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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張?仁(Dr. Ching-Jin Chang) | |
| dc.contributor.author | Ya-Han Yu | en |
| dc.contributor.author | 游雅涵 | zh_TW |
| dc.date.accessioned | 2021-06-15T13:00:37Z | - |
| dc.date.available | 2019-07-26 | |
| dc.date.copyright | 2016-07-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-11 | |
| dc.identifier.citation | 1. Cheadle, C., et al., Stability regulation of mRNA and the control of gene expression. Ann N Y Acad Sci, 2005. 1058: p. 196-204.
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Blackshear, Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling. J Biol Chem, 2002. 277(13): p. 11606-13. 32. Carman, J.A. and S.G. Nadler, Direct association of tristetraprolin with the nucleoporin CAN/Nup214. Biochem Biophys Res Commun, 2004. 315(2): p. 445-9. 33. Brook, M., et al., Posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase pathways. Mol Cell Biol, 2006. 26(6): p. 2408-18. 34. Taylor, G.A., et al., Mitogens stimulate the rapid nuclear to cytosolic translocation of tristetraprolin, a potential zinc-finger transcription factor. Mol Endocrinol, 1996. 10(2): p. 140-6. 35. Baou, M., A. Jewell, and J.J. Murphy, TIS11 family proteins and their roles in posttranscriptional gene regulation. J Biomed Biotechnol, 2009. 2009: p. 634520. 36. Hitti, E., et al., Mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element. Mol Cell Biol, 2006. 26(6): p. 2399-407. 37. Bourcier, C., et al., Constitutive ERK activity induces downregulation of tristetraprolin, a major protein controlling interleukin8/CXCL8 mRNA stability in melanoma cells. Am J Physiol Cell Physiol, 2011. 301(3): p. C609-18. 38. Graham, J.R., et al., mRNA degradation plays a significant role in the program of gene expression regulated by phosphatidylinositol 3-kinase signaling. Mol Cell Biol, 2010. 30(22): p. 5295-305. 39. Johnson, B.A., et al., Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms. J Biol Chem, 2002. 277(20): p. 18029-36. 40. Sun, L., et al., Tristetraprolin (TTP)-14-3-3 complex formation protects TTP from dephosphorylation by protein phosphatase 2a and stabilizes tumor necrosis factor-alpha mRNA. J Biol Chem, 2007. 282(6): p. 3766-77. 41. Schichl, Y.M., et al., Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2). J Biol Chem, 2011. 286(44): p. 38466-77. 42. Deleault, K.M., S.J. Skinner, and S.A. Brooks, Tristetraprolin regulates TNF TNF-alpha mRNA stability via a proteasome dependent mechanism involving the combined action of the ERK and p38 pathways. Mol Immunol, 2008. 45(1): p. 13-24. 43. Liang, J., et al., RNA-destabilizing factor tristetraprolin negatively regulates NF-kappaB signaling. J Biol Chem, 2009. 284(43): p. 29383-90. 44. Schichl, Y.M., et al., Tristetraprolin impairs NF-kappaB/p65 nuclear translocation. J Biol Chem, 2009. 284(43): p. 29571-81. 45. Rosen, E.D. and B.M. Spiegelman, Molecular regulation of adipogenesis. Annu Rev Cell Dev Biol, 2000. 16: p. 145-71. 46. Bost, F., et al., The role of MAPKs in adipocyte differentiation and obesity. Biochimie, 2005. 87(1): p. 51-6. 47. Owens, D.M. and S.M. Keyse, Differential regulation of MAP kinase signalling by dual-specificity protein phosphatases. Oncogene, 2007. 26(22): p. 3203-13. 48. Sakaue, H., et al., Role of MAPK phosphatase-1 (MKP-1) in adipocyte differentiation. J Biol Chem, 2004. 279(38): p. 39951-7. 49. Prusty, D., et al., Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes. J Biol Chem, 2002. 277(48): p. 46226-32. 50. Bost, F., et al., The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes, 2005. 