請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37604
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 翁啟惠(Chi-Huei Wong) | |
dc.contributor.author | Shao-Hsuan Huang | en |
dc.contributor.author | 黃劭軒 | zh_TW |
dc.date.accessioned | 2021-06-13T15:34:35Z | - |
dc.date.available | 2011-07-18 | |
dc.date.copyright | 2008-07-18 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-11 | |
dc.identifier.citation | 1.Willis, Simon N. and Adams, Jerry M., Life in the
balance: how BH3-only proteins induce apoptosis. Current Opinion in Cell Biology 17 (6), 617 (2005); Gross, Atan, McDonnell, James M., and Korsmeyer, Stanley J., BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13 (15), 1899 (1999). 2.Strasser, Andreas, The role of BH3-only proteins in the immune system. Nat Rev Immunol 5 (3), 189 (2005). 3.Danial, Nika N. and Korsmeyer, Stanley J., Cell Death: Critical Control Points. Cell 116 (2), 205 (2004). 4.Danial, Nika N., BCL-2 Family Proteins: Critical Checkpoints of Apoptotic Cell Death. Clin Cancer Res 13 (24), 7254 (2007). 5.Reed, John C, Bcl-2 family proteins. Oncogene 17, 3225 (1998). 6.T Sato, M Hanada, S Bodrug, S Irie, N Iwama, L H Boise, C B Thompson, E Golemis, L Fong, H G Wang, et al., Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci U S A. 91, 9238 (1994). 7.Bernhard Gillissen, Frank Essmann, Vilma Graupner, Lilian Stärck, Silke Radetzki, Bernd Dörken, Klaus Schulze- Osthoff and Peter T. Daniel, Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway. EMBO 22, 3580 (2003). 8.Chen, Lin et al., Differential Targeting of Prosurvival Bcl-2 Proteins by Their BH3-Only Ligands Allows Complementary Apoptotic Function. Molecular Cell 17 (3), 393 (2005). 9.Muchmore, Steven W. et al., X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381 (6580), 335 (1996). 10.Petros, Andrew M. et al., Solution structure of the antiapoptotic protein bcl-2. Proceedings of the National Academy of Sciences 98 (6), 3012 (2001). 11.Sattler, Michael et al., Structure of Bcl-xL-Bak Peptide Complex: Recognition Between Regulators of Apoptosis. Science 275 (5302), 983 (1997). 12.O'Neill, Jason W., Manion, Michael K., Maguire, Brendan, and Hockenbery, David M., BCL-XL Dimerization by Three- dimensional Domain Swapping. Journal of Molecular Biology 356 (2), 367 (2006). 13.Liu, Xinqi et al., The Structure of a Bcl-xL/Bim Fragment Complex: Implications for Bim Function. Immunity 19 (3), 341 (2003). 14.Oberstein, Adam, Jeffrey, Philip D., and Shi, Yigong, Crystal Structure of the Bcl-XL-Beclin 1 Peptide Complex: BECLIN 1 IS A NOVEL BH3-ONLY PROTEIN. J. Biol. Chem. 282 (17), 13123 (2007). 15.Czabotar, Peter E. et al., Structural insights into the degradation of Mcl-1 induced by BH3 domains. Proceedings of the National Academy of Sciences 104 (15), 6217 (2007). 16.Huang, Ziwei, Bcl-2 family proteins as targets for anticancer drug design. Oncogene 19, 6627 (2000). 17.Oltersdorf, Tilman et al., An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435 (7042), 677 (2005). 18.Hajduk, Philip J. and Greer, Jonathan, A decade of fragment-based drug design: strategic advances and lessons learned. Nat Rev Drug Discov 6 (3), 211 (2007). 19.van Delft, Mark F. et al., The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 10 (5), 389 (2006). 20.Konopleva, Marina et al., Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 10 (5), 375 (2006). 21.Tse, Christin et al., ABT-263: A Potent and Orally Bioavailable Bcl-2 Family Inhibitor. Cancer Res 68 (9), 3421 (2008). 