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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82287完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 曾秀如(Shiou-Ru Tzeng) | |
| dc.contributor.author | Yu-Jung Chen | en |
| dc.contributor.author | 陳又榕 | zh_TW |
| dc.date.accessioned | 2022-11-25T06:35:02Z | - |
| dc.date.copyright | 2021-11-09 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-27 | |
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Adv Biol Regul, 2017. 63: p. 107-114. 19.Morley, S.C., The actin-bundling protein L-plastin: a critical regulator of immune cell function. International journal of cell biology, 2012. 2012(935173). 20.Su Kim, D., et al., Composite three-marker assay for early detection of kidney cancer. Cancer Epidemiol Biomarkers Prev, 2013. 22(3): p. 390-8. 21.Li, J. and R. Zhao, Expression and clinical significance of L-plastin in colorectal carcinoma. J Gastrointest Surg, 2011. 15(11): p. 1982-8. 22.Lommel, M.J., et al., L-plastin Ser5 phosphorylation in breast cancer cells and in vitro is mediated by RSK downstream of the ERK/MAPK pathway. The FASEB journal, 2015. 30(3): p. 1218-1233. 23.de Arruda, M.V., et al., Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins. Journal of Cell Biology, 1990. 111(3): p. 1069-1079. 24.Mruk, K., et al., Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering. J Gen Physiol, 2014. 144(1): p. 105-14. 25.Ishida, H., et al., The Calcium-Dependent Switch Helix of L-Plastin Regulates Actin Bundling. Sci Rep, 2017. 7: p. 40662. 26.Klein, M.G., et al., Structure of the actin crosslinking core of fimbrin. Structure, 2004. 12(6): p. 999-1013. 27.Matsudaira, P., Modular organization of actin crosslinking proteins. Trends Biochem Sci, 1991. 16(3): p. 87-92. 28.Lebart, M.-C., et al., Biochemical characterization of the L-plastin-actin interaction shows a resemblance with that of alpha-actinin and allows a distinction to be made between the two actin-binding domains of the molecule. Biochemistry, 2004. 43(9): p. 2428-2437. 29.Giganti, A., et al., Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement. J Cell Sci, 2005. 118(Pt 6): p. 1255-65. 30.Schwebach, C.L., et al., The Roles of Actin-Binding Domains 1 and 2 in the Calcium-Dependent Regulation of Actin Filament Bundling by Human Plastins. J Mol Biol, 2017. 429(16): p. 2490-2508. 31.Janji, B., et al., Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells. J Cell Sci, 2006. 119(Pt 9): p. 1947-60. 32.Park, T., Z.P. Chen, and J. Leavitt, Activation of the Leukocyte Plastin Gene Occurs in Most Human Cancer Cells. Cancer Research, 1994. 54(7): p. 1775-1781. 33.Lapillonne, A., et al., Expression patterns of L-plastin isoform in normal and carcinomatous breast tissues. Anticancer Res, 2000. 20(5A): p. 3177-82. 34.Zheng, J., et al., Steroid hormone induction and expression patterns of L-plastin in normal and carcinomatous prostate tissues. Am J Pathol, 1997. 150(6): p. 2009-18. 35.Zheng, J., et al., Suppression of prostate carcinoma cell invasion by expression of antisense L-plastin gene. Am J Pathol, 1999. 