54(2): p. 402-11. 51. Engelman, J.A., M.P. Lisanti, and P.E. Scherer, Specific inhibitors of p38 mitogen-activated protein kinase block 3T3-L1 adipogenesis. J Biol Chem, 1998. 273(48): p. 32111-20. 52. Aouadi, M., et al., Inhibition of p38MAPK increases adipogenesis from embryonic to adult stages. Diabetes, 2006. 55(2): p. 281-9. 53. Hirosumi, J., et al., A central role for JNK in obesity and insulin resistance. Nature, 2002. 420(6913): p. 333-6. 54. Jaeschke, A., M.P. Czech, and R.J. Davis, An essential role of the JIP1 scaffold protein for JNK activation in adipose tissue. Genes Dev, 2004. 18(16): p. 1976-80. 55. 謝欣蕙, 3T3-L1脂肪前驅細胞分化前期中TTP受ERK磷酸化之調控. 2015, 台灣大學 生化科學所. 56. de Ruijter, A.J., et al., Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J, 2003. 370(Pt 3): p. 737-49. 57. Yoon, H.G., et al., Purification and functional characterization of the human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1. EMBO J, 2003. 22(6): p. 1336-46. 58. Guenther, M.G., O. Barak, and M.A. Lazar, The SMRT and N-CoR Corepressors Are Activating Cofactors for Histone Deacetylase 3. Molecular and Cellular Biology, 2001. 21(18): p. 6091-6101. 59. Sanduja, S., et al., The role of tristetraprolin in cancer and inflammation. Front Biosci (Landmark Ed), 2012. 17: p. 174-88. 60. Maryati, M., B. Airhihen, and G.S. Winkler, The enzyme activities of Caf1 and Ccr4 are both required for deadenylation by the human Ccr4-Not nuclease module. Biochem J, 2015. 469(1): p. 169-76. 61. Sandler, H. and G. Stoecklin, Control of mRNA decay by phosphorylation of tristetraprolin. Biochem Soc Trans, 2008. 36(Pt 3): p. 491-6. 62. Chrestensen, C.A., et al., MAPKAP kinase 2 phosphorylates tristetraprolin on in vivo sites including Ser178, a site required for 14-3-3 binding. J Biol Chem, 2004. 279(11): p. 10176-84. 63. Bermudez, O., et al., Post-transcriptional regulation of the DUSP6/MKP-3 phosphatase by MEK/ERK signaling and hypoxia. J Cell Physiol, 2011. 226(1): p. 276-84. 64. 林念儀, 脂肪細胞分化過程中TTP、MKP-1與MAPK訊息傳遞路徑間之調控機制. 2009, 台灣大學. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50825 | - |
| dc.description.abstract | Tristetraprolin (TTP)是一個核糖核酸(RNA)結合蛋白，可以調控含有多腺嘌呤尿嘧啶序列(AU-rich element)的核糖核酸的降解，且知TTP的功能受到結合蛋白的不同及本身蛋白磷酸化程度所調控。TTP會藉由CCR4-NOT脫腺苷化酶複合蛋白(CCR4-NOT deadenylase complex)進行核糖核酸的poly(A)降解。然而我們發現在CCR4-NOT腺苷化酶複合蛋白中的兩個腺苷化酶對於poly(A)降解是必要的。TTP的磷酸化除了已被廣泛研究的p38路徑外，ERK訊息傳遞路徑也可以去磷酸化TTP進而調控其穩定性、細胞內的位置和功能。先前利用質譜分析得知TTP的第316號絲氨酸會因ERK訊息傳遞路徑的活化而被磷酸化，且知第316號絲氨酸的磷酸化會降低TTP與CCR4-NOT腺苷化酶複合蛋白之間的結合。接著，我們進一步去研究TTP第316號絲氨酸磷酸化在生理條件下的功能及影響。在受到脂多醣(lipopolyssacharide,LPS)刺激的小鼠巨噬細胞RAW264.7中觀察到除了TTP的表現量增加外也伴隨著第316號絲氨酸被磷酸化，且此磷酸化降低了TTP和Cnot1蛋白的結合反而增加了和14-3-3的結合。另外，在脂肪前驅細胞3T3-L1的分化過程中利用MAPK去磷酸化酶(MKP-1)的抑制劑sanguinarine去促進ERK的活化和TTP第316號絲氨酸的磷酸化，進而抑制了3T3-L1脂肪前驅細胞的分化。此外同時也在NIH3T3胚胎纖維母细胞觀察到第316號絲氨酸的磷酸化使TTP較不容易進入細胞核。另外一方面，質譜分析發現和TTP結合的蛋白之一的組蛋白去乙酰酶(histone deacetylases3)是轉譯抑制分子，已知兩者皆參與負調控NF-κB訊息傳遞路徑的活化。我們發現TTP負調控NF-κB訊息傳遞路徑的可能因素，包含TTP會增加HDAC3的蛋白穩定性、TTP的磷酸化會使其較不易進細胞核還有TTP蛋白的不同區域對於轉譯調控有不同的影響。綜合以上研究結果，我們提供證據證明TTP的磷酸化和結合蛋白改變在基因調控上的分子機制。 | zh_TW |
| dc.description.abstract | Tristetraprolin (TTP) is an RNA-binding protein that mediates the degradation of AU-rich element containing mRNA. The function of TTP is tightly regulated by its protein phosphorylation status and protein-protein interaction. TTP-mediated mRNA decay is through the recruitment of CCR4-NOT deadenylase complex, which consists two deadenylases Cnot6 and Cnot7. Our result reveal that both enzymes of Cnot7 and Cnot6 are required for in vitro deadenylation. In addition to extensively studied p38 pathway, previous reports showed that ERK signaling also can regulate the