線蟲dapk-1執行細胞凋亡的功能與遺傳分析及細胞凋亡和自噬間的關係

Yi-Ju Ko; 柯怡如

請用此 Handle URI 來引用此文件： http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31730

完整後設資料紀錄

DC 欄位	值	語言
dc.contributor.advisor	吳益群(Yi-Chun Wu)
dc.contributor.author	Yi-Ju Ko	en
dc.contributor.author	柯怡如	zh_TW
dc.date.accessioned	2021-06-13T03:18:40Z	-
dc.date.available	2007-07-31
dc.date.copyright	2006-07-31
dc.date.issued	2006
dc.date.submitted	2006-07-27
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(2003). Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase. J. Cell Sci. 116:4777-90. Llambi F., Lourenco F.C., Gozuacik D., Guix C., Pays L., Del Rio G., Kimchi A., and Mehlen P. (2005). The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase. EMBO J. 24:1192-201. Melendez A., Talloczy Z., Seaman M., Eskelinen E.L., Hall D.H., and Levine B. (2003). Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301:1387-91. Pelkmans L., Fava E., Grabner H., Hannus M., Habermann B., Krausz E., and Zerial M. (2005). Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436:78-86. Pettmann B., and Henderson C.E. (1998). Neuronal cell death. Neuron 20:633-47. Raveh T., Berissi H., Eisenstein M., Spivak T., and Kimchi A. (2000). A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity. Proc. Natl. Acad. Sci. 97:1572-7. Scott S.V., Nice III D.C., Nau J.J., Weisman L.S., Kamada Y., Keizer-Gunnink I., Funakoshi T., Veenhuis M., Ohsumi Y., and Klionsky D.J. (2000). Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting. J. Biol. Chem. 275:25840-9. Shintani T. and Klionsky D.J. (2004). Autophagy in health and disease: a double-edged sword. Science 306:990-5. Shohat G., Spivak-Kroizman T., Cohen O., Bialik S., Shani G., Berrisi H., Eisenstein M., and Kimchi A. (2001). The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autophosphorylation-based mechanism. J. Biol. Chem. 276:47460-7. Takacs-Vellai K., Vellai T., Puoti A., Passannante M., Wicky C., Streit A., Kovacs A.L., and Muller F. (2005). Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Curr. Biol. 15:1513-7. Yuan J., and Yankner B.A., (2000). Apoptosis in the nervous system. Nature 407:802-9. Yukawa K., Hoshino K., Kishino M., Mune M., Shirasawa N., Kimura A., Tsubota Y., Owada-Makabe K., Tanaka T., Ichinose M., Maeda M., Takeda K., Akira S. (2004). Deletion of the kinase domain in death-associated protein kinase attenuates renal tubular cell apoptosis in chronic obstructive uropathy. Int. J. Mol. Med. 13:515-20.
dc.identifier.uri	http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31730	-
dc.description.abstract	根據我們實驗室的研究發現在線蟲計劃性死亡中，dapk-1會促進細胞凋亡。為了了解dapk-1如何作用於計劃性死亡，我們對dapk-1進行結構與功能分析。如同其人類同源蛋白DAPk，DAPK-1也包含kinase domain, ankyrin repeats, and death domain等區域。我們發現DAPK-1的kinase domain以及death domain對於其促進細胞凋亡的功能是必要的。就我們所知這是第一手證據證明在線蟲中death domain會參與計劃性死亡，隱含了外生性死亡途徑可能參與其中的可能性。更進一步地，根據線蟲資料庫的搜尋及遺傳分析，我們發現UNC-5 (the netrin receptor)可能會作用於DAPK-1所調控的外生性死亡途徑並作為死亡接收器。.然而其他區域如cytoskeleton binding region對於DAPK-1促進細胞凋亡的功能則不必要；至於ankyrin repeats對其功能則並非絕對必要。此結果和先前人類的研究發現不同，在人類中發現這些區域為DAPk促進細胞凋亡的活性所必須。更複雜的是，人類DAPk不僅會促進細胞凋亡，也會影響細胞自噬體的形成，隱含了細胞凋亡和細胞自噬死亡途徑間可能的交互作用。我們希望能利用線蟲這種簡單的模式動物來研究細胞自噬基因參與計劃性死亡的情形。我們觀察到細胞自噬基因如bec-1和unc-51會對計劃性死亡產生抑制的影響，而且至少有部分bec-1的突變所造成的細胞死亡不須經由egl-1誘發。更進一步地，根據遺傳分析定位我們發現dapk-1除了可以促進發育所調控的細胞死亡外，也會調控細胞自噬相關的死亡途徑。	zh_TW
