陳瑞華臺灣大學：分子醫學研究所林裕敏Lin, Yu-MinYu-MinLin2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51384hCed-X is an evolutionary conserved protein found in several eukaryotes including human, mouse, C. elegans, Arabidopsis, Drosophila, and rice. In this thesis, we demonstrate that hCed-X localizes to the cytokeratin 8/18 (K8/K18) intermediate filaments and nucleolus in non-apoptotic epithelial cells. During apoptosis induced by various agents, hCed-X relocates to intracellular inclusions where it colocalizes with K8 and caspase cleaved-K18 proteins in a caspase-dependent manner. Furthermore, physical interaction between hCed-X and K8/18 was demonstrated by fluorescence resonance energy transfer technique. To explore the biological function of hCed-X, we employed antisense and RNA interference technologies. We found that specific knockdown of endogenous hCed-X protein protects cell from apoptosis induced by the TNF? indicating that hCed-X functions as a positive regulator in this process of apoptosis. Furthermore, we provide evidence suggesting that hCed-X lies downstream of caspase-8 and upstream of caspase-9 in TNFTable of content 1 Abstract 3 中文摘要 4 Introduction 5 Apoptosis 5 Keratin intermediate filaments 8 Keratin intermediate filaments and apoptosis 9 hCed-X 12 Materials and methods 15 Cell culture, transfection, and Constructs 15 Expression and purification of GST-fusion proteins 15 Immunization procedure 16 Antibody 17 Western blotting 17 Immnuofluorescence 17 FRET (Fluorescence resonance energy transfer) 18 Flow Cytometry Analysis 18 Results 19 Generate polyclonal antibody of hCed-X 19 hCed-X localizes to the cytokeratin 8/18 filaments and nucleolus 20 Identification of hCed-X as a cytokeratin 8/18-interacting protein using coimmunoprecipitation and FRET imaging analyses 21 hCed-X localizes to granular structures with cytokeratin 8/18 in apoptotic cells 23 Granular structures of hCed-X during apoptosis is caspase-dependent 24 hCed-X is involved in TNF?induced apoptosis 25 hCed-X acts downstream of caspase-8 and upstream of caspase-9 in TNF?mediated signaling pathway 26 Discussion 27 Evolutionary conservation of hCed-X 27 Function of hCed-X in apoptosis 28 hCed-X is a subunit of the eukaryotic translation initiation factor 3 30 Reference List 32 Figures and Tables 38 Figure 1. Alignment of hCed-X proteins. 38 Figure 2. Northern analysis of human hCed-X. 39 Figure 3. A proposed model for the function of Ced-X. 40 Figure 4. Characteristics of anti-hCed-X polyclonal antibody. 41 Figure 5. The subcellular localization of hCed-X in various cell lines. 42 Figure 6. hCed-X is not colocalized with microtubules. 43 Figure 7. Colocalization of hCed-X and K8/K18 filaments in HeLa cells. 44 Figure 8. Colocalization of hCed-X and K8/K18 filaments in HaCaT cells. 45 Figure 9. hCed-X localizes to the nucleolus. 46 Figure 10. Colocalization of YFP-hCed-X with CFP-K8. 47 Figure 11. Use of FRET methodology to assess interaction between various pairs of proteins. 48 Figure 12. Recruitment of hCed-X to cytoplasmic granules with K8/K18 during apoptosis. 49 Figure 13. hCed-X and K8/K18 are recruited to apoptotic body in late-phase of apoptosis. 50 Figure 14. Recruitment of hCed-X and K18 to granular structure in various apoptotic systems. 51 Figure 15. hCed-X colocalizes with caaspase-cleaved K18 during apoptosis. 52 Figure 16. The hCed-X granular structures are caspase-dependent. 53 Figure 17. hCed-X protein remains intact during apoptosis. 54 Figure 18. hCed-X antisense RNA downregulates hCed-X protein level. 55 Figure 19. hCed-X is involved in TNF?induced apoptosis. 56 Figure 20. hCed-X siRNA knockdowns the endogenous level of hCed-X.. 57 Figure 21. hCed-X siRNA attenuates TNF?induced apoptosis.. 58 Figure 22. hCed-X siRNA reduces the active caspase 9 in cells treated with TNF? 59 Figure 23. hCed-X siRNA does not affect the level of active caspase 8 in cells treated with TNF?60 Table 1. C. elegans apoptosis-regulating genes and their human homologs (Peter et al., 1997) 61 Appendix 62 Appendix I. hCed-X interacts with K18 in vivo 626250667 bytesapplication/pdfen-US細胞凋亡腫瘤壞死因子apoptosisTNFhCed-Xotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51384/1/ntu-93-R91448006-1.pdf