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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李財坤 | |
| dc.contributor.author | Yi-Fei Lee | en |
| dc.contributor.author | 李沂霏 | zh_TW |
| dc.date.accessioned | 2021-06-15T05:07:30Z | - |
| dc.date.available | 2015-09-09 | |
| dc.date.copyright | 2010-09-09 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-26 | |
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Neilson, D.E., Adams, M.D., Orr, C.M., Schelling, D.K., Eiben, R.M., Kerr, D.S., Anderson, J., Bassuk, A.G., Bye, A.M., Childs, A.M., et al. (2009). Infection-triggered familial or recurrent cases of acute necrotizing encephalopathy caused by mutations in a component of the nuclear pore, RANBP2. Am J Hum Genet 84, 44-51. 30. Patrick, G.N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P., and Tsai, L.H. (1999). Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615-622. 31. Pemberton, L.F., Blobel, G., and Rosenblum, J.S. (1998). Transport routes through the nuclear pore complex. Curr Opin Cell Biol 10, 392-399. 32. Perry, R.B., and Fainzilber, M. (2009). Nuclear transport factors in neuronal function. Semin Cell Dev Biol 20, 600-606. 33. Purkiss, J.R., and Willars, G.B. (1996). Ionomycin induced changes in intracellular free calcium in SH-SY5Y human neuroblastoma cells: sources of calcium and effects on [3H]-noradrenaline release. Cell Calcium 20, 21-29. 34. Saidak, Z., Boudot, C., Abdoune, R., Petit, L., Brazier, M., Mentaverri, R., and Kamel, S. (2009). Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor. Exp Cell Res 315, 2072-2080. 35. Saido, T.C., Sorimachi, H., and Suzuki, K. (1994). Calpain: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J 8, 814-822. 36. Seewald, M.J., Korner, C., Wittinghofer, A., and Vetter, I.R. (2002). RanGAP mediates GTP hydrolysis without an arginine finger. Nature 415, 662-666. 37. Seewald, M.J., Kraemer, A., Farkasovsky, M., Korner, C., Wittinghofer, A., and Vetter, I.R. (2003). Biochemical characterization of the Ran-RanBP1-RanGAP system: are RanBP proteins and the acidic tail of RanGAP required for the Ran-RanGAP GTPase reaction? Mol Cell Biol 23, 8124-8136. 38. Sheffield, L.G., Miskiewicz, H.B., Tannenbaum, L.B., and Mirra, S.S. (2006). Nuclear pore complex proteins in Alzheimer disease. J Neuropathol Exp Neurol 65, 45-54. 39. Sorimachi, H., Ishiura, S., and Suzuki, K. (1997). Structure and physiological function of calpains. Biochem J 328 ( Pt 3), 721-732. 40. Suzuki, K., Hata, S., Kawabata, Y., and Sorimachi, H. (2004). Structure, activation, and biology of calpain. Diabetes 53 Suppl 1, S12-18. 41. Takahashi-Fujigasaki, J., Arai, K., Funata, N., and Fujigasaki, H. (2006). SUMOylation substrates in neuronal intranuclear inclusion disease. Neuropathol Appl Neurobiol 32, 92-100. 42. Tompa, P., Buzder-Lantos, P., Tantos, A., Farkas, A., Szilagyi, A., Banoczi, Z., Hudecz, F., and Friedrich, P. (2004). On the sequential determinants of calpain cleavage. J Biol Chem 279, 20775-20785. 43. Traglia, H.M., Atkinson, N.S., and Hopper, A.K. (1989). Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes. Mol Cell Biol 9, 2989-2999. 44. Vosler, P.S., Brennan, C.S., and Chen, J. (2008). Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 38, 78-100. 45. Wu, J., Matunis, M.J., Kraemer, D., Blobel, G., and Coutavas, E. (1995). Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region. J Biol Chem 270, 14209-14213. 46. Yagisawa, H. (2006). Nucleocytoplasmic shuttling of phospholipase C-delta1: a link to Ca2+. J Cell Biochem 97, 233-243. 47. Yokoyama, N., Hayashi, N., Seki, T., Pante, N., Ohba, T., Nishii, K., Kuma, K., Hayashida, T., Miyata, T., Aebi, U., et al. (1995). A giant nucleopore protein that binds Ran/TC4. Nature 376, 184-188. 