請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17768
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 曾秀如 | |
dc.contributor.author | Cheng-Yu Feng | en |
dc.contributor.author | 馮政諭 | zh_TW |
dc.date.accessioned | 2021-06-08T00:43:14Z | - |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-13 | |
dc.identifier.citation | 1. M. L. Breuer, H. T. Cuypers , A. Berns, A Evidence for the involvement of pim- 2, a new common proviral insertion site, in progression of lymphomas. Embo J 8, 743–748 (1989). 2. D. Hoover, M. Friedmann, R. Reeves, N. S. Magnuson, Recombinant human pim‑1 protein exhibits serine/threonine kinase activity. J. Biol. Chem. 266, 14018–14023 (1991). 3. R. Padma, L. Nagarajan, The human PIM‑1 gene product is a protein serine kinase. Cancer Res. 51, 2486–2489 (1991). 4. B. A. Carmen, Pim kinases in cancer: Diagnostic, prognostic and treatment opportuneities. J. BCP. 85, 629-643 (2013). 5. M.C. Nawijn, A. Alendar , A. Berns, For better or for worse: the role of Pim oncogenes in tumorigenesis. Nat Rev Cancer . 11, 23–34 (2011). 6. H. Mikkers, J. Allen, P. Knipscheer, L. Romeijn, A. Hart, E. Vink, Highthroughput retroviral tagging to identify components of specific signaling pathways in cancer. Nat Genet. 32, 153–159(2002). 7. N. Zhu, CD40 signaling in B cells regulates the expression of the Pim‑1 kinase via the NF‑κB pathway. J. Immunol. 168, 744–754 (2002). 8. T. Shirogane, Synergistic roles for Pim‑1 and c‑Myc in STAT3‑mediated cell cycle progression and antiapoptosis. Immunity. 11, 709–719 (1999). 9. A. Castro, T. K. Sengupta, D. C. Ruiz, E. Yang, L. B. Ivashkiv, IL‑4 selectively inhibits IL‑2‑triggered Stat5 activation, but not proliferation, in human T cells. J. Immunol. 162, 1261–1269 (1999). 10. A. T. Wierenga, E. Vellenga, J. J. Schuringa, Maximal STAT5‑induced proliferation and self‑renewal at intermediate STAT5 activity levels. Mol. Cell. Biol. 28, 6668–6680 (2008). 11. H. Mikkers, Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol. Cell. Biol. 24, 6104–6115 (2004). 12. J. Domen, Comparison of the human and mouse PIM‑1 cDNAs: nucleotide sequence and immunological identification of the in vitro synthesized PIM‑1 protein. Oncogene Res. 1, 103–112 (1987). 13. G. Selten, H. T. Cuypers, A. Berns, Proviral activation of the putative oncogene Pim‑1 in MuLV induced T‑cell lymphomas. EMBO J. 4, 1793–1798 (1985). 14. Y. Xie, 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 (2006). 15. C. J. Fox, The serine/threonine kinase Pim‑2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev. 17, 1841–1854 (2003) 16. K. C. Qian, Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim‑1 kinase. J. Biol. Chem. 280, 6130–6137 (2005). 17. O. Kim, Synergism of cytoplasmic kinases in IL6‑induced ligand‑independent activation of androgen receptor in prostate cancer cells. Oncogene. 23, 1838–1844 (2004). 18. A. N. Bullock, J. Debreczeni, A. L. Amos, S. Knapp, B. E. Turk, Structure and substrate specificity of the Pim‑1 kinase. J. Biol. Chem. 280, 41675–41682 (2005) 19. J. A. Losman, X. P. Chen, B. Q. Vuong, S. Fay, P. B. Rothman, Protein phosphatase 2A regulates the stability of Pim protein kinases. J. Biol. Chem. 278, 4800–4805 (2003). 20. J. Ma, H. K. Arnold, M. B. Lilly, R. C. Sears, A. S. Kraft, Negative regulation of Pim‑1 protein kinase levels by the B56β subunit of PP2A. Oncogene. 26, 5145–5153 (2007). 21. B. Yan, The PIM‑2 kinase phosphorylates BAD on serine 112 and reverses BAD‑induced cell death. J. Biol. Chem. 278, 45358–45367 (2003). 