陳瑞華臺灣大學：分子醫學研究所李宜蒨Lee, Yi-ChienYi-ChienLee2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51348BTB-kelch蛋白KLEIP (Kelch-like ECT2 interacting protein)，為一個大小64 kDa的蛋白。其蛋白質結構中包含了一個BTB/POZ區塊，一個BACK區塊,以及六個kelch重複片段。在本實驗室先前的研究中，我們用死亡相關激酶 (DAPK)之死亡區塊進行酵母菌雙雜合系統分析，發現KLEIP為其交互作用蛋白。進一步的實驗發現，KLEIP的功能在於作為Cul3泛素連接酶複合體作用時的受質辨認者，KLEIP對DAPK做專一性的辨認之後，透過這樣的複合體對DAPK進行泛素化修飾，進而使DAPK被送至蛋白酶體降解。在本篇論文中，我們發現KLEIP在不同的細胞株及不同的人類組織中均有表現。此外，利用Triton生化萃取分析，發現KLEIP分布在細胞中Triton無法溶解的部份。在此之後，我們更進一步證明細胞中內生的KLEIP以及Cul3與BTB蛋白交互作用的能力皆在DAPK泛素化扮演不可或缺的角色，證實了DAPK為KLEIP/Cul3/Roc1泛素連接酶複合體之受質。另外，我們進一步探究是否KLEIP本身會被轉譯後修飾及調控。參考以酵母菌雙雜合系統預測KLEIP之交互蛋白與KLEIP蛋白質序列分析結果，我們推測KLEIP 會進行類泛素化修飾。之後我們證明了KLEIP的確會進行類泛素化修飾，且這個修飾在缺氧環境下會受到正向調控。此外，我們也發現了KLEIP還會與SUMO1有非共價之交互作用。綜合以上結果，我們發現了KLEIP的轉譯後類泛素修飾以及其與SUMO1之非共價交互作用能力，並且推測這兩種特性可能會調節KLEIP之功能或其在細胞內之位置，進而影響DAPK之泛素化修飾。The BTB-kelch protein KLEIP (Kelch-like ECT2 interacting protein) is a 64 kDa protein which contains a BTB/POZ domain, a BACK domain, and six kelch repeats. Previous study in our laboratory identified KLEIP as a binding partner of DAPK (Death -associated protein kinase) and revealed a function of KLEIP as the substrate adaptor of Cul3 ubiquitin E3 ligase complex to promote DAPK ubiquitination and proteasomal degradation. In this thesis, we first found that KLEIP was ubiquitously expressed in various tissues and cell lines and present in detergent insoluble compartment of cells. We also demonstrated the essential roles of endogenous KLEIP and the KLEIP binding ability of Cul3 to promote DAPK ubiquitination, further confirming DAPK as a substrate of KLEIP/Cul3/Roc1 E3 ligase complex. In addition, we explored whether the substrate adaptor KLEIP itself would be modified and/or regulated. Based on the results of yeast two-hybrid screen for putative KLEIP-interacting proteins and sequence analysis of KLEIP protein, we postulated that KLEIP may undergo SUMO modification. Indeed, we demonstrated that KLEIP was sumoylated in vivo, and this modification was up-regulated under hypoxia conditions. Furthermore, we found that KLEIP can bind SUMO1 non-covalently through its consensus SUMO interacting motifs (SIMs). Together, we revealed the post-translational SUMO modification and SUMO1-binding ability of KLEIP, and we speculated that sumoylation and SUMO1-interaction of KLEIP may regulate its function and/or subcellular localization to influence the ubiquitination of DAPK.Table of Content 中文摘要 4 Abstract 5 Introduction 6 Ubiquitination 6 BTB proteins 9 SUMOylation 12 KLEIP 18 Materials and Methods 19 Cell culture and transient transfection 19 Plasmid 19 Antibodies and reagents 20 Western blotting 20 In vivo ubiquitination assay 20 In vivo sumoylation assay 21 Detergent extraction assay 21 RT-PCR 22 Lentivirus production 22 Results 24 KLEIP mRNA is ubiquitously expressed in various human cell lines and tissues 24 Endogenous KLEIP is present in the detergent-insoluble fraction in cells 24 The BTB-binding ability of Cul3 is required for Cul3-KLEIP-mediated DAPK ubiquitination 25 Establishment of KLEIP knockdown cell lines 25 KLEIP is required for ubiquitination of DAPK in vivo 26 KLEIP is sumoylated in vivo 27 Lys 278 residue in KLEIP is crucial for SUMO-1 modification of KLEIP 28 The sumoylation of KLEIP is up-regulated under hypoxia conditions 28 KLEIP associates with SUMO1 in vivo 29 IIe34 residue in SUMO1 is important for the non-covalent binding between KLEIP and SUMO1 30 KLEIP interacts with SUMO1 through its conserved SIMs. 30 Discussion 32 Reference 38 Figures 46 Figure 1. KLEIP mRNA expression in different human cell lines. 46 Figure 2. KLEIP mRNA expression of in different human tissues. 47 Figure 3. Endogenous KLEIP is part of the detergent-insoluble fraction in cells. 48 Figure 4. The BTB-binding mutants of Cul3 cannot efficiently bind KLEIP and promote DAPK ubiquitination. 49 Figure 5. Establish the KLEIP knockdown cell lines. 50 Figure 6. KLEIP knockdown attenuates the ubiquitination of DAPK in vivo. 51 Figure 7. KLEIP can be sumoylated in vivo. 52 Figure 8. KLEIP K278R mutant is less sumoylated in vivo. 53 Figure 9. The sumoylation of KLEIP is not affected in KLEIPK49R mutant. 54 Figure 10. Endogenous KLEIP protein level is not changed under hypoxia conditions. 55 Figure 11. KLEIP sumoylation is apparently increased under hypoxia conditions. 56 Figure 12. KLEIP can bind SUMO1 non-covalently. 57 Figure 13. KLEIP interacts with SUMO1 through its conserved SUMO interacting motifs. 58 Appendix 59 Figure S1. KLEIP can be sumoylated in vivo. 59 Figure S2. The non-covalent binding between KLEIP and SUMO1 is largely diminished in SUMOI34E mutant. 601808029 bytesapplication/pdfen-US類泛素化泛素化轉譯後修飾SumoylationUbiquitinationPost-translational modificationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51348/1/ntu-96-R94448004-1.pdf