48. Yudin, D., Hanz, S., Yoo, S., Iavnilovitch, E., Willis, D., Gradus, T., Vuppalanchi, D., Segal-Ruder, Y., Ben-Yaakov, K., Hieda, M., et al. (2008). Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve. Neuron 59, 241-252. 49. Zhang, J., Ito, H., Wate, R., Ohnishi, S., Nakano, S., and Kusaka, H. (2006). Altered distributions of nucleocytoplasmic transport-related proteins in the spinal cord of a mouse model of amyotrophic lateral sclerosis. Acta Neuropathol 112, 673-680. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46407 | - |
| dc.description.abstract | Ran GTP 酶活化蛋白質 (簡稱 RanGAP1) 是一個對於細胞核與細胞質之間物質的運輸 (nucleocytoplasmic transport) 相當重要的蛋白質，同時也是第一個發現會被類泛素小型修飾因子 SUMO (small-ubiquitin-like modifier) 所修飾的蛋白質。過去研究已知 SUMO 修飾能使細胞質中的 RanGAP1 聚集至細胞核邊緣 (nulcear rim)，藉此維持正常的細胞核內外物質運輸，然而對於 RanGAP1 是否有其他轉譯後修飾與生物功能仍尚未知曉。在本篇論文中，我們證實當鈣離子流入胞內時 (intracellular Ca2+ influx) 會使 RanGAP1 發生限制性的蛋白酶水解，並可能藉由此現象調控核質間的物質運輸。為了探究此現象，我們首先利用鈣離子螯合物，發現其能減弱此水解現象，顯示 RanGAP1 水解確實由鈣離子所調控。接著，透過生化、藥理以及基因調控等方式，我們證實 RanGAP1 水解現象是透過鈣離子所活化的一種半胱氨酸蛋白酶 (cysteine protease) calpain 2蛋白酶所主導。經由將純化的RanGAP1蛋白質與 calpain 2 蛋白酶反應所得之 RanGAP1 水解產物進行蛋白質 N 端定序 (N-terminal sequencing)，進一步確認 calpain 2 蛋白酶在 RanGAP1 上的兩個主要切位：其一是位於 RanGAP1 N 端的第 60 個胺基酸蘇氨酸 (Threonin) 與第 61 個胺基酸纈草胺酸 (Valine) 之間，其二則位於 RanGAP1 酸性區塊 (acidic domain) 與 C 端交界處的第 427 個胺基酸絲氨酸 (Serine) 與第428 個胺基酸絲氨酸之間。我們進一步發現 calpain 2 蛋白酶對 RanGAP1 的水解作用會導致 RanGAP1 自細胞核邊緣離開，很可能是經由移除 C 端可被 SUMO 修飾的區塊；我們也觀察到經 calpain 2 蛋白酶切去 N 端的 RanGAP1 亦會改變其胞內位置而聚集至細胞核內，也許是經由移除 N 端的岀核訊息 (nuclear export signal)。综合本篇研究結果，我們歸納出一個新的 RanGAP1 蛋白質轉譯後修飾機制：透過鈣離子活化 calpain 2 蛋白酶所調控的 RanGAP1 蛋白酶水解現象。此現象不僅改變 RanGAP1 在胞內的位置，也極可能因而影響到 RanGAP1 所調控的核質運輸，關於此鈣離子調控之 RanGAP1 水解現象的生物功能與其生理意義皆有待更深入的探討。 | zh_TW |
| dc.description.abstract | The GTPase activating protein 1 of Ran (RanGAP1), which plays a vital role in nucleocytoplasmic transport, is the first modification substrate for small-ubiquitin-like-modifier (SUMO) identified. SUMOylation targets RanGAP1 to nuclear rim and has been suggested as an important regulation for its nucleocytoplasmic transport function. Nevertheless, other post-translational modification(s) on RanGAP1 and their influences on cellular function(s) of RanGAP1 remained largely unknown. In this thesis, we demonstrated that intracellular Ca2+ influx induces limited proteolysis on RanGAP1 and thereby potentially modulates nucleocytoplasmic transport of substrate proteins. First, this proteolytic event could be attenuated by Ca2+ chelators, indicating the involvement of Ca2+ in RanGAP1 proteolysis. Using biochemical, pharmacological and genetic approaches, we showed that calpain 2, a Ca2+-dependent cysteine protease, is mainly responsible for this Ca2+-mediated RanGAP1 cleavage. Through in vitro calpain 2 cleavage assay with recombinant RanGAP1proteins, and followed with the N-terminal sequencing, two major calpain 2-cleaved sites were identified: (i) Thr60-Val61 on N’-terminal leucine-rich-repeat and (ii) Ser427-Ser428 between acidic domain and C’-terminal tail. Further studies indicated that calpain 2-mediated proteolytic cleavages of RanGAP1 potentially caused its translocation from nuclear rim to cytoplasm, probably through proteolytic removal of its C’-terminus where SUMOylation occurred. Similarly, the N’-terminal truncation of RanGAP1 by calpain 2 seemed to delocalize RanGAP1 to nucleus possibly due to lost of domains containing nuclear export sequence (NES). In sum, our results suggest a potential proteolytic modification of RanGAP1 via calpain 2 and its influences on cellular locations of RanGAP1 and nucleocytoplasmic transport of reporter substrate proteins. The implications of our study for the RanGAP1-mediated biological functions therefore need further investigation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T05:07:30Z (GMT). No. of bitstreams: 1 ntu-99-R97445110-1.pdf: 3847180 bytes, checksum: c49a498403c0ad693ac8d0035e9f95b2 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 口試委員會審定書
