2026-09-23T10:11:14Zhttps://repository.ajou.ac.kr/oai/request

oai:repository.ajou.ac.kr:201003/130262024-10-11T02:21:25Zcom_201003_14372com_201003_14368col_201003_14374

바이러스 감염시 미토콘드리아 E3 ubiquitin ligase 인 MARCH5의 새로운 생체 기능 유, 영석 조, 혜성 대학원 의생명과학과 107397 유, 영석 MARCH5 Mitochondria have a central role in preserving cellular homeostasis. Ubiquitin proteasome system (UPS) on the mitochondria is one of quality control systems that works as a first line barrier against aggregated or misfolded proteins. In this study, I investigated functional role of mitochondrial E3 ligase, MARCH5, against protein aggregated at the mitochondria membranes. First, the mitochondrial antiviral signaling (MAVS) protein forms aggregates that elicit robust type-I interferon induction on viral infection, but persistent MAVS signaling leads to host immunopathology; it remains unknown how these signaling aggregates are resolved. Here I identify the mitochondria resident E3 ligase, MARCH5, as a negative regulator of MAVS aggregates. March5+/- mice and MARCH5-deficient immune cells exhibited low viral replication and elevated type-I interferon responses to RNA viruses. MARCH5 bound MAVS only during viral stimulation when MAVS forms aggregates, and these interactions required the RING domain of MARCH5 and the CARD of MAVS. MARCH5, but not its RING mutant of MARCH5H43W, reduced the level of MAVS aggregates. MARCH5 transferred ubiquitin to Lys7 and Lys500 of MAVS and promoted its proteasome-mediated degradation. Second, the hepatitis B virus X (HBx) is non-structural protein encoded by hepatitis B virus (HBV) genome. Highly expressed HBx accumulates at the mitochondria and induces mitochondrial dysfunction that contributes to the pathogenesis of liver cancer. I found that MARCH5 was co-immunoprecipitated with HBx, which was subsequently ubiquitinated through interaction with N-terminal RING domain of MARCH5, whereas MARCH5H43W, which has no ligase activity failed to ubiquitinate HBx. In vitro and in vivo ubiquitination assays revealed that MARCH5 facilitated polyubiquitin chain formation at HBx protein and its proteasome-dependent degradation. Taken together, MARCH5 is a crucial mitochondria-resident regulator that plays a protective role against accumulation of aggregated MAVS and HBx. I. INTRODUCTION 1 II. MATERIALS AND METHODS 3 A. Mice and in vivo viral infection 3 B. Cell culture and transfections 3 C. Plasmid construction 3 D. RNA interference 4 E. Luciferase reporter assays 4 F. Virus replication assay 4 G. Type-I interferon and pro-inflammatory cytokines bioassay 5 H. In vivo and in vitro ubiquitination assay 5 I. Immunoblot analysis and immuneprecipitation assay 5 J. Cycloheximide chase assay 6 K. Immunofluorescence 6 L. Quantitative real-time PCR and RT-PCR 6 III. RESULTS A. March5+/- mice exhibited low viral replication and reduced morbidity in response to RNA viral infection 9 B. MARCH5-deficient immune cells produce elevated levels of type-I interferon and proinflammatory cytokines upon infection with RNA viruses 13 C. The E3 ligase activity of MARCH5 is necessary for inhibition of RLR-mediated antiviral signaling 17 D. MARCH5 suppresses the MAVS-mediated type-I IFN signaling 20 E. MARCH5 binding to MAVS is necessary for the inhibition of type-I IFN responses 22 F. MARCH5 reduced the MAVS aggregates via the proteasome-mediated degradation pathway 24 G. MARCH5 transfers ubiquitin to Lys7 and Lys500 residues of MAVS and accelerates MAVS degradation 28 IV. DISCUSSION 32 V. CONCLUSION 35 PART2. I. INTRODUCTION 37 II. MATERIALS AND METHODS 40 A. Cell culture and transfection 40 B. siRNA and plasmids 40 C. Immunoprecipitation and immunoblotting 40 D. Antibodies 41 E. Immunocytochemistry and confocal microscopy 41 F. Analysis of intracellular ROS levels by flow cytometry 42 G. In vivo and in vitro ubiquitination assay 42 III. RESULTS A. MARCH5 facilitates the degradation of mitochondrial HBx 44 B. MARCH5 interacts and polyubiquitinates mitochondrial HBx 49 C. MARCH5 attenuates Cycloocygenase-2 induction by HBx 52 IV. DISCUSSION 55 V. CONCLUSION 58 VI. REFERENCES 59 VII. 국문요약 62 Doctor 2016-11-27T23:48:13Z 2016-11-27T23:48:13Z 2016 2016 Thesis Theses http://repository.ajou.ac.kr/handle/201003/13026 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000021837 000000021837 en