22.Gemin X Inc. website: http://www.geminx.com/en/research/gx15070.php; Kenza Daïri, Y. Y., Michael Faley, Sasmita Tripathy, Elise Rioux, Xavier Billot, Daniel Rabouin, Gerson Gonzalez, Jean-François Lavallée, and Giorgio Attardo, A Scalable Process for the Synthesis of the Bcl Inhibitor Obatoclax. Organic Process Research & Development 11, 1051 (2007). 23.Wang, G. et al., Structure-Based Design of Potent Small- Molecule Inhibitors of Anti-Apoptotic Bcl-2 Proteins. J. Med. Chem. 49 (21), 6139 (2006). 24.Becattini, Barbara et al., Rational Design and Real Time, In-Cell Detection of the Proapoptotic Activity of a Novel Compound Targeting Bcl-XL. Chemistry & Biology 11 (3), 389 (2004). 25.Klasa, Richard J., Gillum, Amanda M., Klem, Robert E., and Frankel, Stanley R., Oblimersen Bcl-2 Antisense: Facilitating Apoptosis in Anticancer Treatment. Antisense and Nucleic Acid Drug Development 12 (3), 193 (2002). 26.Shimazu, Tsutomu et al., NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev. 21 (8), 929 (2007). 27.Klumpp, Susanne and Krieglstein, Josef, Serine/threonine protein phosphatases in apoptosis. Current Opinion in Pharmacology 2 (4), 458 (2002). 28.Verma, Sulekha, Zhao, Ling-jun, and Chinnadurai, G., Phosphorylation of the Pro-apoptotic Protein BIK. MAPPING OF PHOSPHORYLATION SITES AND EFFECT ON APOPTOSIS. J. Biol. Chem. 276 (7), 4671 (2001). 29.Li, Yan M. et al., Enhancement of Bik Antitumor Effect by Bik Mutants. Cancer Res 63 (22), 7630 (2003). 30.Li, Zheng et al., Suppression of pancreatic tumor progression by systemic delivery of a pancreatic-cancer- specific promoter driven Bik mutant. Cancer Letters 236 (1), 58 (2006); Xie, Xiaoming et al., Targeted Expression of BikDD Eradicates Pancreatic Tumors in Noninvasive Imaging Models. Cancer Cell 12 (1), 52 (2007). 31.Michael P. Weiner , G. L. C., Warren Schoettlin, Janice Cline, Eric Mathur and John C. Bauer, Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Genes 151, 119 (1994). 32.D'Arcy, Martin Zulauf and Allan, Light scattering of proteins as a criterion for crystallization. Journal of Crystal Growth 122, 102 (1992). 33.Faham, Salem and Bowie, James U., Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. Journal of Molecular Biology 316 (1), 1 (2002). 34.Cherezov, Vadim et al., High-Resolution Crystal Structure of an Engineered Human 2-Adrenergic G Protein Coupled Receptor. Science 318 (5854), 1258 (2007). 35.Prive Gilbert, G., Detergents for the stabilization and crystallization of membrane proteins. Methods 41 (4), 388 (2007). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37604 | - |
dc.description.abstract | Bcl-2家族蛋白在程序性細胞死亡過程中扮演相當重要的角色。Bcl-2 蛋白家族主要可分為兩大類。一類為Pro-apoptotic,另一類則為Anti-apoptotic。Bik 屬於前者,而Bcl-XL屬於後者。他們在細胞中互相拮抗,並調控細胞凋亡的程序。
過去的研究發現,Bik 可被CKII(Casein Kinase 2)磷酸化。為了要模擬此磷酸化過程,洪明奇院士團隊將Bik上之Thr33和Ser35位置定點突變成Aspartate,並將此突變株取名為BikDD。在試管試驗中,BikDD 增強了其原本對Bcl-XL之親和力,另外在不同之癌症細胞株(乳癌、前列腺癌、胰臟癌、肺癌),BikDD 皆展現其增強之毒殺效果。最近他們設計了一種只會針對胰臟癌表現的啟動子(CCKAR-VISA),利用其專一性,BikDD 蛋白特定地表現在帶有胰臟癌的老鼠身上,並根除了胰臟腫瘤, 此一成果提供了未來基因療法用於人體試驗的可能性。 本篇論文的目標為研究BikDD和Bcl-XL複合體,希望透過對結構之瞭解,提供更多資訊給BikDD基因療法用於人體試驗基礎,以及癌症新藥的開發。BikDD和Bcl-XL皆屬於膜蛋白,因其疏水物理特性,增加了純化及長晶的困難度。我們首先利用大腸桿菌大量表現出蛋白複合體,再利用不同的純化方法,將此複合體從水溶液狀態或膜上純化出來。為了要拿到符合長晶條件的樣品,我們亦測試了不同之介面活性劑,不同片段之表現,以及Bicelle 結晶法,並利用DLS和AUC來驗證其均質度。在拿到夠均質的樣品之後,我們進行了結晶條件測試。 此外,我們也利用FRET技術,設計了一種可用來篩選Bcl-XL抑制物的平台,希望藉此找出癌症藥物前驅物,提供治療癌症的契機。 | zh_TW |
dc.description.abstract | Bik and Bcl-XL are Bcl-2 family members. They play crucial roles in the regulation of programmed cell death. Bcl-2 family can be broadly categorized into two groups. One is pro-apoptotic proteins, the other is anti-apoptotic proteins. Pro-apoptotic and anti-apoptotic proteins antagonize each other and mediate apoptosis. Bik belongs to the pro-apoptic type , Bcl-XL belongs to the anti-apoptotic type.
