吳益群臺灣大學：分子與細胞生物學研究所柯怡如Ko, Yi-JuYi-JuKo2007-11-252018-07-062007-11-252018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/49898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。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.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…………………………………………………………….58666857 bytesapplication/pdfen-US細胞凋亡細胞自噬計劃性死亡apoptosisautophagyprogrammed cell deathdapk-1bec-1otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49898/1/ntu-95-R93b43005-1.pdf