155(1): p. 115-22. 36.Otsuka, M., et al., Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis. Biochem Biophys Res Commun, 2001. 289(4): p. 876-81. 37.Klemke, M., et al., Phosphorylation of ectopically expressed L-plastin enhances invasiveness of human melanoma cells. Int J Cancer, 2007. 120(12): p. 2590-9. 38.C S Lin, R.H.A., et al., Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts. Molecular and Cellular Biology, 1988. 8(11): p. 4659-4668. 39.Galkin, V.E., et al., High-resolution cryo-EM structure of the F-actin-fimbrin/plastin ABD2 complex. Proc Natl Acad Sci U S A, 2008. 105(5): p. 1494-8. 40.Cooper, D.R., et al., Protein crystallization by surface entropy reduction: optimization of the SER strategy. Acta Crystallogr D Biol Crystallogr, 2007. 63(Pt 5): p. 636-45. 41.Rao, J.N., Y. Madasu, and R. Dominguez, Mechanism of actin filament pointed-end capping by tropomodulin. Science, 2014. 345(6195): p. 463-7. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82287 | - |
| dc.description.abstract | L-plastin是鈣離子負向調節的肌動蛋白結合蛋白，在造血細胞和大多數轉移性癌細胞中表達。這些細胞類型是可移動的，需要對肌動蛋白細胞骨架進行不斷重塑，其中L-plastin能夠將絲狀的肌動蛋白捆綁成微絲束。L-plastin之N端是由和鈣調蛋白calmodulin（CaM）同源的EF-hands motifs組成，作為鈣離子的傳導器。其C端為結合肌動蛋白絲的兩個肌動蛋白結合區（ABD）。目前已知EF-hand motifs和鈣離子結合時會使L-plastin N端連接到C端之間一段無構型的序列轉變為穩定的α螺旋結構稱為H5，進而與EF-hand motifs和C端肌動蛋白結合區交互作用而影響肌動蛋白捆綁活性。全長的L-plastin結構尚未被解出，因此調節L-plastin的肌動蛋白捆綁活性依然是不夠清楚的。為了探討L-plastin的表徵及如何調節肌動蛋白捆綁的活性，於是我們利用大腸桿菌大量表達L-plastin及不同長度的L-plastin，經固定化金屬離子管柱層析及膠體過濾層析純化後獲得蛋白，不同長度之L-plastin以單體、雙聚體及多聚體三種形式存在，這三種形式並不會互相轉換。微絲束形成試驗發現不含EF-hand但包含H5區域和肌動蛋白結合區之L-plastin H5-ABD12的單體具有很好的捆綁活性，而僅含有肌動蛋白結合區的L-plastin ABD12使肌動蛋白絲捆綁成微絲束之活性非常差，推論H5區域對於調控捆綁的活性是至關重要的。為了進一步探討L-plastin和肌動蛋白之間的交互作用，我們使用核磁共振光譜進行分析發現含有H5區域及一個肌動蛋白結合區的L-plastin H5-ABD1及僅含有一個肌動蛋白結合區的L-plastin ABD1，他們都能與肌動蛋白結合。透過表面電漿共振實驗顯示L-plastin H5-ABD1和肌動蛋白之結合能力比L-plastin ABD1強4倍。除了功能性測試及核磁共振實驗之外，我們也嘗試利用X射線蛋白質晶體繞射法分析全長L-plastin及L-plastin H5-ABD12，但目前尚未成功計算出L-plastin及L-plastin H5-ABD12的結構。綜合上述，本研究透過微絲束形成試驗、核磁共振實驗、表面電漿共振等實驗顯示L-plastin的H5區域對於肌動蛋白絲捆綁成微絲束之活性是扮演非常重要的角色。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T06:35:02Z (GMT). No. of bitstreams: 1 U0001-2409202115400500.pdf: 6992408 bytes, checksum: f04bf33d8b8bfc60c0300ab52a6077b5 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "致謝………………………………………………………………………………...…. Ⅱ 摘要……………………………………………………………………………...…….Ⅲ Abstract…………………………………………………………………………..……Ⅴ 縮寫表………………………………………………………………………...……. XIV 一、前言……………………………………………………………………...………….1 1.1 人類Plastin異構體（isoform）…………………………………….……………….1 1.2 L-plastin之功能…………………………………………………………………….2 1.3 L-plastin之結構…………………………………………………………………….2 1.4 L-plastin之調控機制…………………………………….…………………………3 1.5 L-plastin與惡性腫瘤…………………………………….…………………………4 1.6 研究動機…………...……………………………...……….………………………5 二、實驗材料與方法…………………………………………………...……………….6 2.1 實驗材料………………………...……………………………………………...….6 2.1.1 菌株 (表1)、質體 (表2)………………………………………………….……..6 2.1.2 引子 (表3)………………………………………………………………..……..6 2.1.3 培養基、培養液 (表4) 及緩衝溶液 (表5)…………………………….....……6 2.1.4 蛋白質表現質體……………………………………………...……………….…6 2.2 實驗方式……………………………………………………………...