簡易檢索 / 詳目顯示
| 研究生： |
蔡孟儒 Meng-Ju Tsai |
|---|---|
| 論文名稱： |
硫磺礦硫化葉菌程序性細胞死亡蛋白5晶體結構分析及其與DNA的相互作用 Crystal Structure Analysis of Sulfolobus solfataricus Programmed Cell Death Protein 5 and Its Interaction with DNA |
| 指導教授： |
陳青諭
Chin-Yu Chen |
| 口試委員: | |
| 學位類別： |
碩士 Master |
| 系所名稱： |
生醫理工學院 - 生命科學系 Department of Life Science |
| 論文出版年： | 2019 |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 76 |
| 中文關鍵詞： | 硫磺礦硫化葉菌 、程序性細胞死亡蛋白5 、X光繞射 、蛋白質結晶學 、DNA結合蛋白 |
| 外文關鍵詞： | Sulfolobus solfataricus, the programmed cell death protein 5, crystal structure, X-ray crystallography, DNA-binding protein |
| 相關次數： | 點閱：11 下載：0 |
| 分享至: |
| 查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
中文摘要
程序性細胞死亡蛋白5 (PDCD5)在真核生物的細胞凋亡途徑中扮演著重要信號蛋白的角色，在現有的研究中，已知人類PDCD5蛋白中存在著三螺旋束和兩個游離的氮端區域，然而對於古生菌中PDCD5的研究仍尚不清楚。本研究中，我們鑑定了來自硫磺礦硫化葉菌的PDCD5同源蛋白(Sso-PDCD5)，並且以1.55Å的高解析度呈現Sso-PDCD5的晶體結構，Sso-PDCD5為PDCD5同源蛋白中第一個解析出來的晶體結構，此蛋白質具有低柔韌性的緊密核心，氮端區域具有四個α-螺旋，以及具有彈性的碳端尾端。而位於碳端區域的色胺酸(W117)在激發螢光的實驗中發現色胺酸(W117)發散出的螢光可以被20bp的雙股DNA給遮蔽，螢光強度隨DNA濃度的增加而減少，這顯示Sso-PDCD5可以透過碳端尾端與DNA進行相互作用。在等溫滴定量熱的結果顯示碳端截斷蛋白(Sso-PDCD5_CTT，刪除K108-K118序列)顯著降低了與DNA結合的親和力，更進一步證明Sso-PDCD5利用動態的碳端尾端與雙股DNA進行結合。除此之外，電子顯微鏡(EM)的圖像顯示，Sso-PDCD5透過橋接的方式與雙股DNA相互作用結合。總而言之，經過結構以及生化方面的實驗數據表明Sso-PDCD5具有與雙股DNA結合的能力，是一種DNA結合蛋白。
關鍵字：硫磺礦硫化葉菌、程序性細胞死亡蛋白5、X光繞射、蛋白質結晶學、DNA結合蛋白
Abstract
The Programmed Cell Death protein 5 (PDCD5) is an important signal protein of apoptosis pathway in eukaryotes. In previous research, it is known there are triple-helix bundle and two dissociated N-terminal regions in human PDCD5 protein. However, the study of PDCD5 in hyperthermophile archaea remains unclear. Here, we identify a PDCD5 homolog from Sulfolobus solfataricus (Sso_PDCD5) and present the crystal structure of Sso_PDCD5 at a high resolution of 1.55 Å. This is the first crystal structure of a PDCD5 homolog to be solved, showing that the protein has a compact core of low flexibility with four alpha-helices at N-terminal region and a flexible unstructured C-terminal tail. The fluorescence of C-terminal tryptophan (W117) can be quenched by 20 bp double-strand DNA which indicates PDCD5 may interact with DNA by the C-terminal tail. The isothermal titration calorimety (ITC) results show C-terminal truncated protein (PDCD5_CTT, detection of K108-K118) significantly reduced the DNA-binding affinity, further demonstrated that the flexible C-terminus of Sso_PDCD5 involved in binding dsDNA. In addition, Sso_PDCD5 binds dsDNA through bridging interactions as shown in electron microscopy (EM) images. In conclusion, the structural and biochemical data suggest that Sso_PDCD5 may function as a DNA-binding protein.