誌謝……………………………………………………………………………………....I 中文摘要………………………………………………………………………………...II ABSTRACT…………………………………………………………………………...IV INTRODUCTION……………………………………………………………………...1 1. RanGAP1 (GTPase activating protein 1 for Ran) ………………………………….1 1-1. Structure………………………………………………………………………...2 1-2. Subcellular localization and SUMO modification……………………………...3 1-3. Functions………………………………………………………………………..4 1-3-1. Nucleocytoplasmic transport……………………………………………….4 1-3-2. Mitotic spindle formation…………………………………………………..6 2. Ca2+ homeostasis and Ca2+ signaling……………………………………………......6 2-1. Ca2+ homeostasis…………………………………………………………......…7 2-2-1. Ionomycin….................................................................................................8 2-2. Ca2+ signaling.......................................................................................................8 2-2-1. Calpain family………………………………………………………….......8 SPECIFIC AIMS……………………………………………………………………...10 MATERIAL AND METHODS……………………………………………………….11 - Cell lines, cell culture and plasmid transfection…………………………………….11 - Plasmids……………………………………………………………………………..11 - Reagents and antibodies…………………………………………………………….11 - Preparation of total cell lysates, cytosolic and nuclear extracts for blot analysis…..12 - Western blot analysis………………………………………………………………..14 - Cloning, protein expression and protein purification……………………………….14 - In vitro calpain 2 cleavage and N-terminal sequencing………………………….....16 - Fluorescence microscopy & confocal microscopy…….............................................16 - Generation of stable FL-RanGAP1-V5-His, ΔN-RanGAP1-myc-His, and M-RanGAP1-myc-His expressing cell lines……………………………………….17 RESULTS……………………………………………………………………………...18 - Ionomycin, a Ca2+ ionophore, induces limited proteolysis on RanGAP1…………..18 - Ca2+ is the key regulator of ionomycin-induced RanGAP1 cleavage………………19 - Calpain 2 is responsible for Ca2+-mediated cleavage of RanGAP1………………...20 - Calpain 2 cleaves RanGAP1 at T60-V61 and S427-S428………………………………21 - Calpain 2-mediated RanGAP1 cleavage alters RanGAP1 subcellular localization...24 - The effect of ectopic expression of FL-,ΔN-, and M-RanGAP1 in nuclear transport of reporter GFP-fused protein containing nuclear localization signal (NLS)………27 DISCUSSION………………………………………………………………………….29 - Ca2+ influx had an effect on both RanGAP1 proteolysis and SUMOylation……….29 - The evolutionary point of view of calpain 2 cleavage sites among different species30 - The subcellular localization of cleaved products of RanGAP1…………………….31 - RanGAP1 proteolysis might affect on its ability of nucleocytoplasmic transport through Ran cycle…………………………………………………………………...32 - Specificity of Ca2+-mediated RanGAP1 proteolysis in different cell lines…………33 - Potential biological implication of Ca2+-induced RanGAP1 proteolysis: Ca2+, calpain, and nucleocytoplasmic transport………………………………….....34 FIGURES……………………………………………………………………………...37 - Figure 1: Ionomycin treatment induces proteolysis of RanGAP1.............................38 - Figure 2: Intra- and extra- cellular Ca2+ chelators attenuated ionomycin-induced proteolysis of RanGAP1.............................................................................39 - Figure 3: Calpain is involved in the Ca2+-mediated RanGAP1 cleavage...................40 - Figure 4: Calpain 2 contributes to the ionomycin-induced RanGAP1 cleavage........41 - Figure 5: Schematic illustration for His-tagged truncated RanGAP1 expression constructs for protein expression in