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研究生:陳佳筠
研究生(外文):Jia-Yun Chen
論文名稱:線蟲同源基因-死亡相關蛋白激酶與肌肉相關蛋白在線蟲計劃性死亡中扮演正向促進之功能
論文名稱(外文):cDAPK and cM9 act as positive mediators during programmed cell death in Caenorabditis elegans
指導教授:陳瑞華陳瑞華引用關係
指導教授(外文):Ruey-Hwa Chen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:計劃性細胞死亡死亡相關蛋白激酶線蟲
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計劃性細胞死亡(programmed cell death, apoptosis)是多細胞生物用以去除不必要或可能有害的細胞所必須的正常程序。近來的研究顯示在計劃性細胞死亡的分子機制上,不同的物種間具有高度的保守性。死亡相關蛋白激酶(DAPK)是一個能促使細胞進行計劃性死亡,並且廣泛的參與於各種刺激所造成的計劃性細胞死亡過程。一個未知功能的蛋白-M9-在酵母菌雙雜合系統(yeast two hybrid screen)中顯示與死亡相關蛋白激酶的死亡相關功能區塊(death domain)有強烈的交互作用。我們在線蟲(Caenorhabditis elegans)找到了死亡相關蛋白激酶與M9的同源蛋白-cDAPK和cM9。cDAPK和cM9與他們在人類的同源蛋白在胺基酸序列上有著高度的一致性(identity)與相似性(similarity)。為了探討cDAPK在線蟲體內的功能,我們利用一種斷絕基因表現的方法-稱為核醣核酸干擾(RNA interference)。我們發現在cDAPK的核醣核酸干擾突變線蟲中,在胚胎發育過程中由於計劃性細胞死亡所產生的細胞屍體(corpse)數目有顯著的減少,並且我們也觀察到在線蟲咽部(pharynx)因為計劃性細胞死亡受抑制而出現的多餘細胞(extra cell)。此外我們發現在cDAPK的核醣核酸干擾突變線蟲中,因為外源性表現(ectopic expression)一些殺手蛋白-如egl-1,ced-4-所造成觸感神經元(touch neruon)的死亡情形有受到抑制,但對另一個殺手蛋白-ced-3-而言,卻不能產生相同的抑制作用。人為的大量表現cDAPK於線蟲體內並不能觀察到計劃性細胞死亡的增加。在cM9的研究方面,我們觀察螢光標定的cM9融合蛋白(cM9::YFP fusion protein)發現它能表現在大量進行計劃性死亡的發育時期並且座落於細胞的細胞質(cytoplasm)中。正如cDAPK的核醣核酸干擾突變線蟲,cM9的突變線蟲在胚胎發育過程中由於計劃性細胞死亡所產生的細胞屍體(corpse)數目也有顯著的減少,由實驗的結果顯示,這種減少的現象與ced-8是無關的。同時我們也發現cM9的突變能夠更加強一些殺手蛋白-如ced-3,ced-4-輕微突變(weak mutant)所產生的計劃性細胞死亡障礙。在cM9突變線蟲中,因為外源性表現egl-1所造成觸感神經元的死亡情形有受到抑制,但對ced-3而言,卻不能產生相同的抑制作用。綜合以上所言,本篇論文的結果顯示cDAPK和cM9在線蟲計劃性死亡的訊息傳遞鏈中,是位於egl-1的下游亦或是平行的位階,但位於ced-3的上游亦或是平行的位階。過量表現cDAPK和cM9蛋白並不足以引起計劃性細胞死亡,但它們的基因產物對於計劃性細胞死亡的進行確有其必要性。此外,本篇論文也提供了一個活體(in vivo)的證據確認cDAPK和cM9在計劃性細胞死亡的重要性,進而為日後的研究者提供一個新的方向。

Programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary or potentially harmful cells in multicellular organisms. Recent studies demonstrate that the molecular control of this process is evolutionally conserved in animals. DAPK, a death domain containing calcium/calmodulin- regulated serine/threonine kinase, is a proapoptotic protein and participates in a wide array of apoptotic systems. A novel protein M9 has been shown to interact strongly with the death-domain of DAPK in a yeast-two-hybrid screen. We have identified DAPK and M9 homologs in Caenorhabditis elegans, termed cDAPK and cM9. The cDAPK and cM9 proteins share high identity and similarity to their human counterparts and possess all structural/functional domains. Using RNA interference (RNAi), a method specifically disrupting gene expression, we found that cDAPK(RNAi) mutant showed decreased numbers of cell corpses throughout embryogenesis. Furthermore, extra surviving cells were observed in their anterior pharynx. The cDAPK(RNAi) mutant also suppressed ectopic cell death caused by overexpression of the killer gene egl-1 or ced-4, but not by overexpression of ced-3 under the touch-neuron or the heat-shock promoters. Overexpression of cDAPK, however, did not result in ectopic cell deaths. With respect to the studies of cM9, we found that cM9::YFP translational fusion is expressed in cells where programmed cell deaths extensively occur in embryonic stages, and is localized to the cytoplasm. Similar to cDAPK(RNAi), the cM9 deletion mutant displayed decreased numbers of cell corpses throughout embryogenesis, which