…………....6 2.2.1 轉型作用（Transformation）………………………………………….……..……6 2.2.2 點突變及選殖（Site-directed mutagenesis and cloning）……………………….6 2.2.3 重組蛋白之大量表現………………………………………...………………….8 2.2.4 重組蛋白之純化………………………………………………………...……….9 2.2.5 蛋白質緩衝溶液之置換與濃縮………………………..………………………10 2.2.6 蛋白質濃度測定………………………………………………….…………….11 2.2.7 微絲束形成試驗（Actin bundling assay）…………………………..…………..11 2.2.8 表面電漿共振（Surface Plasmom Resonance, SPR）…………..………………11 2.2.9 蛋白質核磁共振（protein NMR）………………………………………………13 2.2.10 蛋白質結晶實驗………………………………………………………………13 三、結果………………………………………………………………………………..17 3.1 重組蛋白之純化結果………………………………………………………….…17 3.1.1全長L-plastin、L-plastin ABD12、L-plastin 98-ABD12、L-plastin H5-ABD12之純化結果…………………………………………………………………………....17 3.1.2 L-plastin H5-ABD1、L-plastin 98-ABD1、L-plastin ABD1之純化結果……….19 3.1.3 L-plastin NTD97、L-plastin NTD112之純化結果………………………….......19 3.1.4 L-plastin K96A+K97A+E98A、L-plastin H5-K96A+K97A+E98A、L-plastin E327A+K328A之純化結果…………………………………………………………..20 3.2 L-plastin單體、雙聚體及多聚體之差異探討………………………………….....20 3.3 蛋白質活性測試………………………………………………………………….21 3.4 核磁共振（NMR）光譜分析…………………………………………………...…22 3.4.1 L-plastin H5-ABD12、L-plastin 98-ABD12與L-plastin ABD12之二維1H-15N TROSY-HSQC光譜比較……………………………………………………………..23 3.4.2 L-plastin H5-ABD1之二維1H-15N TROSY-HSQC光譜 ……………..………23 3.4.3 L-plastin ABD1之二維1H-15N TROSY-HSQC光譜……………………..…….24 3.5 L-plastin H5-ABD1、L-plastin ABD1與肌動蛋白之表面電漿共振（SPR）分析..25 3.6 L-plastin蛋白質結晶實驗…………………………………………………...……26 3.6.1 L-plastin H5-ABD12、Se-L-plastin及Se-L-plastin H5-ABD12之結晶條件篩選.. ………………………………………………………………………………………....26 3.6.2不同長度之L-plastin蛋白之微調結果…………………..……..………………27 3.6.2.1全長L-plastin蛋白晶體…………………………..…………...………………27 3.6.2.2 L-plastin H5-ABD12蛋白晶體…………………………….…………………27 3.6.2.3 SER全長L-plastin及L-plastin H5-ABD12突變體蛋白質晶體…………….28 3.6.2.4 Se-L-plastin H5-ABD12蛋白晶體…………………………………………....28 3.6.3 X射線繞射數據收集與結構分析……………………………………...………29 四、討論………………………………………………………………………….…….30 五、圖……………………………………………………………………………..…....33 六、表………………………………………………………………...……………….100 七、參考文獻………………………………………………………….……………...108 " | |
| dc.language.iso | zh-TW | |
| dc.subject | 核磁共振實驗 | zh_TW |
| dc.subject | L-plastin | zh_TW |
| dc.subject | 蛋白質晶體 | zh_TW |
| dc.subject | 表面電漿共振 | zh_TW |
| dc.subject | 微絲束形成試驗 | zh_TW |
| dc.subject | X-ray crystallography | en |
| dc.subject | L-plastin | en |
| dc.subject | actin bundling assay | en |
| dc.subject | NMR | en |
| dc.subject | SPR | en |
| dc.title | 肌動蛋白結合蛋白L-plastin之結構特性 | zh_TW |
| dc.title | Structural Characterization of the Actin-Bundling Protein L-plastin | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 詹迺立(Hsin-Tsai Liu),徐駿森(Chih-Yang Tseng) | |
| dc.subject.keyword | L-plastin,微絲束形成試驗,核磁共振實驗,表面電漿共振,蛋白質晶體, | zh_TW |
| dc.subject.keyword | L-plastin,actin bundling assay,NMR,SPR,X-ray crystallography, | en |
| dc.relation.page | 111 | |
| dc.identifier.doi | 10.6342/NTU202103342 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2021-09-27 | |
| 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 | 2026-09-24 | - |
| 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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