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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 曾秀如 | |
dc.contributor.author | Yu-Ting Ma | en |
dc.contributor.author | 馬瑜廷 | zh_TW |
dc.date.accessioned | 2021-07-09T15:53:58Z | - |
dc.date.available | 2029-02-14 | |
dc.date.copyright | 2019-03-11 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2019-02-14 | |
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J Cell Sci 123, 40-50, doi:10.1242/jcs.055889 (2010). 13 Chua, H. H. et al. RBMY, a novel inhibitor of glycogen synthase kinase 3beta, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma. Hepatology 62, 1480-1496, doi:10.1002/hep.27996 (2015). 14 Tsuei, D. J. et al. Male germ cell-specific RNA binding protein RBMY: a new oncogene explaining male predominance in liver cancer. PLoS One 6, e26948, doi:10.1371/journal.pone.0026948 (2011). 15 Le, B. T. et al. Targeting Pim kinases for cancer treatment: opportunities and challenges. Future Med Chem 7, 35-53, doi:10.4155/fmc.14.145 (2015). 16 Bachmann, M. & Moroy, T. The serine/threonine kinase Pim-1. Int J Biochem Cell Biol 37, 726-730, doi:10.1016/j.biocel.2004.11.005 (2005). 17 Blanco-Aparicio, C. & Carnero, A. Pim kinases in cancer: diagnostic, prognostic and treatment opportunities. Biochem Pharmacol 85, 629-643, doi:10.1016/j.bcp.2012.09.018 (2013). 18 Brault, L. et al. PIM serine/threonine kinases in the pathogenesis and therapy of hematologic malignancies and solid cancers. Haematologica 95, 1004-1015, doi:10.3324/haematol.2009.017079 (2010). 19 Warfel, N. A. & Kraft, A. S. PIM kinase (and Akt) biology and signaling in tumors. Pharmacol Ther 151, 41-49, doi:10.1016/j.pharmthera.2015.03.001 (2015). 20 Saris, C. J., Domen, J. & Berns, A. The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG. EMBO J 10, 655-664 (1991). 21 Xie, Y. et al. The 44 kDa Pim-1 kinase directly interacts with tyrosine kinase Etk/BMX and protects human prostate cancer cells from apoptosis induced by chemotherapeutic drugs. Oncogene 25, 70-78, doi:10.1038/sj.onc.1209058 (2006). 22 Zemskova, M., Sahakian, E., Bashkirova, S. & Lilly, M. The PIM1 kinase is a critical component of a survival pathway activated by docetaxel and promotes survival of docetaxel-treated prostate cancer cells. 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Mol Cancer Res 3, 170-181 (2005). 28 Semenza, G. L. Hypoxia-inducible factors in physiology and medicine. Cell 148, 399-408, doi:10.1016/j.cell.2012.01.021 (2012). 29 Zhu, N. et al. CD40 signaling in B cells regulates the expression of the Pim-1 kinase via the NF-kappa B pathway. J Immunol 168, 744-754 (2002). 30 Leung, C. O. et al. PIM1 regulates glycolysis and promotes tumor progression in hepatocellular carcinoma. Oncotarget 6, 10880-10892, doi:10.18632/oncotarget.3534 (2015). 31 Qian, K. C. et al. Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase. J Biol Chem 280, 6130-6137, doi:10.1074/jbc.M409123200 (2005). 32 Ogawa, N., Yuki, H. & Tanaka, A. Insights from Pim1 structure for anti-cancer drug design. Expert Opin Drug Dis 7, 1177-1192, doi:10.1517/17460441.2012.727394 (2012). 33 Thaimattam, R., Banerjee, R., Miglani, R. & Iqbal, J. Protein kinase inhibitors: Structural insights into selectivity. Curr Pharm Design 13, 2751-2765, doi:Doi 10.2174/138161207781757042 (2007). 34 Muraski, J. A. et al. Pim-1 regulates cardiomyocyte survival downstream of Akt. Nat Med 13, 1467-1475, doi:10.1038/nm1671 (2007). 35 Liu, D. et al. Pim-3 protects against cardiomyocyte apoptosis in anoxia/reoxygenation injury via p38-mediated signal pathway. Int J Biochem Cell Biol 41, 2315-2322, doi:10.1016/j.biocel.2009.05.021 (2009). 