dc.description.abstract	Recent studies in our lab have revealed the pro-apoptotic function of dapk-1 during programmed cell death. To understand how dapk-1 acts in programmed cell death, we performed structure-function analysis of dapk-1. Like its human homolog DAPk, DAPK-1 contains multiple domains including kinase domain, ankyrin repeats, and death domain. We found that kinase and death domains of DAPK-1 are required for its pro-apoptotic function. To our knowledge, this is the first evidence showing the involvement of death domain in C. elegans programmed cell death, implying the potential role of an extrinsic cell death pathway. Taking a candidate gene approach, we showed that the netrin receptor unc-5 might be the death receptor acting in the DAPK-1 mediated extrinsic cell death pathway. Other regions such as cytoskeleton binding region is dispensable and ankyrin repeats are not absolutely necessary for DAPK-1 pro-apoptotic function. This result is distinct from the previous finding that these regions are required for the apoptosis-promoting activity of human DAPk. More complicatedly, human DAPk is also known as a positive mediator of autophagy, implying the potential interplay of apoptotic and autophagic pathways. Therefore, we tried to include the autophagic genes in C. elegans programmed cell death. In this study we observed that autophagic genes such as bec-1 and unc-51 bring inhibitory effects to C. elegans programmed cell death and at least some bec-1 mediated cell deaths do not require egl-1. Moreover, from double-mutant analysis we found that in addition to developmentally regulated cell death, dapk-1 may also mediate autophagy-dependent cell death.	en
dc.description.provenance	Made available in DSpace on 2021-06-13T03:18:40Z (GMT). No. of bitstreams: 1 ntu-95-R93b43005-1.pdf: 666857 bytes, checksum: f74aede482dd9e7cb7286277f2ede979 (MD5) Previous issue date: 2006	en
dc.description.tableofcontents	Table of Contents Table of Contents……………………………………………………………………..1 Abstract……………………………………………………………………………….4 中文摘要………………………………………………………………………………5 Introduction…………………………………………………………………………..6 Materials and Methods……………………………………………………………..15 Strains and Genetics……………………………………………………………….15 Transgenic Animals………………………………………………………………..16 Heat Shock Experiment……………………………………………………………17 Western Blotting…………………………………………………………………...17 Results……………………………………………………………………………….19 Ectopic expression of full-length DAPK-1 by let-858 promoter completely rescues the defect of programmed cell death in dapk-1(ju469)……………….…………...19 Constitutively active form of DAPK-1 does not exhibit stronger killing potential.20 Kinase domain is required for the pro-apoptotic activity of DAPK-1…………….21 The importance of death domain in C. elegans programmed cell death is first identified…………………………………………………………………………...21 Ankyrin repeats are not absolutely required and cytoskeleton binding region is dispensable for the pro-apoptotic function of DAPK-1…………………………...23 Human DAPk can replace DAPK-1 to promote apoptosis in C. elegans…………23 Autophagic genes, bec-1 and unc-51, contribute inhibitory effects to C. elegans programmed cell death…………………………………………………………….24 At least some bec-1 mediated cell deaths do not require egl-1 and bec-1 acts upstream of ced-4………………………………………………………………….25 bec-1 possibly acts upstream of dapk-1…………………………………………...26 unc-51 acts upstream of or in parallel to ced-3 and dapk-1……………………….26 Discussion……………………………………………………………………………28 dapk-1 may encode short and long protein………………………………………..28 DAPK-1 possibly interacts with other apoptotic mediators through the death domain and then phosphorylates them through the catalytic activity to promote programmed cell death in C. elegans……………………………………………...29 The contribution of the proper localization to actin cytoskeleton from ankyrin repeats and cytoskeleton binding region is of minor importance to the pro-apoptotic function of DAPK-1………………………………………………………………31 The netrin receptor