1………………………………….. 40 4-3. The ITC analysis of Pim1 WT…………………………………………………. 40 4-4. The DSC analysis of Pim1 WT and SGI-1776 bound form……………………. 41 4-5. The structural biology analysis of mutant types of Pim1………………………. 41 4-5-1. The study of Pim1 P311T…………………………………………………….. 41 4-5-1-1. The NMR spectra analysis of Pim1 P311T………………………………… 41 4-5-1-2. The crystal structure analysis of Pim1 P311T……………………………… 42 4-5-1-3. The ITC analysis of Pim1 P311T…………………………………………... 43 4-5-2. The study of Pim1 N82K……………………………………………………... 43 4-5-2-1. The NMR spectra analysis of Pim1 N82K…………………………………. 43 4-5-3. The study of Pim1 L193F…………………………………………………….. 44 4-5-3-1. The NMR spectra analysis of Pim1 L193F………………………………… 44 4-5-4. The study of other mutant types of Pim1…………………………………….. 45 5. Discussion………………………………………………………………………… 45 5-1. The NMR analysis of Pim1 WT………………………………………………... 46 5-2. The DSC analysis of Pim1……………………………………………………… 47 5-3. The analysis of mutant types of Pim1. …………………………………………. 47 5-4. The future work………………………………………………………………… 48 6. Figures……………………………………………………………………………. 48 Figure 1. Molecular regulation of PIM1 expression and functions…………………. 49 Figure 2. The crystal structure of Pim1 with ATP analog…………………………… 50 Figure 4. pET28a, pETM44 vector and sequences………………………………….. 52 Figure 5. The graphic illustration of NMR time scale………………………………. 53 Figure 6. The NMR backbone assignments…………………………………………. 54 Figure 7. The metabolic pathway of IVL precursors………………………………... 55 Figure 8. Protein Crystallization and drug soaking………………………………….. 56 Figure 9. Designed of target protein…………………………………………………. 57 Figures 10. Pim1 and MBP-Pim1 expression and purification in protonated M9 medium………………………………………………………………………………. 59 Figure 11. MBP-Pim1 affinity purification and size-exclusion chromatography results………………………………………………………………………………… 60 Figure 12. The purification results of TEV protease……………………………....... 61 Figure 13. The 1H-15N HSQC spectra in different buffer condition………………… 62 Figure 14. Overlay of 1H-15N HSQC NMR spectra in different condition………….. 63 Figure 15. Overlay of 1H-15N HSQC spectra of Pim1 apo-form and inhibitor bound form………………………………………………………………………………….. 64 Figure 16. The 1H-15N HSQC spectra of Pim1 in pH 8 at different temperature…… 65 Figure 17. The 1H-15N HSQC spectra of Pim1 in pH 7 at different temperature……. 66 Figure 18. Sequential assignment of Pim1 WT……………………………………… 67 Figure 19. The spectra of Pim1 kinase with specific labeling……………………….. 69 Figure 20. The spectra assignment of Pim1…………………………………………. 70 Figure 21. Thermodynamic parameters for the binding of the Pim1 WT to SGI-1776…………………………………………………………………………….. 71 Figure 22. Thermodynamic parameters for the binding of the Pim1 WT to SD-169……………………………………………………………………………….. 72 Figure 23. DSC thermograms of Pim1 WT apoform and SGI-1776 bound form…… 73 Figure 24. 1H-15N HSQC spectra of Pim1 P311T in pH 8 at different temperature. …………………………………………………………………………. 74 Figure 25. 1H-15N HSQC spectra of Pim1 P311T are in comparison with Pim1 WT………………………………………………………………………………….... 75 Figure 26. The crystals in the buffer optimization…………………………………... 76 Figure 27. Overall crystal structures of Pim1 WT and P311T…………………......... 77 Figure 28. Crystal structures of Pim1 P311T compare with Pim1 WT……………... 78 Figure 29. Thermodynamic parameters for the binding of the Pim1 P311T to SGI-1776…………………………………………………………………………….. 79 Figure 30. 1H-15N HSQC spectra of N82K with drugs……………………………… 80 Figure 31. Overlay of 1H-15N HSQC spectra of Pim1 WT and SGI-1776…………... 81 Figure 32. The results of Pim1 L193F purification………………………………...... 82 Figure 33. 1H-15N-1H HSQC spectra of Pim1 L193F apo-form at different temperature…………………………………………………………………………... 84 Figure 34. The SDS-PAGE image of other Pim1 mutant types purification………... 85 7. Table……………………………………………………………………………… 86 Table 1. The types of competent cell………………………………………………... 86 Table 2. Amino acid composition of Pim1…………………………………………... 87 Table 3. The NMR buffer optimization……………………………………………… 88 Table 4. The Pim1 crystal buffer optimization………………………………………. 89 8. Reference.………………………………………………………………………… 91 | |
| dc.language.iso | en | |
| dc.title | Pim1激酶在原態和配體結合狀態下的特性研究 | zh_TW |
| dc.title | Characterization of PIM1 Kinase in the Apo and Ligand-Bound States | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 徐駿森(Chun-Hua Hsu),詹迺立(Nei-Li Chan) | |
| dc.subject.keyword | Pim1激?,NMR判定,激?抑制物, | zh_TW |
| dc.subject.keyword | Pim1 kinase,NMR assignment,kinase inhibitor, | en |
| dc.relation.page | 93 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2015-08-04 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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