22. N. N. Danial, BAD: undertaker by night, candyman by day. Oncogene. 27 (Suppl. 1), S53–S70 (2008). 23. A. A. Alizadeh, Distinct types of diffuse large B‑cell lymphoma identified by gene expression profiling. Nature. 403, 503–511 (2000). 24. G. Wright, A gene expression‑based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc. Natl Acad. Sci. USA 100, 9991–9996 (2003). 25. E. D. Hsi, Ki67 and PIM1 expression predict outcome in mantle cell lymphoma treated with high dose therapy, stem cell transplantation and rituximab: a Cancer and Leukemia Group B 59909 correlative science study. Leuk. Lymphoma. 49, 2081–2090 (2008). 26. M. Eilers, R. N. Eisenman, Myc’s broad reach. Genes Dev. 22, 2755–2766 (2008). 27. N. B. Alex, R. Santina, A, Ann, Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor. PloS ONE. 4, (2009). 28. I. Alexey, L. Zhang, F. Junhua, Structure-based design of low-nanomolar PIM kinase inhibitors. BMCL. 25, 474-480 (2015) | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/17768 | - |
dc.description.abstract | PIM蛋白的致癌效果主要是透過增生機制使細胞不斷的生長,如透過增強MYC蛋白的轉錄活性、增強cap-dependent translation以及細胞週期的進行、透過活化MCL1的基因以及磷酸化Bad蛋白來達到抑制細胞凋亡。PIM蛋白是一種經由轉譯後即有活性的絲氨酸/蘇氨酸激酵素,且PIM1經常廣泛的表現於造血細胞,而PIM2則表現於血液惡性腫瘤,包括了急性骨髓性白血病、慢性淋巴細胞白血病、彌漫性大B細胞淋巴瘤。重要的是,在已含有抗藥性的癌症當中,PIM2的抑制可以使得癌細胞恢復細胞凋亡,這是目前癌症治療當中極具潛力的療法之一。而近日來與PIM蛋白相關的抑制劑設計以及合成,均是以PIM1為主要目標,主要是因為PIM1不管是脫輔基態或是抑制劑結合態的結構均廣為人知。然而PIM2目前僅只有兩篇抑制劑結合態的結構解出,而根據此兩篇解出之結構與PIM1相比,發現確實有些許的不同可以用來作為PIM2專一抑制劑的參考。因此我們的研究目標便是解出PIM2的脫輔基態以及抑制劑結合態的結構,以便更進一步提供日後藥物設計的重要參考。首先我們使大腸桿菌成功的表現出PIM2,並利用鎳離子親和性樹脂以及膠體過濾法純化出PIM2蛋白。接著便使用前結晶測試法來測出PIM2適合的結晶濃度,並利用機械手臂測試了市售的結晶試劑,然而並未發現任何晶體,因此改用其它策略。一:利用TEV酵素切去PIM2蛋白N端的his-tag,以減少可動端。二:根據隨機微晶種法,利用PIM1晶體做為PIM2養晶時之晶種。三:利用多種截短型之PIM2來幫助晶體篩選(如N端截短型以及N,C端截短型)。至此為止,我們仍致力於上述養晶實驗。我們同時也利用了恆溫滴定微卡計以及差式掃描量熱儀來探測SGI-1776、5-carboxamidoindole以及Imatinib mesylate是否會和PIM2進行結合,藉此即可利用可結合之藥物進行共結晶。測試結果出來僅有SGI-1776以及5- carboxamidoindole可和PIM2進行結合。因此我們便將此兩種抑制劑與上述各種型式之PIM2蛋白進行共結晶,以期結出多種型式下的PIM2結構而利於日後人們對於PIM2的藥物設計。 | zh_TW |
dc.description.abstract | The tumorigenic effects of PIM kinases are perpetuated through proliferative mechanisms including potentiation of MYC transcriptional activity, enhancement of cap-dependent translation, and cell cycle progression, as well as antiapoptotic effects with up-regulation of MCL1 and Bad phosphorylation. The PIM proteins are constitutively active serine/threonine kinases that are ubiquitously expressed with predominance for PIM1 in hematopoietic cells and for PIM2 in hematological malignancies including acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and diffuse large B-cell. Importantly, PIM2 inactivation can restore apoptosis to otherwise drug-resistant cancers and is therefore an attractive therapy to supplement current drug regimen. Several recent publications report the design and synthesis of new PIM kinase inhibitors targeting mainly PIM1. The availability of PIM1 kinase crystal structures at apo and drig-bound statues is playing a key role in the identification of new inhibitors. However, there were only two crystal structures of PIM2 kinase in complex with inhibitor until now and it revealed some differences to PIM1 which may be explored further to generate isoform selective inhibitors. Therefore, we decide to solve the structure of PIM-2 at apo and drug-bound states and it will provide significant structural and biological insights that should greatly benefit a structure-based drug design. We have successfully