36 Mondello, P., Cuzzocrea, S. & Mian, M. Pim kinases in hematological malignancies: where are we now and where are we going? J Hematol Oncol 7, doi:ARTN 9510.1186/s13045-014-0095-z (2014). 37 Morishita, D. et al. Cell-permeable carboxyl-terminal p27(Kip1) peptide exhibits anti-tumor activity by inhibiting Pim-1 kinase. J Biol Chem 286, 2681-2688, doi:10.1074/jbc.M109.092452 (2011). 38 Lee, C. R., Park, Y. H., Kim, Y. R., Peterkofsky, A. & Seok, Y. J. Phosphorylation-Dependent Mobility Shift of Proteins on SDS-PAGE is Due to Decreased Binding of SDS. B Korean Chem Soc 34, 2063-2066, doi:10.5012/bkcs.2013.34.7.2063 (2013). 39 Wang, Z. et al. Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. J Vet Sci 2, 167-179 (2001). 40 Friedmann, M., Nissen, M. S., Hoover, D. S., Reeves, R. & Magnuson, N. S. Characterization of the proto-oncogene pim-1: kinase activity and substrate recognition sequence. Arch Biochem Biophys 298, 594-601 (1992). 41 Palaty, C. K., Clark-Lewis, I., Leung, D. & Pelech, S. L. Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase. Biochem Cell Biol 75, 153-162 (1997). 42 Peng, C. et al. Pim kinase substrate identification and specificity. J Biochem 141, 353-362, doi:10.1093/jb/mvm040 (2007). 43 Bullock, A. N., Debreczeni, J., Amos, A. L., Knapp, S. & Turk, B. E. Structure and substrate specificity of the Pim-1 kinase. J Biol Chem 280, 41675-41682, doi:10.1074/jbc.M510711200 (2005). 44 Montrose, K., Yang, Y., Sun, X., Wiles, S. & Krissansen, G. W. Xentry, a new class of cell-penetrating peptide uniquely equipped for delivery of drugs. Sci Rep 3, 1661, doi:10.1038/srep01661 (2013). 45 Claveria-Gimeno, R., Vega, S., Abian, O. & Velazquez-Campoy, A. A look at ligand binding thermodynamics in drug discovery. Expert Opin Drug Dis 12, 363-377, doi:10.1080/17460441.2017.1297418 (2017). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76536 | - |
dc.description.abstract | 肝細胞癌在全世界是高發生率及高死亡率的癌症之一,男性不論年齡皆是罹患肝細胞癌的高危險群,先前的研究資料指出,男性特有之Y染色體上核糖核酸結合模體蛋白 (RBMY) 會於人類和嚙齒動物的肝臟細胞發育初期表現,於成人的肝臟細胞中消失,但RBMY在肝癌組織的表現量會異常上升並與罹患肝細胞癌病人的預後不佳以及五年存活率的下降有所關連。作為一個有生殖細胞特異性的RNA選擇性剪接調控子,RBMY蛋白含有RNA識別模體用來與將被剪接的RNA結合以及四個富含絲胺酸、精胺酸、蘇胺酸和酪胺酸之高重複片段 (SRGY boxes) 負責與剪接因子形成交互作用。目前研究已指出RBMY的活性和功能表現取決於蛋白本身磷酸化的狀態,且我們的合作者藉由人類蛋白質微陣列分析,指出Pim1為最有可能與RBMY交互作用的激酶,然而對Pim1與RBMY之間磷酸化的修飾不甚瞭解,為此,我們透過生化和生物物理的方法深入探討他們之間的交互作用。首先,本實驗室已成功的使用大腸桿菌系統大量表現Pim1激酶,但是全長RBMY表現量不足,因此合成了多段SRGY boxes上序列之RBMY胜肽以進行實驗。等溫滴定量熱法的結果顯示,Pim1在RBMY的SRGY boxes上有多重結合位,而透過體外激酶測定搭配基質輔助雷射脫附游離-飛行式質譜儀 (MALDI-TOF MS),顯示RBMY上亦有多重磷酸位點。為了發展防止RBMY被磷酸化活化的胜肽抑制劑,我們選其中三條RBMY胜肽在其N端接上細胞穿透胜肽,經證實若給予Hep1細胞RBMY胜肽可有效抑制其轉移活性。最後,我們成功解出RBMY胜肽與Pim1激酶複合體晶體結構,發現胜肽會與Pim1激酶的C端葉產生廣泛的極性交互作用,且從結構的角度分析,Pim1偏好辨認磷酸化位點上游-5及-3位置為鹼性胺基酸 (尤其是精胺酸) 之受質。綜合以上結果,本篇研究呈現了RBMY與Pim1交互作用之細節,且提供了結構資訊給未來在設計胜肽抑制劑時參考。 | zh_TW |
dc.description.abstract | Hepatocellular carcinoma (HCC) is one of the cancers with high incidence and mortality. Scientists have speculated RNA-Binding Motif on Y chromosome (RBMY) plays an important role in male specific HCC progression. As a germ-cell specific RNA alternative splicing regulator, RBMY contains an RNA recognition motif and four repetitive segments rich in serine, arginine, glycine and tyrosine residues (SRGY boxes) for protein interaction. In previous studies, Provirus Integration in Moloney-1 (Pim1) kinase, a proto-oncogene protein, was identified to interact with RBMY by protein microarray analysis. However, the phosphorylation pattern and binding sites between RBMY and Pim1 kinase remains unclear. To this end, we use a combination of biochemical and biophysical approaches to deeply investigate the interaction between RBMY and Pim1 kinase. Due to the poor expression of full-length