is independent of ced-8. Moreover, cM9 mutant can interact synergistically with weak ced-4 and ced-3 mutations to suppress cell killing. The cM9 mutant also suppressed ectopic cell death caused by overexpression of the killer gene egl-1 but not by overexpression of ced-3 under the touch-neuron promoter. Overexpression of cM9 did not show ectopic cell deaths. Altogether, our results suggest that cDAPK and cM9 both act downstream of or in parallel to egl-1, and upstream of or in parallel to ced-3. They are not sufficient to induce cell-killing, but are required for promoting the proper execution of programmed cell death in C. elegans. Our observations provide the first in vivo evidence of cDAPK and cM9 in programmed cell death, and may open an avenue for further studies of their functions in the model organism C. elegans.

Table of contents
Table of contents 1
Abstract 4
中文摘要 5
Experimental Procedures 14
Strains and Genetics 14
Molecular Constructs 14
Transgenic Animals 17
Heat Shock Experiments 18
Inducible RNAi 19
Bacteria-mediated RNAi. 19
Antibodies and Western Blot 20
Results 22
Identification of a C. elegans DAPK homolog 22
cDAPK (RNAi) mutant causes significantly decreased numbers of cell corpses throughout embryogenesis 23
cDAPK (RNAi) mutant generated by in vivo expression of heritable cDAPK inverted-repeat (IR) phenocopies cDAPK (RNAi) mutant generated by feeding 25
cDAPK (RNAi) mutant prevents cell deaths and generates extra ‘undead’ cells in the anterior pharynx of mutant animals 26
Ectopic expression of cDAPK does not result in extra cell deaths 27
cDAPK acts downstream of or in parallel to egl-1 28
Overexpression of cDAPK or its constitutively active form does not rescue the defect of programmed cell death in egl-1 loss-of-function mutant background 30
cDAPK acts downstream of or in parallel to ced-4 31
cDAPK acts upstream of or in parallel to ced-3 31
Identification of a C. elegans M9 homolog 32
cM9 mutant causes significantly decrease numbers of cell corpses throughout embryogenesis 33
Ectopic expression of cM9 does not result in extra cell deaths 34
cM9 interacts with ced-3 and ced-4 to affect programmed cell death 34
cM9 functions in parallel of ced-8 35
cM9 acts downstream of or in parallel to egl-1 and functions in a cell- autonomous fashion 36
cM9 acts upstream of or in parallel to ced-3 37
cM9 is widely expressed and is localized to the cytoplasm during embryogenesis 37
Discussion 39
Function of cDAPK in programmed cell death 39
Evolutionary conservation of cDAPK 42
Function of cM9 in programmed cell death 45
References 49
Figures and tables 57
Figure 1 : The genetic pathway of apoptosis in C. elegans. 57
Figure 2. Schematic representation of the domain structure of DAPK protein. 58
Figure 3. cDAPK encodes a hDAPK homolog 59
Figure 4. Alignment of cDAPK with human DAPK. 60
Figure 5. RNAi mediated by feeding efficiently eliminate cDAPK protein products 61
Figure 6. Strategy for generation of heritable and inducible RNAi. 62
Figure 7. Overexpression of cDAPK or its constitutive active form (dCaM) results in arrested embryos with aberrant epidermal shape changes. 63
Figure 8. Embryonic lethality caused by egl-1-induced ectopic cell deaths is suppressed by cDAPK(RNAi) mutant 64