protein stability, subcellular localization, and function of TTP. By mass spectrometry analysis Ser-316 of TTP was identified to be phosphorylated in response to ERK signaling. Ser-316 phosphorylation of TTP would decrease its interaction with the CCR4-NOT deadenylase complex. Next, we aim to investigate the functional effect of Ser-316 phosphorylation under the physiological condition. We demonstrates that ERK signals induce TTP expression and phosphorylation, which result in changing the binding protein of TTP in LPS-stimulated RAW264.7 and inhibiting 3T3-L1 adipogenesis. Additionally, we observed that Ser-316 phosphorylation can affect the subcellular localization of TTP. On the other hand, transcription co-repressor HDAC3 was identified as TTP-associated proteins in the mass spectrometry analysis. Both of TTP and HDAC3 have been known can negatively regulate NF-κB signaling-mediated transcriptional activation. We demonstrate possible molecular mechanism including that TTP deletion mutants has Transcriptional activity; TTP increases Hdac3 protein stability; and p38-mediated TTP phosphorylation affects its subcellular localization. Collectively, our results might provide evidence to demonstrate the molecular mechanism of TTP phosphorylation and protein-protein interaction on gene regulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T13:00:37Z (GMT). No. of bitstreams: 1 ntu-105-R03b46026-1.pdf: 3283092 bytes, checksum: 89554647a2c171e48a58d88cb5ce6c2f (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書..........................................i
摘要...................................................ii Abstract..............................................iii Content.................................................v Abbreviations..........................................ix 1. Introduction....................................1 1.1 ARE-mediated mRNA decay.........................1 1.2 Tristetraprolin (TTP) family....................2 1.3 Functions of TTP proteins in mRNA destabilization.........................................2 1.4 Phosphorylation and subcellular localization of TTP.....................................................3 1.5 RNA-binding independent function of the TTP.....5 1.6 Molecular regulation of adipogenesis............5 1.7 Specific aims...................................7 2. Materials and Methods...........................8 2.1 Plasmid Constructs..................................8 2.2 Cell Culture........................................9 2.3 Oil Red O Staining.................................10 2.4 Preparation Whole Cell Extracts and Cytoplasmic/Nuclear Extracts...........................10 2.5 Western Blot Assay.................................11 2.6 Antibody and Chemicals.............................12 2.7 Immunoprecipitation Assay..........................12 2.8 In vitro deadenylase assay.........................13 2.9 Dual Luciferase Reporter Assay.....................14 2.10 Indirect Immunofluorescence Microscopy............14 2.11 GST-tagged recombinant protein purification and GST pull-down assay..................................................15 2.12 RNA Extraction and Reverse-transcription..........15 2.13 Real-time PCR.....................................16 2.14 Statistical Analysis..............................17 3. Results........................................18 3.1 The enzyme activity of Cnot6 and Cnot6 are both required for in vitro deadenylation....................18 3.2 TTP phosphorylation at serine 316 by ERK signaling regulates its protein-binding activity in LPS-stimulated RAW264.7 cells..............................19 