Previous studies showed that Bik can be phosphorylated by protein kinase CKII (Casein Kinase 2). In order to mimic the post-translational phosphorylation process of Bik, Prof. Min-Chie Hung’s group generated a Bik mutant, BikDD (T33D/S35D). BikDD enhanced not only the binding affinity with Bcl-XL in vitro but also cancer cell killing ability in different cancer cell lines. Recently, they designed a pancreatic cancer specific promoter (CCKAR-VISA). By taking advantage of the specificity of this promoter, BikDD targeted pancreatic cancer cell in the pancreatic tumor-bearing mouse models. Pancreatic cancer cells were effectively killed and eradicated by BikDD. The significant anti-tumor results provide hopes for future cancer gene therapy in human clinical trials. The goal of this project is to determine the BikDD-BclXL complex structure. The structural information will provide solid evidence for Dr. Min-Chie Hung’s liposome-mediated BikDD cancer gene therapy. Both BikDD and Bcl-XL are membrane proteins. Their hydrophobic properties make themselves difficult to be purified and crystallized. We successfully over-expressed BikDD-BclXL complex in the E. coli system and tried different approaches to purify them from soluble or membrane fraction. In order to get a homogeneous sample for crystallization, we systematically screened different detergents, expressed different fragments, and tried bicelle crystallization methods. We utilized AUC and DLS technique to measure the protein homogeneity. After we obtained a homogeneous sample, we performed crystallization trials. In addition, we designed a platform for searching Bcl-XL inhibitors by using FRET. We hope this platform can help us to discover potential candidates for novel anti-cancer drug development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T15:34:35Z (GMT). No. of bitstreams: 1 ntu-97-R95b46025-1.pdf: 5382003 bytes, checksum: b34657b581bca837d2588f4e81da611c (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | ACKNOWLEDGEMENTS...........................................I
CHINESE ABSTRACT..........................................II ENGLISH ABSTRACT.........................................III TABLE OF CONTENTS.........................................IV LIST OF FIGURES...........................................VI LIST OF TABLES...........................................VII LIST OF APPENDIX.........................................VII LIST OF ABBREVIATIONS................................. VIII Chapter1. INTRODUCTION.....................................1 1.1 Roles of Bcl-2 Family in apoptosis.....................1 1.2 Homo- or Hetero- dimerization of Bcl-2 family proteins.2 1.3 Structural basis of Bcl-2 family proteins..............2 1.4 Anti-apoptotic proteins as targets for anticancer drug design.................................................3 1.5 Bik as a therapeutic agent for cancer gene therapy.....4 1.6 Goal of solving the BikDD - BclXL complex structure..5 Chapter 2. MATERIALS and METHODS...........................6 Materials 2.1 Reagents............................................6 2.2 Instruments............................................7 Methods 2.3 Plasmid construction.................................8 2.4 Site-directed mutagenesis.............................8 2.5 Deletion of the loop region...........................8 2.6 Protein over-expression..............................9 2.7 Large-scale protein production......................9 2.8 Protein purification...........................9 2.9 Detergent tests......................................10 2.10 Crystallization................................10 2.11 Analytical Ultracentrifuge (AUC)....................10 2.12 Dynamic light scattering (DLS)......................11 2.13 Bicelle crystallization.............................11 2.14 FRET(Förster resonance energy transfer).............12 Chapter 3 RESULTS and DISCUSSION.........................13 3.1 BikDD and Bcl-XL can be co-expressed in the E. coli system...............................................13 3.2 Purification of the soluble BikDD-BclXL complex......13 3.3 Purification of BikDD-BCLXL complex from E.coli membrane.............................................14 3.4 Detergent exchange by using Gel-filtration column....16 3.5 Homogeneity analysis by using AUC and DLS............17 3.6 Deletion of the Bcl-XL loop region...................17 3.7 Crystallization......................................18 3.8 Bicelle crystallization method.......................18 3.9 Co-expression of BikDD fragments with Bcl-XL△TM.....19 Chapter 4 CONCLUSION.....................................21 REFERENCE................................................24 FIGURE...................................................26 TABLE....................................................45 APPENDIX.................................................48 | |
dc.language.iso | en | |
dc.title | BikDD與BclXL蛋白複合體的純化與結晶 | zh_TW |
dc.title | Purification and Crystallization of BikDD-BclXL complex | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 馬徹(Che Ma) | |
dc.contributor.oralexamcommittee | 李宗璘(Tsung-Lin Li) | |
dc.subject.keyword | Bcl-2 蛋白家族,細胞凋亡,膜蛋白,BikDD,BclXL, | zh_TW |
dc.subject.keyword | Bcl-2 family,BikDD,BclXL,Apoptosis,Membrane protein, | en |
dc.relation.page | 49 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-11 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-97-1.pdf 目前未授權公開取用 | 5.26 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。