expressed PIM2 in E. coli and purified it with Ni column and gel-filtration. Then we used Pre-Crystallization Test (PCT) to test the suitable concentration of PIM2 for crystallization and crystal screening kit to find the suitable crystal condition. However, it did not grow any crystals. So we adopted other strategies to obtain crystal: 1. His-tag of PIM2 N-terminal was removed by TEV protease so it can reduce flexible part; 2. we used PIM1 crystal as seed to co-crystallize with PIM2 (random Microseed Matrix Screening, rMMS); 3. the various truncated constructs of PIM2 including N-terminal truncated forms and N,C-terminal truncated forms were used to crystal screening . Up to now, we still exert our best to get crystal. We also used Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) to detect which kinds of inhibitors (SGI-1776、5-carboxamidoindole and Imatinib mesylate) will interact with PIM2 kinase, so we can use theses inhibitors to co-crystallize with PIM2 kinase. Then Only SGI-1776 and 5-carboxamidoindole can interact with PIM2 kinase. Then we adopted various PIM2 (including full length、N-terminal truncated form and N,C-terminal truncated form) to co-crystallize with SGI-1776 and 5- carboxamidoindole. We hope to get more kinds of PIM2 structure then assist people to generate further isoform selective inhibitors. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T00:43:14Z (GMT). No. of bitstreams: 1 ntu-104-R02442027-1.pdf: 7068405 bytes, checksum: a007913c33067839d0ddc196754e2aa7 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 摘要…………………………………………………………………………………….i Abstract……………………………………………………………………………….iii 目錄…………………………………………………………………………………...v 表目錄……………………………………………………………………………....vii 圖目錄…………………………………………………………………………...….viii 附錄…………………………………………………………………………………...ix 一.前言……………………………………………………………………………….1 1.1 人類PIM蛋白介紹……………………………………………………………….1 1.2 PIM蛋白的轉錄轉譯….………………………………………………………….1 1.3 PIM蛋白轉譯後調控….………………………………………………………….2 1.4 PIM蛋白活化之訊號傳遞….…………………………………………………….3 1.5 PIM蛋白之下游路徑….………………………………………………………….4 1.6 PIM蛋白與癌症….……………………………………………………………….5 1.7 PIM2蛋白之構型….……………………………………………………………...6 1.8 PIM2的藥物設計….……………………………………………………………...7 1.9 研究目的….……………………………………………………………………....8 第二章 實驗材料….……………………………………………………………..….10 2.1 大腸桿菌及質體……………………………………………………………..….10 2.2 藥品….……………………………………………………………………….….10 2.3 試劑組….……………………………………………………………..………....11 2.4 儀器裝置….……………………………………………………………..……....12 第三章 實驗方法….……………………………………………………………..….13 3.1 點突變及選殖 (Site Directed Mutagenesis and cloning) ….………13 3.1.1 引子設計 (primer design) ….………………………………….……….....13 3.1.2 聚合酵素連鎖反應 (Polymerase Chain Reaction, PCR) ….……………15 3.1.3 洋菜膠體電泳(Agarose gel electrophoresis) ….………………………….16 3.1.4 選殖(cloning) ….……………………………………………………..….16 3.1.5 製備勝任細胞(competent cells) ….………………………….……….….17 3.1.6 轉型作用(Transformation) ….………………………..……………….....18 3.2 重組蛋白之表現與純化….……………………….……………………………19 3.2.1 重組蛋白之表現….……………………….………………………...……19 3.2.2 重組蛋白之純化….……………………….………………………......…20 3.2.3 膠體過濾層析(Gel-filtration chromatography) ….……………………....21 3.2.4 重組蛋白定量….……………………….………………………........…..23 3.2.5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)…23 3.3 蛋白質養晶條件篩選 (screening of protein crystallization) ….……………....26 3.4 隨機微晶種篩選 (random microseed matrix screening) ….…………………..28 3.5 差式掃描量熱儀 (Differential scanning calorimetry, DSC) ….……………....28 3.6 恆溫滴定微卡計 (Isothermal Titration Calorimetry, ITC) ….………………...29 第四章 結果……………………………………………………………………..….31 4.1 全長型PIM2表現以及純化………………………………………………..