the bacterial system...........................42 - Figure 6: Expression of RanGAP1 NTD and CTD variants in E. coli.......................43 - Figure 7: RanGAP1 cleavage by calpain 2 in vitro....................................................44 - Figure 8: Determination of calpain 2 cleavage sites on RanGAP1 by N-terminal sequencing..................................................................................................45 - Figure 9: Identified calpain 2-mediated cleavage sites on RanGAP1........................46 - Figure 10: Schematic illustration of the anticipated RanGAP1-related products after calpain 2 cleavage.....................................................................................47 - Figure 11: The schematic illustration for tagged N’-terminal truncated RanGAP1 (ΔN) and both N’- and C’-terminal truncated (M) RanGAP1 constructs.....48 - Figure 12: The ectopic expression of FL-RanGAP1 and TR-RanGAP1 proteins in HEK293T cells..........................................................................................49 - Figure 13: Ionomycin-induced proteolytic cleavage of ectopic expressed FL-RanGAP1 and TR-RanGAP1 proteins................................................50 - Figure 14: Expression of FL-RanGAP1 and TR-RanGAP1 proteins in HeLa cells..51 - Figure 15: Calpain 2-mediated RanGAP1 cleavage contributed partially to the ionomycin-induced RanGAP1 delocalization..........................................52 - Figure 16: Determination of the subcellular localizations of ectopic expressed RanGAP1 in HeLa cells by immunofluorescence analysis......................53 - Figure 17: Statistical analysis for the subcellular localizations of ectopic expressed FL-RanGAP1, ΔN-RanGAP1, and M-RanGAP1 proteins....................54 - Figure 18: Determination of the subcellular localizations of ectopic expressed RanGAP1 proteins by fractionation approach..........................................55 - Figure 19: Confocal microscopy analysis of the cellular distributions of ectopic expressed full length and truncated RanGAP1 proteins...........................56 - Figure 20: The effect of ectopic expression of FL-,ΔN-, and M-RanGAP1 in nuclear transport of reporter YFP-fused protein containing nuclear localization signal (NLS)..............................................................................................57 APPENDIXES................................................................................................................58 - Appendix Figure 1: The establishment of FL-RanGAP1 and TR-RanGAP1 stable clones in HCT116 cells...............................................................59 - Appendix Table 1: Disorders of Ca2+/calpain system or nucleocytoplasmic transport mechanisms in neuronal diseases and pathological settings.........60 REFERENCES………………………………………………………...……………...61 | |
| dc.language.iso | en | |
| dc.subject | 水解 | zh_TW |
| dc.subject | Ran GTP 酶 | zh_TW |
| dc.subject | 活化蛋白質 | zh_TW |
| dc.subject | calpain 2 蛋白酶 | zh_TW |
| dc.subject | 蛋白酶 | zh_TW |
| dc.subject | calpain 2 | en |
| dc.subject | RanGAP1 | en |
| dc.subject | calcium | en |
| dc.subject | nucleocytoplasmic transport | en |
| dc.title | 鈣離子活化 calpain 蛋白酶水解 RanGAP1 之分子機制與其生物意義 | zh_TW |
| dc.title | Calcium Induces Proteolytic Cleavage of RanGAP1 through Calpain | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄,李明學 | |
| dc.subject.keyword | Ran GTP 酶,活化蛋白質,calpain 2 蛋白酶,蛋白酶,水解, | zh_TW |
| dc.subject.keyword | RanGAP1,calcium,nucleocytoplasmic transport,calpain 2, | en |
| dc.relation.page | 64 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-07-27 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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