Title Page
ABSTRACT
국문 초록
PREFACE
Contents
ABBREVIATIONS 16
PRELIMINARY BACKGROUND 19
1. Ubiquitination 19
2. Linear ubiquitin chain assembly complex (LUBAC) 22
3. LUBAC-associated deubiquitinases 25
4. Canonical NF-κB pathway 28
5. TNFR1 signaling complex (TNF-RSC) 31
AIM AND OBJECTIVES 34
MAIN STUDY : TSG101 Physically Interacts with Linear Ubiquitin Chain Assembly Complex (LUBAC) and Upregulates the TNFα-Induced NF-κB Activation 35
1. Abstract 36
2. Introduction 37
3. Material and Methods 39
3.1. Yeast two-hybrid screening 39
3.2. Cell culture and DNA transfection 39
3.3. Plasmids 39
3.4. Antibodies and reagents 40
3.5. Co-immunoprecipitation and immunoblot analysis 40
3.6. Ubiquitination assay 40
3.7. RNA interference 41
3.8. Pull-down analysis of endogenous linear ubiquitin conjugates 41
3.9. Analysis of TNF-RSC 42
3.10. Reverse transcription followed by quantitative PCR (RT-qPCR) 42
3.11. ELISA 42
3.12. Statistical analysis 43
4. Results 44
4.1. TSG101 physically associates with LUBAC 44
4.2. TSG101 promotes the E3 ligase activity of LUBAC 51
4.3. TSG101 enhances the E3 activity of LUBAC within TNF-RSC 60
4.4. Interactions of HOIP with HOIL-1L, SHARPIN, or DUBs are not altered by TSG101 63
4.5. TSG101 positively regulates the TNFα-induced stimulation of the NF-κB signaling pathway 66
5. Discussion 71
CONCLUSION 73
REFERENCES 76
PRELIMINARY BACKGROUND 21
Figure 1. Enzymatic cascades of ubiquitination and structure of ubiquitin. 21
Figure 2. Components and catalytic mechanism of LUBAC. 24
Figure 3. Regulation of linear ubiquitination by LUBAC-DUB complexes. 27
Figure 4. Canonical NF-κB pathway. 30
Figure 5. Formation and signaling cascades of TNF-RSC. 33
MAIN STUDY 47
Figure 6. TSG101 interacts with HOIP. 47
Figure 7. TSG101 interacts with HOIL-1L, but not with SHARPIN. 50
Figure 8. TSG101 enhances the E3 ligase activity of LUBAC. 55
Figure 9. Depletion of TSG101 by siRNA mitigates the TNFα-induced E3 ligase activity of LUBAC. 57
Figure 10. Knockdown of TSG101 by siRNA does not further decrease the levels of the linear ubiquitination by the siRNA-mediated depletion of HOIL-1L. 59
Figure 11. Effect of siRNA-mediated TSG101 depletion on the E3 activity of LUBAC in TNF-RSC. 62
Figure 12. TSG101 does not affect the interaction of HOIP with HOIL-1L, SHARPIN, or DUBs. 65
Figure 13. TSG101 promotes TNFα-induced NF-κB activation. 68
Figure 14. TSG101 deficiency impairs TNFα-induced gene induction. 70
Figure 15. Proposed mechanism regulating LUBAC-mediated NF-κB activation by TSG101. 75