Figure 9. cDAPK(RNAi) mutant suppresses egl-1-mediated neuronal deaths 65
Figure 10. cDAPK(RNAi) mutant suppresses ced-4-mediated neuronal deaths 66
Figure 11. cDAPK(RNAi) mutant does not enhance touch neurons surviving by ced-3 ectopic expression 67
Figure 12. Cloning of cM9 68
Figure 13.Alignment of M9 proteins 69
Figure 14. cM9 mutant causes significantly decreased numbers of cell corpses throughout embryogenesis 70
Figure 15. cM9(gk126) further reduces cell corpse numbers in weak ced-4(n2273) mutation 71
Figure 16. cM9(gk126) causes reduction of total corpse numbers without delaying in weak ced-3(n2427) mutation 72
Figure 17. cM9(gk126) further reduces cell corpse numbers in null ced-8(n1891) mutant background. 73
Figure 18. cM9(gk126) mutant suppresses egl-1-mediated neuronal deaths in a cell autonomous way 74
Figure 18. cM9(gk126) mutant suppresses egl-1-mediated neuronal deaths in a cell autonomous way 74
Figure 19. cM9(gk126) mutant does not enhance touch neurons surviving by ced-3 ectopic expression 75
Figure 20. Expression of PcM9cM9::YFP 76
Figure 21. A proposed model for the functions of cDAPK and cM9. 77
Table 1. cDAPK(RNAi) generated by in vivo expression of cDAPK dsRNA hairpin under the control of heat- shock promoter hsp16-2 reduces cell-killing 78
Table 2. Overexpression of cDAPK or dCaM under the control of heat-shock promoters slightly enhances embryonic lethality compared to wild-type 79
Table 3. Overexpression of constitutively active cDAPK (dCaM) under the control of heat shock promoters does not result in ectopic cell deaths 80
Table 4. Overexpression of cDAPK or its constitutively active form dCaM does not rescue the defect of programmed cell death in egl-1(n1084 n3082) loss-of-function mutant background 81
Table 5. Overexpression of a cM9 cDNA rescues the cM9 cell-killing defect 82
Table 6. Overexpression of cM9 under the control of heat-shock promoters slightly enhances embryonic lethality compared to wild-type 83
Appendix 84
Appendix 1. cDAPK(RNAi) mutant causes significantly decreased numbers of cell corpes throughout embryogenesis 84
Appendix 2. cDAPK(RNAi) mutant enhances cell survival 85

References
Chalfie M., Tu Y., Euskirchen G., Ward W.W., Prasher D.C. (1994) Green fluorescent protein as a marker for gene expression.Science. 263:802-5.
Chen F., Hersh B.M., Conradt B., Zhou Z., Riemer D., Gruenbaum Y., Horvitz H.R. (2000) Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science. 287:1485-9.
Chinnaiyan A.M., Chaudhary D., O'Rourke K., Koonin E.V., Dixit V.M. (1997) Role of CED-4 in the activation of CED-3. Nature. 388:728-9.
Cohen O., and Kimchi A. (2001) DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ. 8:6-15.
Cohen O., Feinstein E., Kimchi A. (1997) DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 16:998-1008.
Cohen O., Inbal B., Kissil J.L., Raveh T., Berissi H., Spivak-Kroizaman T., Feinstein E., Kimchi A. (1999) DAP-kinase participates in TNF-alpha- and Fas-induced apoptosis and its function requires the death domain. J Cell Biol. 146:141-8.
Conradt B. (2002) With a little help from your friends: cells don't die alone. Nat Cell Biol. 4:E139-43.
Conradt B., and Horvitz H.R. (1998) The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell. 93:519-29.