3.3 Analysis of subcellular localization of TTP with Ser-316 phosphorylation................................20 3.4 MKP-1 inhibitor Sanguinarine enhances Ser-316 phosphorylation of TTP and affects TTP subcellular localization during 3T3-L1 differentiation.............21 3.5 MKP-1 inhibitor sanguinarine inhibited 3T3-L1 adipogenesis...........................................22 3.6 TTP interacts with Hdac3 through TZF motif and as a modulator of NF-κB signaling.........................22 3.7 Transcriptional activity analysis of TTP deletion mutants................................................24 3.8 Hdac3 is more stable in the presence of TTP....24 3.9 Transcriptional effect of p38-mediated TTP phosphorylation........................................25 4. Discussion.............................................27 5. Figures........................................32 Figure 1. The enzyme activity of Cnot6 and Cnot7 are both required for in vitro deadenylation....................32 Figure 2. TTP phosphorylation at serine 316 by ERK signaling regulates its protein-binding activity in LPS-stimulated RAW264.7 cells..............................35 Figure 3. Analysis of subcellular localization of TTP with Ser-316 phosphorylation...........................38 Figure 4. MKP-1 inhibitor Sanguinarine enhances Ser-316 phosphorylation of TTP and affects TTP subcellular localization during 3T3-L1 differentiation.............41 Figure 5. MKP-1 inhibitor sanguinarine inhibited 3T3-L1 adipogenesis...........................................44 Figure 6. TTP interacts with Hdac3 through TZF motif and as a modulator of NF-κB signaling......................47 Figure 7. Transcriptional activity analysis of TTP deletion mutants.......................................52 Figure 8. Hdac3 is more stable in the presence of TTP..55 Figure 9. Transcriptional effect of p38-mediated TTP phosphorylation........................................57 6. Tables.........................................60 Table 1. Primers for PCR...............................60 Table 2. Primers for real-time PCR.....................61 Table3. TTP-associated proteins identified by MS/MS analysis...............................................62 7. Appendix.......................................63 8. References.....................................64 | |
| dc.language.iso | en | |
| dc.subject | 鋅指蛋白36(TTP) | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | ERK訊息傳遞路徑 | zh_TW |
| dc.subject | NF-κB訊息傳遞路徑 | zh_TW |
| dc.subject | 鋅指蛋白36(TTP) | zh_TW |
| dc.subject | 磷酸化 | zh_TW |
| dc.subject | ERK訊息傳遞路徑 | zh_TW |
| dc.subject | NF-κB訊息傳遞路徑 | zh_TW |
| dc.subject | Phosphorylation | en |
| dc.subject | Tristetraprolin | en |
| dc.subject | Phosphorylation | en |
| dc.subject | ERK signaling | en |
| dc.subject | NF-κB signaling | en |
| dc.subject | Tristetraprolin | en |
| dc.subject | ERK signaling | en |
| dc.subject | NF-κB signaling | en |
| dc.title | Tristetraprolin磷酸化與結合蛋白之研究 | zh_TW |
| dc.title | Functional regulation of tristetraprolin through protein phosphorylation and protein-protein interaction | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張震東(Dr. Geen-Dong Chang),張茂山(Dr. Mau-Sun Chang),果伽蘭(Dr. Chia-lam Kuo) | |
| dc.subject.keyword | 鋅指蛋白36(TTP),磷酸化,ERK訊息傳遞路徑,NF-κB訊息傳遞路徑, | zh_TW |
| dc.subject.keyword | Tristetraprolin,Phosphorylation,ERK signaling,NF-κB signaling, | en |
| dc.relation.page | 68 | |
| dc.identifier.doi | 10.6342/NTU201600810 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-12 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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