指導教授：張世宗臺灣大學：生化科技學系黃珮慈Huang, Pei-TzuPei-TzuHuang2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261636蛋白酶體 (proteasome) 由20S core particle (CP) 和19S regulatory particle (RP)所組成。而19S regulatory particle的基座(base)由六個Rpt次單元體Rpt1~6共同組成。Rpt5 (19S regulatory particle ATPase 5) 屬於AAA-ATPase family，具有辨識泛素鏈、對蛋白質進行去摺疊、以及運送目標蛋白質等功能。本實驗室先前的研究發現Rpt5在COS7細胞株中會受到SUMO2的修飾，而且也可以利用大腸桿菌系統將Rpt5進行SUMO1跟SUMO2的修飾。本研究更進一步以 in vitro sumoylation assay證明，在試管中Rpt5重組蛋白可以被SUMO1修飾。 在蛋白酶體降解蛋白質的過程中，ATP的結合與水解扮演重要的角色，因此本研究想要探討SUMO修飾是否會影響Rpt5的ATPase活性。實驗結果顯示，在受到SUMO修飾之後，Rpt5的ATPase活性會下降。此外，Rpt5上有六個可能為SUMO-interacting motif (SIM) 的序列。先前的研究指出將Rpt5的SIM3突變之後，會使Rpt5在大腸桿菌系統中被SUMO1修飾的情形顯著下降；但在HEK293T細胞中重複此實驗時，卻發現Rpt5的SUMO修飾並未下降。為了排除HEK293T內生性Rpt5的干擾，本研究嘗試使用shRNA來knockdown 內生性Rpt5的表現。但是實驗結果顯示Rpt5 knockdown的細胞株無法存活，因此仍無法充分證實SIM3是否參與Rpt5之SUMO修飾作用。The 26S proteasome is composed of 20S core particle (CP) capped with 19S regulatory particle (RP). Regulatory particle triple-A ATPase 5 (Rpt5) is one of the subunits of the 19S RP, which forms the base of 19S RP together with Rpt1, Rpt2, Rpt3, Rpt4 and Rpt6. The 19S base performs several functions, such as polyubiquitin chain recognition, substrate unfolding, gate opening and also translocation of target proteins into 20S CP. Our previous study has revealed that Rpt5 was modified by small ubiquitin-like modifier 2 (SUMO2) in COS7 cells, and modified by SUMO1 and SUMO2 in the E. coli sumoylation system. In the present study, the in vitro sumoylation assay further demonstrated that recombinant Rpt5 can be modified by SUMO1. Because ATP binding and hydrolysis play critical roles in the regulation of proteasome function, this study was aimed to examine whether SUMO modification may affect the ATPase activity of Rpt5. The result showed that the ATPase activity of Rpt5 was reduced by SUMO2 modification. Furthermore, Rpt5 was found to contain several putative SUMO interacting motifs (SIMs). Although previous in vitro experimental results showed that sumoylation of Rpt5 by SUMO1 was markedly reduced while SIM3 was mutated, the level of Rpt5 sumoylation was not lowered when SIM3 was mutated in HEK293T cells. To rule out the possibility that endogenous Rpt5 might interfere with the observation of sumoylation pattern, shRNAs were applied to knockdown the expression level of endogenous Rpt5. However, the cells with inhibited Rpt5 expression were not viable. Therefore, whether SIM3 is involved in Rpt5 sumoylation remain elusive.Contents Introduction 1 1. The ubiquitin-proteasome system 1 2. The proteasome 1 2.1 20S core particle 2 2.2 19S regulatory particle 3 2.3 Regulatory particle triple-A ATPase 5 (Rpt5) 6 3. The ubiquitination pathway 8 4. The sumoylation modification 10 4.1 The SUMO Protein 12 4.2 The sumoylation pathway 13 4.3 SUMO consensus motif 15 4.4 SUMO interacting motif 16 4.5 Crosstalk between SUMO and ubiquitin pathway 18 5. Specific aims of the study 19 Materials and Methods 22 1. Bacterial system 22 2. Cell system 23 3. Plasmid DNA preparation 24 4. Preparation of competent cell 25 5. Expression of heterologous Rpt5 protein in E.coli transformation 25 6. IPTG induction 26 7. Cell disruption by FRENCHR Press (high-pressure homogenizer) 27 8. Purification of recombinant protein 27 9. Bradford protein quantification 30 10. SDS-PAGE 31 11. Native PAGE 31 12. Western blotting (Immunoblotting) 32 13. Antibody stripping and reprobing 32 14. ATPase activity assay 33 15. In vitro sumoylation assay 33 16. Mammalian cell culture 34 17. Cryopreservation 34 18. Preparation of shRNA 34 19. Transfection 35 20. Cell lysis 35 Results 37 1. Examine whether sumoylation will affect the ATPase activity of Rpt5 37 1.1 Expression of Rpt5 and Rpt5-SUMO2 37 1.2 Purification of Rpt5 and Rpt5-SUMO2 38 1.3 Rpt5 and Rpt5-SUMO2 ATPase activity assay 39 2. Use in vitro sumoylation system to observe the sumoylation of Rpt5 40 2.1 Purification of Sae1/2, Ubc9, SUMO1 and SUMO2 40 2.2 In vitro sumoylation assay of Rpt5m9ok9okokooooooooooook 41 3. Examine Rpt5 sumoylation site in HEK293T cell 41 Discussion 42 1. Addition of glycylglycine did not enhance the expression of Rpt5 protein in E. coli 42 2. Purification of Rpt5 and Rpt5-SUMO2 protein by anion exchange column and affinity chromatography 43 3. Sumoylation may affect the ATPase activity of Rpt5 44 4. Rpt5 can be modified by SUMO1 45 5. Knockdown of Rpt5 by shRNA may not be viable in cells 47 References 61 Appendix 7265381403 bytesapplication/pdf論文使用權限：不同意授權ProteasomeRpt5SUMOsumoylationSIMthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261636/1/ntu-103-R01b22001-1.pdf