Deiss L.P., Feinstein E., Berissi H., Cohen O., Kimchi A. (1995) Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev. 9:15-30.
del Peso L., Gonzalez V.M., Inohara N., Ellis R.E., Nunez G. (2000) Disruption of the CED-9.CED-4 complex by EGL-1 is a critical step for programmed cell death in Caenorhabditis elegans. J Biol Chem. 275:27205-11.
Derry W.B., Putzke A.P., Rothman J.H. (2001) Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance. Science. 294:591-5
Deveraux Q.L., Roy N., Stennicke H.R., Van Arsdale T., Zhou Q., Srinivasula S.M., Alnemri E.S., Salvesen G.S., Reed J.C. (1997) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 17:2215-23.
Deveraux Q.L., Takahashi R., Salvesen G.S., Reed J.C. (1997) X-linked IAP is a direct inhibitor of cell-death proteases. Nature. 388:300-4
Du C., Fang M., Li Y., Li L., Wang X. (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell. 102:33-42.
Ellis H.M., and Horvitz H.R. (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell. 44:817-29.
Ellis R.E., and Horvitz H.R. (1991) Two C. elegans genes control the programmed deaths of specific cells in the pharynx. Development. 112:591-603.
Ellis R.E., Jacobson D.M., Horvitz H.R. (1991) Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. Genetics. 129:79-94.EMBO J. 11:2885-93.
Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Fraser A.G., Kamath R.S., Zipperlen P., Martinez-Campos M., Sohrmann M., Ahringer J. (2000) Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 408:325-30.
Gozani O., Boyce M., Yoo L., Karuman P., Yuan J. (2002) Life and death in paradise Nat. Cell. biol. 4:E159-62.
Gonczy P., Echeverri G., Oegema K., Coulson A., Jones S.J., Copley R.R., Duperon J., Oegema J., Brehm M., Cassin E., Hannak E., Kirkham M., Pichler S., Flohrs K., Goessen A., Leidel S., Alleaume A.M., Martin C., Ozlu N., Bork P., Hyman A.A. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature. 408:331-6.
Hamelin M., Scott I.M., Way J.C., Culotti J.G. (1992) The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons.
Hedgecock E.M., Sulston J.E., Thomson J.N. (1983) Mutations affecting programmed cell deaths in the nematode Caenorhabditis elegans. Science. 220:1277-9
Hengartner M.O., and Horvitz H.R. (1994) Activation of C. elegans cell death protein CED-9 by an amino-acid substitution in a domain conserved in Bcl-2. Nature. 369:318-20.
Hengartner M.O., and Horvitz H.R. (1994) Programmed cell death in Caenorhabditis elegans. Curr Opin Genet Dev. 4:581-6.
Hengartner M.O., Ellis R.E., Horvitz H.R. (1992) Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature. 356:494-9.
Hoeppner D.J., Hengartner M.O., Schnabel R. (2001) Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans. Nature. 412:202-6.
Horvitz H.R. (1999) Genetic control of programmed cell death in the nematode. Caenorhabditis elegans. Cancer Res. 59:1701s-1706s.
Inbal B., Bialik S., Sabanay I., Shani G., Kimchi A. (2002) DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J Cell Biol. 157:455-68.
Inbal B., Cohen O., Polak-Charcon S., Kopolovic J., Vadai E., Eisenbach L., Kimchi A. (1997) DAP kinase links the control of apoptosis to metastasis. Nature. 390:180-4.
Jang C.W., Chen C.H., Chen C.C., Chen J.Y., Su Y.H., Chen R.H. (2002) TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nat Cell Biol. 4:51-8.
Kaufmann S.H., and Hengartner M.O.(2001) Programmed cell death: alive and well in the new millennium. Trends Cell Biol. 11:526-34.
Liu Q.A., Hengartner M.O. (1998) Candidate adaptor protein CED-6 promotes the engulfment of apoptotic cells in C. elegans. Cell. 93:961-72.
Metzstein M.M., Stanfield G.M., Horvitz H.R. (1998) Genetics of programmed cell death in C. elegans: past, present and future. Trends Genet. 14:410-6.
Nature. 391:806-11.
