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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 何孟樵(Meng-Chiao Ho) | |
dc.contributor.author | Min-Chi Yeh | en |
dc.contributor.author | 葉敏琪 | zh_TW |
dc.date.accessioned | 2021-07-10T21:35:03Z | - |
dc.date.available | 2021-07-10T21:35:03Z | - |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-16 | |
dc.identifier.citation | Adams, Josephine, Reed Kelso, and Lynn Cooley. 'The kelch repeat superfamily of proteins: propellers of cell function.' Trends in cell biology 10.1 (2000): 17-24.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76685 | - |
dc.description.abstract | 由KLHL20所參與的泛素化作用已經被發現會跟腫瘤增生跟細胞自噬終止作用有極大的關係。KLHL20是一個E3連接酶,會去抑制PML和UKL1,PML是一個可以抑制腫瘤的蛋白,而ULK1是參與在細胞自噬的蛋白。所以KLHL20在PML跟ULK1的作用機制中扮演一個重要的角色。當PML的Ser518被磷酸化且Pro519被異構化時就會被KLHL20所辨識,被磷酸化的ULK1已知會被KLHL20所認,因此KLHL20被認為會用不同種的方式辨識其他蛋白。但是目前KLHL20的被辨識機制並不是很清楚,我們合作的實驗室已經發現KLHL20會跟不同種類的蛋白作用產生不同的路徑,目前已知的是KLHL20會跟Cul3和Roc1作用形成複合體再跟其他蛋白作用。但是像是KLHL20的受質PML也是一個蛋白載體,因此用Pull-down的方式很難去找出其他KLHL20的受質蛋白。所以我們的目的是要去找出其他會跟KLHL20作用的蛋白,利用從可能的受質蛋白序列合成胜肽,再將KLHL20直接跟胜肽做反應,找出特定的序列,再從中找到其他受質蛋白。再進一步的解出蛋白和胜肽的複合體結構跟發展治療癌症藥物。 | zh_TW |
dc.description.abstract | KLHL20-mediated ubiquitination is highly related to tumor progression and autophagy termination. KLHL20, an E3 ligase down regulates PML and ULK1, which are tumor suppressor and autophagy proteins, respectively. Hence, KLHL20 is a key substrate adaptor for both PML and ULK1 pathways. The recognition of PML by KLHL20 requires both serine phosphorylation at Ser518 and the isomerization at Pro519. ULK1 autophosphorylation is required for KLHL20 recruitment but the isomerization is not identified. These findings indicate that KLHL20 can interact with protein substrates in different fashions. However, the molecular recognition mechanism of KLHL20 is not yet clear. Our collaborator has found that KLHL20 may be involved in different cellular functions by interacting with other protein substrates. KLHL20 is known to interact with Cullin3 (Cul3) and Roc1 and often forms complexes with other proteins. Some of KLHL20 substrates such as PML also interact with other proteins. Hence, it is very difficult to identify KLHL20 substrates by conventional pull-down assays. Currently, we plan to identify KLHL20 by biophysical approaches. We aim to develop direct binding assays to examine if the peptide sequences from the potential substrates can interact with KLHL20 directly. We also hope to identify the critical sequence for KLHL20 binding using one bead-one compound combinatorial chemistry (OBOC) approach. Our results can facilitate KLHL20 substrate identification. Our ultimate goals are to determine the structures of KLHL20-substrate complexes and develop the potential drugs for cancer therapy. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:35:03Z (GMT). No. of bitstreams: 1 ntu-105-R03b46029-1.pdf: 1661416 bytes, checksum: 15dfe158af2c7f08059aa19906687eb6 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | List of Contents
論文口試委員審定書……………………………………………………………………i 中文摘要 .......................................................................................................................... ii Abstract............................................................................................................................ iii List of Contents .................................................................................................................v List of Figures…………………………………………………………………………...ix List of Tables…………………………………………………………………………...xii Introduction: Literature review..........................................................................................1 1. Ubiquitination ................................................................................................... 1 1.1 The cascade of ubiquitination ............................................................... 1 1.2 The different topologies of ubiquitination ............................................ 2 1.3 The diversity of E3 ubiquitin ligases .................................................... 2 2 BTB-Kelch protein family ................................................................................ 4 2.1 BTB-Kelch protein family .................................................................... 4 2.2 KLHL20 (Kelch-like protein 20) .......................................................... 6 3 Tumor progression ............................................................................................ 9 3.1 Tumor progression ................................................................................ 9 3.2 PML ...................................................................................................... 9 4 Autophagy ....................................................................................................... 11 4.1 The types of autophagy ....................................................................... 11 4.2 The Initiation and Nucleation of autophagosome............................... 12 4.3 The termination of autophagosome .................................................... 13 4.4 The regulation of ULK1 ..................................................................... 13 5 Combinatorial peptide library ......................................................................... 17 5.1 Combinatorial peptide library ............................................................. 17 5.2 Solid phase peptide synthesis ............................................................. 17 5.3 One-beads-one-compound .................................................................. 19 6 Fluorescence polarization ............................................................................... 20 6.1 Fluorescence assays ............................................................................ 20 6.2 Fluorescence polarization ................................................................... 20 Introduction: Aims...........................................................................................................22 Results .............................................................................................................................24 Cloning, expression and purification of KLHL20302-609 ......................................... 24 Condon optimization ...................................................................................... 26 Thermal-denaturation assays .......................................................................... 27 Crystallization of the KLHL20302-609 .............................................................. 28 NMR studies of PML peptide and KLHL20302-609 ................................................. 29 PML peptide (with/without Pin 11-167) ............................................................ 29 KLHL20302-609 ................................................................................................. 30 Pull down assay ...................................................................................................... 