UNC-5 might be the death receptor acting in the DAPK-1 mediated extrinsic cell death pathway…………………………………………….32 In addition to developmentally regulated cell death, dapk-1 may mediate autophagy-dependent cell death as well…………………………………………..33 References…………………………………………………………………………..34 Figures and Tables………………………………………………………………….39 Figure 1. Polyclonal rabbit anti-DAPK-1 antibodies recognize endogenous and over-expressed DAPK-1 in worm lysates………………………………………...39 Figure 2. Ectopic expression of full-length DAPK-1 by the let-858 promoter rescues the defect of programmed cell death in dapk-1(ju469)…………………..40 Figure 3. Ectopic expression of constitutively active form of DAPK-1 rescues the defect of programmed cell death in dapk-1(ju469)……………………………….41 Figure 4. Ectopic expression of mutant DAPK-1 which delete kinase domain could not rescue the defect of programmed cell death in dapk-1(ju469)………………...42 Figure 5. Ectopic expression of mutant DAPK-1 which delete death domain could not rescue the defect of programmed cell death in dapk-1(ju469)………………...43 Figure 6. Over-expression of death domain alone by the heat-shock promoter decreases the cell corpse number in wild type…………………………………….44 Figure 7. UNC-5, a death domain-containing membrane protein, acts in the same genetic pathway with DAPK-1……………………………………………………45 Figure 8. Ectopic expression of mutant DAPK-1 which delete all ankyrin repeats could partially rescue the defect of programmed cell death in dapk-1(ju469)……46 Figure 9. Ectopic expression of mutant DAPK-1 which delete cytoskeleton binding region rescues the defect of programmed cell death in dapk-1(ju469)……………47 Figure 10. Ectopic expression of human DAPk rescues the defect of programmed cell death in dapk-1(ju469)………………………………………………………...48 Figure 11. The deletion mutation of bec-1 results in more cell deaths at early embryonic stages than wild type…………………………………………………..49 Figure 12. The unc-51 mutation slightly affects the kinetics of killing…………...50 Figure 13. ced-3 is epistatic to unc-51 during programmed cell death……………51 Figure 14. Possible dapk-1-mediated extrinsic cell death pathway and the roles of bec-1 and unc-51, autophagic genes, in programmed cell death…………………..52 Table 1. Genetics analysis of bec-1 and pro-apoptotic genes in C. elegans programmed cell death…………………………………………………………….53 Table 2. Genetics analysis of unc-51, bec-1, and pro-apoptotic genes in C. elegans programmed cell death…………………………………………………………….54 Appendix 1. Alignment of DAPK-1 with human DAPk…………………………..55 Appendix 2. Over-expressed GFP proteins drove by the heat shock promoter increase the cell corpse number in dapk-1(ju469)…………………………………56 Appendix 3. Over-expression of constitutively active dapk-1(dCaM) under the control of heat shock promoters does not result in ectopic cell deaths……………57 Appendix 4. The ced-3(n2452) mutation blocks almost all cell deaths in unc-51(e369) mutation…………………………………………………………….58
dc.language.iso	en
dc.title	線蟲dapk-1執行細胞凋亡的功能與遺傳分析及細胞凋亡和自噬間的關係	zh_TW
dc.title	Functional and genetic analysis of dapk-1 during apoptosis and the construction of connection between apoptosis and autophagy in C. elegans	en
dc.type	Thesis
dc.date.schoolyear	94-2
dc.description.degree	碩士
dc.contributor.oralexamcommittee	黃偉邦(Wei-Pang Huang),陳瑞華(Ruey-Hwa Chen)
dc.subject.keyword	細胞凋亡,細胞自噬,計劃性死亡,	zh_TW
dc.subject.keyword	apoptosis,autophagy,programmed cell death,dapk-1,bec-1,	en
dc.relation.page	58
dc.rights.note	有償授權
dc.date.accepted	2006-07-30
dc.contributor.author-college	生命科學院	zh_TW
dc.contributor.author-dept	分子與細胞生物學研究所	zh_TW
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