Parrish J., Li L., Klotz K., Ledwich D., Wang X., Xue D. (2001) Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature. 412:90-4.
Pelled D., Raveh T., Riebeling C., Fridkin M., Berissi H., Futerman A.H., Kimchi A. (2002) Death-associated protein (DAP) kinase plays a central role in ceramide-induced apoptosis in cultured hippocampal neurons. J Biol Chem. 277:1957-61
Priess J.R., and Hirsh D.I. (1986) Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo. Dev Biol. 1986 117:156-73.
Raveh T., Droguett G., Horwitz M.S., DePinho R.A., Kimchi A. (2001) DAP kinase activates a p19ARF/p53-mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol. 3:1-7.
Reddien P.W., Cameron S., Horvitz H.R. (2001) Phagocytosis promotes programmed cell death in C. elegans. Nature. 412:198-202.
Rinkenberger J.L., and Korsmeyer S.J. (1997) Errors of homeostasis and deregulated apoptosis. Curr Opin Genet Dev. 7:589-96.
Savage C., Hamelin M., Culotti J.G., Coulson A., Albertson D.G., Chalfie M. (1989) mec-7 is a beta-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans. Genes Dev. 3:870-81.
Schumacher B., Hofmann K., Boulton S., Gartner A. (2001) The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr Biol. 11:1722-7.
Seshagiri S., Miller L.K. (1997) Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis. Curr Biol. 7:455-60.
Shaham S., and. Horvitz H.R. (1996) An alternatively spliced C. elegans ced-4 RNA encodes a novel cell death inhibitor. Cell. 86:201-8.
Shaham S., Reddien P.W., Davies B., Horvitz H.R. (1999) Mutational analysis of the Caenorhabditis elegans cell-death gene ced-3.Genetics. 153:1655-71.
Stanfield G.M., and Horvitz H.R. (2000) The ced-8 gene controls the timing of programmed cell deaths in C. elegans. Mol Cell. 5:423-33.
Stringham E.G., Dixon D.K., Jones D., Candido E.P. (1992) Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans. Mol Biol Cell. 3:221-33.
Sulston J.E., and Horvitz H.R. (1977) Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev Biol. 56:110-56.
Sulston J.E., Schierenberg E., White J.G., Thomson J.N. (1983)The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol. 100:64-119.
Tabara H., Grishok A., Mello C.C. (1998) RNAi in C. elegans: soaking in the genome sequence. Science. 282:430-1.
Tavernarakis N., Wang S.L., Dorovkov M., Ryazanov A., Driscoll M. (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet. 24:180-3.
Thompson C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science. 267:1456-62.
Timmons L., Court D.L., Fire A. (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 263:103-12.
Timmons L., Fire A. (1998) Specific interference by ingested dsRNA. Nature. 395:854.
Vaux D.L., and Korsmeyer S.J. (1999) Cell death in development. Cell. 96:245-54.
Verhagen A.M., Ekert P.G., Pakusch M., Silke J., Connolly L.M., Reid G.E., Moritz R.L., Simpson R.J., Vaux D.L. (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell. 102:43-53.
White E. (1996) Life, death, and the pursuit of apoptosis. Genes Dev. 10:1-15.
Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. et al. (1994) 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 368:32-8.
Wu D., Wallen H.D., Inohara N., Nunez G. (1997) Interaction and regulation of the Caenorhabditis elegans death protease CED-3 by CED-4 and CED-9. J Biol Chem. 272:21449-54.
Wu Y.C., and Horvitz H.R. (1998) The C. elegans cell corpse engulfment gene ced-7 encodes a protein similar to ABC transporters. Cell. 93:951-60.
Wu Y.C., Stanfield G.M., Horvitz H.R. (2000) NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis. Genes Dev. 14:536-48.
Yuan J., and Horvitz H.R. (1992) The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death. Development. 116:309-20.
Zhou Q., Snipas S., Orth K., Muzio M., Dixit V.M., Salvesen G.S. (1997) Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem. 272:7797-800.
Zhou Z., Hartwieg E., Horvitz H.R. (2001) CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell. 104:43-56.

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