RBMY in E. coli, several peptides from SRGY boxes of RBMY were synthesized. The results showed that there are multiple Pim1 kinase binding sites on SRGY boxes of RBMY by using isothermal titration calorimetry (ITC). The in vitro kinase assay was performed to investigate whether the candidate peptides could be phosphorylated by Pim1 kinase. Multiple phosphorylation sites were demonstrated by MALDI-TOF Mass Spectrometry. To develop membrane-permeable peptide inhibitors which prevent the phosphorylation-activation of RBMY by Pim1, three of candidate peptides were conjugated cell penetrating peptide (CPP) to their N-terminus. The binding affinity between CPP-RBMY peptides and Pim1 was relatively the same as that between unconjugated peptides and Pim1. And the presences of these peptides in Hep1 cell could effectively suppress its metastatic activity. Furthermore, two crystal structures of Pim1 in complex with two CPP-RBMY peptides were determined at resolution of 1.98 and 2.17 Å, respectively. RBMY-peptides locate at C-lobe of Pim1 and interact with its αD and αF helix. There is no significant conformational change upon binding of RBMY peptides. Instead, peptides make extensive network polar contacts with Pim1. Complex structures revealed a preference for basic residues upstream of the phosphorylation site, specifically arginine at -5 and -3 positions. These data displayed the insight of interaction between RBMY and Pim1 and could provide structural information for Pim1 peptide inhibitors design. | en |
dc.description.provenance | Made available in DSpace on 2021-07-09T15:53:58Z (GMT). No. of bitstreams: 1 ntu-107-R05442010-1.pdf: 26038989 bytes, checksum: 23f0b5fd091b264975632b338f09bdf0 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 摘要 iii Abstract iv 圖目錄 viii 表目錄 ix 縮寫表 x 胺基酸表 xi 一、 引言 1 1.1 肝細胞癌臨床特徵 1 1.2 Y染色體上核糖核酸結合模體蛋白為男性特異性致癌蛋白 2 1.3 Pim1激酶 2 1.4 Pim1激酶的表現與調控 3 1.5 Pim1激酶在肝細胞癌中的角色 4 1.6 Pim1激酶結構之研究 5 1.7 Pim1激酶做為治療癌症標靶的困境 5 1.8 研究動機與目的 7 二、 材料與方法 8 2.1 實驗材料 8 2.1.1 生物材料與試劑 8 2.1.2 蛋白質表現質體 8 2.1.3 RBMY胜肽 (RBMY peptide) 8 2.2 實驗方法 8 2.2.1 轉型作用 8 2.2.2 蛋白質之大量表現 9 2.2.3 蛋白質純化 9 2.2.3.1 蛋白質萃取 9 2.2.3.2 親和層析法 (affinity chromatography) 9 2.2.3.3 膠體過濾色層分析 (gel filtration chromatography) 10 2.2.4 蛋白質定量 10 2.2.5 等溫滴定量熱法 (isothermal titration calorimetry, ITC) 11 2.2.6 激酶受質磷酸化測定 11 2.2.7 蛋白質結晶 11 2.2.7.1 蛋白質結晶實驗 11 2.2.7.2 共結晶 (co-crystallization) 條件篩選 12 2.2.7.3 Pim1結晶條件之微調以及配體浸泡 12 2.2.8 蛋白質晶體之X-ray繞射數據收集與結構解析 13 三、 結果 14 3.1 Pim1激酶蛋白質純化 14 3.2 RBMY胜肽設計 15 3.3 Pim1激酶與RBMY之交互作用 16 3.4 RBMY上有多個可被Pim1激酶磷酸化之位點 16 3.5 RBMY與Pim1交互作用位點 17 3.6 Pim1激酶與CPP-RBMY胜肽之交互作用 18 3.7 Pim1激酶與RBMY胜肽複合體晶體之結構解析 19 3.7.1 Pim1激酶與RBMY胜肽複合體之結晶與結構決定 19 3.7.2 Pim1激酶與RBMY胜肽複合體結構揭露受質辨認胺基酸之特性 20 四、 討論 22 4.1 Pim1激酶與RBMY胜肽之交互作用與胜肽設計 22 4.2 Pim1與受質複合體結晶 23 4.3 Pim1激酶與不同結構比較探討受質特性 24 五、 圖 26 六、 表 48 七、 參考文獻 56 八、 附錄 60 | |
dc.language.iso | zh-TW | |
dc.title | Y染色體上RNA結合模體蛋白與Pim1激酶交互作用之結構表徵 | zh_TW |
dc.title | Structural Characterization of the Interactions between RNA-Binding Motif on Y Chromosome and Pim1 kinase | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹迺立,張美惠 | |
dc.subject.keyword | Pim1激?,Y染色體上RNA結合模體蛋白,肝細胞癌,胜?抑制劑,受質專一性,蛋白質結晶,X-ray繞射分析, | zh_TW |
dc.subject.keyword | Pim1 kinase,RBMY,HCC,peptide inhibitor,substrate specificity,protein crystallography,X-ray diffraction, | en |
dc.relation.page | 61 | |
dc.identifier.doi | 10.6342/NTU201802755 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2019-02-14 | |
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 |
dc.date.embargo-lift | 2029-02-14 | - |
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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