….31 4.2 全長型PIM2之前結晶測試(pre-crystallization test) ……………………..…....31 4.3 全長型PIM2之晶體篩選(crystal screening) ……………………..…………....32 4.4 全長型PIM2之隨機微晶種篩選(random microseed matrix screening)……….32 4.5 全長型PIM2之隨機微晶種篩選之晶體微調(crystal optimization)…………..33 4.6 PIM2之N端截短形式(truncated form) ……………………..…………..…....34 4.7 PIM2之N端截短形式之純化……………………..……………………..…....34 4.8 PIM2之N端截短形式之晶體篩選…………………………………….…..…....35 4.9 PIM2之N端、C端截短形式…………………………………….…..………..35 4.10 PIM2之N端、C端截短形式之純化…………………………………….…....36 4.11 PIM2之N端、C端截短形式之晶體篩選…………………………………....36 4.12 全長型PIM2之恆溫滴定微卡計(Isothermal Titration Calorimetry, ITC)…....37 4.13 全長型PIM2之差式掃描量熱儀(Differential Scanning Calorimetry, DSC)....38 4.14 PIM2截短型與全長型之共結晶(co-crystallization)………………………….39 第五章 討論….……………………………………………………………..……….41 5.1 全長型與截短型之PIM2比較….……………………………………………….41 5.2 全長型與截短型之PIM2晶體篩選….………………………………………….42 參考文獻…………………………………………………………………………….84 表目錄 表1-1 PIM蛋白過表現所導致之惡性腫瘤……………………………………...…43 表1-2 PIM蛋白抑制劑之改良……………………………………...………………44 表4-1 PIM2晶體篩選所使用之結晶試劑……………………………………......…78 表4-2 3D structure 25的條件微調表……………………………………...............…79 表4-3 為3D structure 25的條件微調後PIM2所產生的晶體之抗凍劑…………….80 表4-4 全長型PIM2與SGI-1776之恆溫滴定微卡計測試結果圖表整理…………..81 圖目錄 圖1-1 JAK/STAT 訊號傳遞路徑以及PIM蛋白的活化……………………………45 圖1-2 PIM2蛋白之結構……………………………………………………………46 圖1-3 PIM2蛋白與PIM1激酶之比較………………………………………….…47 圖4-1 全長型PIM2蛋白質之表現 (SDS-PAGE 10%)…………………………….48 圖4-2 PIM2蛋白質之前結晶測試…………………………………...……………...49 圖4-3 PIM2蛋白質與抑制劑之共結晶前結晶測試……………...………………...50 圖4-4 蒸氣擴散法…………………………………………………………………...51 圖4-5 PIM1晶體………………………………………………………......................52 圖4-6 全長型PIM2之隨機微晶種篩選所長出之各種晶型的晶體.........................53 圖4-7 PIM1晶體…………………………………………………………………...54 圖4-8 全長型PIM2之晶體探測…...............................55 圖4-9 3D structure 25的條件微調晶體……………..……………………….…........56 圖4-10 N端截短型………………………………………………………..…….........57 圖4-11 N端截短型式: N1-PIM2蛋白質之表現 (SDS-PAGE 10%)……….……....58 圖4-12 N端截短型式: N2-PIM2蛋白質之表現 (SDS-PAGE 10%)………….…....59 圖4-13 N端截短型式: N3-PIM2蛋白質之表現 (SDS-PAGE 10%)……….……....60 圖4-14 N、C端截短型………………..………….…..…………………………........61 圖4-15 N端、C端截短型式: N1C1-PIM2蛋白質之表現 (SDS-PAGE 10%)………62 圖4-16 N端、C端截短型式: N2C1-PIM2蛋白質之表現 (SDS-PAGE 10%)………63 圖4-17 N端、C端截短型式: N1C2-PIM2蛋白質之表現 (SDS-PAGE 10%)………64 圖4-18 N端、C端截短型式: N2C2-PIM2蛋白質之表現 (SDS-PAGE 10%)………65 圖4-19 N端、C端截短型式: N1C3-PIM2蛋白質之表現 (SDS-PAGE 10%)………66 圖4-20 N端、C端截短型式: N2C3-PIM2蛋白質之表現 (SDS-PAGE 10%)………67 圖4-21預測定之抑制劑……………………..…………………………….................68 圖4-22 全長型PIM2與SGI-1776之恆溫滴定微卡計測試結果…………………....69 圖4-23 全長型PIM2與SGI-1776之恆溫滴定微卡計測試結果…………………....70 圖4-24 全長型PIM2與Imatinib之恆溫滴定微卡計測試結果…………………......71 圖4-25 全長型PIM2與5-carboxamidoindole之恆溫滴定微卡計測試結果…….....72 圖4-26 全長型PIM2與5-carboxamidoindole之恆溫滴定微卡計測試結果…….....73 圖4-27 全長型PIM2之差式掃描量熱儀測試結果………………………………...74 圖4-28 全長型PIM2與SGI-1776之差式掃描量熱儀測試結果……........................75 圖4-29 全長型PIM2與Imatinib之差式掃描量熱儀測試結果……........................76 圖4-30 全長型PIM2與5-carboxamidoindole之差式掃描量熱儀測試結果……....77 附錄 圖1-1 N端截短型所使用之質體pET-28a …………………………………………45 圖1-2可測定物質結合後產生的吸放熱之儀器……………………………………46 | |
dc.language.iso | zh-TW | |
dc.title | PIM2蛋白在脫輔基和抑制劑結合狀態下的結構研究 | zh_TW |
dc.title | Structural studies of human PIM2 in apo- and inhibitor-bound states | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹迺立,徐駿森 | |
dc.subject.keyword | PIM2激蛋白,結晶結構,抑制劑, | zh_TW |
dc.subject.keyword | PIM2 kinase,crystal structure,kinase inhibitor, | en |
dc.relation.page | 87 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2015-08-13 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 6.9 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。