Keywords: Sulfolobus solfataricus, the programmed cell death protein 5, crystal structure, X-ray crystallography, DNA-binding protein
參考文獻
1. Chen Y., Sun R., Han W., Zhang Y., Song Q., Di C., and Ma D. (2001). “Nuclear translocation of PDCD5 (TFAR19): an early signal for apoptosis?” FEBS Lett.
509(2):191-196
2. Li P., Fei H., Wang L., Xu H., Zhang H., and Zhang L. (2018). “PDCD5 regulates cell proliferation, cell cycle progression and apoptosis.” Oncol Lett.15(1), 1177-1183
3. Li, G., D. Ma and Y. Chen (2016). “Cellular functions of programmed cell death 5.” Biochim. Biophys. Acta 1863(4):572-580
4. Salvi, M., D. Xu, Y. Chen, A. Cabrelle, S. Sarno and L. A. Pinna (2009). “Programmed cell death 5 (PDCD5) is phosphorylated by CK2 in vitro and in 293T cells.” Biochem Biophys Res Commun 387(3):606-610
5. Zhuge C., Sun X., Chen Y., and Lei J (2016). “PDCD5 functions as a regulator of p53 dynamics in the DNA damage response.” J Theor Biol. 388:1-10
6. Thomas Ulas., S. Alexander Riemer., Melanie Zaparty., Bettina Siebers., and Dietmar Schomburg (2012). “Genome-Scale Reconstruction and Analysis of the Metabolic Network in the Hyperthermophilic Archaeon Sulfolobus Solfataricus.” PLoS One. 7(8): 43401
7. Hong, J., J. Zhang, Z. Liu, S. Qin, J. Wu and Y. Shi (2009). “Solution structure of S.cerevisiae PDCD5-like protein and its promoting role in H(2)O(2)-induced apoptosis in yeast.” Biochemistry 48(29):6824-6834
8. Reiter J., Herker E., Madeo F., and Schmitt MJ (2005). “Viral killer toxins induce caspase-mediated apoptosis in yeast.” J Cell Biol. 168(3):353-358
9. Ivanovska I., and Hardwick JM (2005). “Viruses activate a genetically conserved cell death pathway in a unicellular organism.” J Cell Biol. 170(3):391-399
10. Suarez MF., Filonova LH., Smertenko A., Savenkov EI., Clapham DH., von Arnold S., Zhivotovsky B., and Bozhkov PV (2004). “Metacaspase-dependent programmed cell death is essential for plant embryogenesis.” Curr Biol. 14(9):R339-340
11. Yang H., Ren Q., and Zhang Z (2008). “Cleavage of Mcd1 by caspase-like protease Esp1 promotes apoptosis in budding yeast.” Mol Biol Cell. 19(5):2127-2134
12. Fuchs Y., and Steller H (2015). “Live to die another way: modes of programmed cell death and the signals emanating from dying cells.” Nat Rev Mol Cell Biol. 16(6):329-344
13. Swamy MJ., Sankhala RS., and Singh BP (2019). “Thermodynamic Analysis of Protein-Lipid Interactions by Isothermal Titration Calorimetry.” Methods Mol Biol. 2003:71-89
14. Matsuo K., and Gekko K (2019). “Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Lipid Environment.” Methods Mol Biol. 2003:253-279
15. Zhuge C., Sun X., Chen Y., and Lei J (2016). “Corrigendum to ''PDCD5 interacts with p53 and functions as a regulator of p53 dynamics in the DNA damage response.” J Theor Biol. 396:210
16. Zhao H., Peng C., Lu X., Guo M., Yang T., Zhou J., and Hai Y (2019). “PDCD5 inhibits osteosarcoma cell metastasis via targeting TGF-β1/Smad signaling pathway and is associated with good prognosis.” Am J Transl Res. 11(2):1116-1128
17. Lin KF., Hsu JY., Hsieh DL., Tsai MJ., Yeh CH., and Chen CY (2019). “Crystal structure of the programmed cell death 5 protein from Sulfolobus solfataricus.” Acta Crystallogr F Struct Biol Commun. 75(Pt 2):73-79
18. Zhou Z., Jian J., Wang Z., Hu J., and Liu W. (2015).J Int Oncol Vol.42, No.5