30 Fluorescence polarization ....................................................................................... 31 The issue of insoluble KLHL20302-609 protein ........................................................ 32 Future works ........................................................................................................... 34 Materials and Methods ....................................................................................................35 Materials ................................................................................................................. 35 Instruments and equipment ..................................................................................... 38 Methods .................................................................................................................. 39 High through Put Cloning and Expression of Recombinant KLHL20 ........... 39 Western Blotting ............................................................................................. 40 KLHL20 Codon Optimization ........................................................................ 41 The Large scale of expression and purification of KLHL20302-609 ................. 41 Mass spectroscopy .......................................................................................... 42 Thermofluor assays to optimize buffer condition ........................................... 43 Crystallization ................................................................................................. 44 NMR spectroscopy ......................................................................................... 44 Pull down assay .............................................................................................. 45 Fluorescence Polarization assay ..................................................................... 45 One-bead-one-compound screening ............................................................... 46 References .......................................................................................................................78 List of Figures Figure 1. The mechanism of Cullin3-KLHL20 ubiquitin ligase-dependent pathway targets PML ............................................................................................................ 48 Figure 2. The mechanism of Cullin3-KLHL20 ubiquitin ligase governs the turnover of ULK1 and VPS34 complexes to control autophagy termination ........ 49 Figure 3. The protein expression of KLHL20302-609 using human cDNA with a Histag ........................................................................................................................... 50 Figure 4. The expression test of the KLHL20302-609 constructs with different fusion tags .......................................................................................................................... 51 Figure 5. The small scale protein expression testing with various induction temperature and the concentration of IPTG. .......................................................... 52 Figure 6. The expression of KLHL20302-609 with various time-course ................... 53 Figure 7. The various E. coli competent cell of KLHL20302-609 ............................. 54 Figure 8. The list of protein constructs and expression vector I have created. ...... 55 Figure 9. The result of high-throughput expression screening ............................... 56 Figure 10. The western Blotting of KLHL20302-609 with four different tags. ......... 57 Figure 11. The purification result of protein with the different induction condition ................................................................................................................................ 58 Figure 12. The purification result of KLHL20302-609 that was induced at 0.1 mM ITPG ....................................................................................................................... 59 Figure 13. The protein purification result of KLHL20302-609 using autoinduction system. .................................................................................................................... 60 Figure 14. The purification result of KLHL20302-609 in presence of ATP or ADP during the protein purification. ............................................................................... 61 Figure 15. The purification result of KLHL20302-609 that MgCl2 was added into the cell culture medium ................................................................................................ 62 Figure 16. The DNA sequence alignment between native human KLHL20302-609 and codon optiimzed KLHL20302-609 ...................................................................... 63 Figure 17. The purification result of KLHL20302-609 using codon optimized gene shows significant improve of protein purity and protein yield. .............................. 64 Figure 18. The 1D-NMR sepctrum of KLHL20at various pH and temperature .... 65 Figure 19. The binding experiments of KLHL20 and peptide using fluoroscence polarization method ................................................................................................ 66 Figure 20. The commassie blue strain and western blotting of KLHL20302-609 production using different glycerol stock ............................................................... 67 Figure 21. The list of culture medium conditions with various IPTG concentration and induction time with/without benzyl alcohol (BA) and magnesium (Mg+). ..... 68 Figure 22. The expression result of protein induced at different O.D. and IPTG concentration .......................................................................................................... 69 Figure 23. The protein purification result of KLHL20 that was induced at various of IPTG concentraion and lyzed by different buffer condition .............................. 71 List of Tables Table 1. The primer for LIC ................................................................................... 73 Table 2. The Tm of buffer (the concentration of salt) ............................................. 75 Table 3. The Tm of buffer (the pH of salt) .............................................................. 76 Table 4. The peptide for binding assay................................................................... 77 | |
dc.language.iso | zh-TW | |
dc.title | KLHL20結構域蛋白質之結構及功能研究 | zh_TW |
dc.title | Structural and Functional Study of KLHL20 Domain Proteins | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳瑞華(Ruey-Hwa Chen),吳世雄(Shih-Hsiung Wu),施修明(Hsiu-Ming Shih) | |
dc.subject.keyword | KLHL20,腫瘤增生,細胞自噬終止作用,OBOC,泛素化, | zh_TW |
dc.subject.keyword | KLHL20,Tumor progression,Autophagy termination,OBOC,protein-protein interactions, | en |
dc.relation.page | 88 | |
dc.identifier.doi | 10.6342/NTU201602778 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-17 | |
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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