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Functional characterization of ubiquitome regulating genomic stability and proteostasis
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 강호철 | - |
| dc.contributor.author | 김소연 | - |
| dc.date.accessioned | 2022-11-29T02:32:46Z | - |
| dc.date.available | 2022-11-29T02:32:46Z | - |
| dc.date.issued | 2021-08 | - |
| dc.identifier.other | 31125 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/20350 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2021. 8 | - |
| dc.description.tableofcontents | I. INTRODUCTION 1 A. DNA damage 1 B. DNA damage response 2 C. DNA repair pathways 3 D. Post-translational modifications for DNA repair signaling 6 E. Ubiquitination for DNA repair signaling 8 F. DDR-coupled Liquid demixing 9 G. Protein quality control by ubiquitin and ubiquitin-like modifiers 11 H. Ubiquitin-like modifiers, NEDD8 14 I. The aims of this study 17 II. MATERIALS AND METHODS 18 1. E3 ubiquitin ligase library cloning 18 2. Plasmids 18 3. Cell lines and cell culture 28 4. Generation of RNF114 knockout (KO) cell lines 29 5. FokI assay 29 6. Immunofluorescence microscopy 30 7. G2/M checkpoint analysis 31 8. Clonogenic cell survival assay 31 9. NHEJ and HR repair assay 32 10. Fixed or live cell imaging with laser micro-irradiation 32 11. Purification of recombinant proteins from Sf9 insect cells 33 12. Protein microarray 34 13. Analysis of binding affinity (KD) between NEDD8 and HDAC6 35 14. Dot-blot assay 36 15. In vitro and in vivo ubiquitination assay 37 16. Mass spectrometric analysis for identification of UnK11Ubs 38 17. Immunoprecipitation assay 38 18. GST pull-down assay 38 19. Preparation of whole cell lysates and cell extracts 39 20. In vitro deNEDDylation assay 40 21. Enrichment of endogenous unanchored ubiquitin chains for SILAC 41 22. Metabolic labeling for SILAC 41 23. In-gel digestion 42 24. LC-MS/MS analysis for SILAC 43 25. In vitro DOT1L activity assay 44 26. Quantitative real-time PCR 44 27. Chromatin fractionation and chromatin immunoprecipitation assay 47 28. Quantification of images 47 29. Statistical analysis 47 III. RESULTS 49 Part A. Unanchored Lys11 ubiquitin chains drive poly(ADP-ribose)-coupled DNA damage signaling. 49 1. Identification of novel DDR-related E3 ubiquitin ligases through systematic HT screening. 49 2. Functional dissection of novel E3s in DDR. 53 3. Novel E3s are translocated to mIR-induced DNA lesions in a PAR-dependent manner. 56 4. DIZELs are crucial factors for the maintenance of genomic integrity. 59 5. DIZELs synthesize unanchored Lys11-linked ubiquitin chains. 62 6. DIZELs as a PAR-binding proteins accelerate UnK11Ubs generation in a PAR-dependent manner. 66 7. Zf-Di19, a new PAR sensor, is an essential domain for UnK11Ubs production and DIZELs translocation to DNA lesions. 69 8. A cysteine residue on C2H2(II) is required for PAR interaction and recruitment of DIZELs at DNA lesions, whereas a tyrosine residue on C2H2(I) accelerates UnK11Ubs accumulation. 73 9. RNF114 and RNF166 generate UnK11Ubs in vivo through tyrosine residue on its C2H2(I) domain. 77 10. UnK11Ubs generation by RNF114 and RNF166 drive PAR-mediated liquid demixing at damaged chromatin. 84 11. UnK11Ubs regulate H3K79 methylation by recruiting DOT1L to facilitate 53BP1 foci formation at damaged chromatin. 91 12. UnK11Ubs generation by RNF114 and RNF166 are required for 53BP1 foci formation at damaged chromatin and cell survival. 95 Part B. Stress-induced NEDDylation promotes cytosolic protein aggregation through HDAC6 in a p62-dependent manner. 101 1. Identification of HDAC6 as a NEDD8 binding protein. 101 2. NEDD8 binds to HDAC6 via a C-terminal di-glycine (di-Gly) motif. 105 3. HDAC6 colocalizes with ALBs containing cytosolic NEDDylated aggregates. 108 4. NEDD8 directly controls cytosolic ALBs formation during ubiquitin stress. 112 5. UBA1-dependent NEDDylation, not the canonical NEDDylation, is crucial for ALBs formation. 116 6. Cytosolic ALBs containing stress-induced NEDDylation are regulated by NEDD8-specific protease NEDP1. 119 7. Stress-induced ALBs formation is mainly achieved by NEDDylation rather than ubiquitination. 122 8. HDAC6 regulates ALB formation through its deacetylase activity during ubiquitin stress. 126 9. Ubiquitin stress-induced ALBs are linked to aggresome-autophagy machinery. 130 10. p62 functions as an adaptor for NEDD8 that regulates stress-induced cytosolic ALBs linked to aggresome- autophagic flux. 134 IV. DISCUSSION 139 REPERENCES 148 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Functional characterization of ubiquitome regulating genomic stability and proteostasis | - |
| dc.title.alternative | 유전체 안정성 및 단백질 항상성 유지를 위한 유비퀴톰의 역할 규명 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2021. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 1227087 | - |
| dc.identifier.uci | I804:41038-000000031125 | - |
| dc.identifier.url | https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000031125 | - |
| dc.subject.keyword | Aggresome-like bodies | - |
| dc.subject.keyword | Ataxia telangiectasia mutated kinase | - |
| dc.subject.keyword | Atypical NEDDylation | - |
| dc.subject.keyword | DNA damage response | - |
| dc.subject.keyword | E3 ubiquitin ligase | - |
| dc.subject.keyword | Poly(ADP-ribose) polymerase 1 | - |
| dc.subject.keyword | Protein aggregates | - |
| dc.subject.keyword | Protein quality control system | - |
| dc.subject.keyword | Ubiquitin stress | - |
| dc.subject.keyword | Unanchored ubiquitin chains | - |
| dc.description.alternativeAbstract | Covalent attachment of ubiquitin and ubiquitin-like modifiers (UBLs), which are share the β-grasp fold, to substrates is the key post-translational modifications (PTMs) that determine the fate, function, and turnover of most cellular proteins. Thus, ubiquitin- and UBLs-linked PTMs in the intracellular context precisely control various cellular processes such as DNA damage response, proteasomal proteolytic pathway and autophagy. Hence, stress-induced aberrations of ubiquitin and UBLs system cause multiple human diseases such as cancers, neurological disorders and aging disorders. In the physiological or pathophysiological context, however, it is unclear that how ubiquitin and UBLs differentially regulate versatile cellular process with high specificity through homologous and sometimes parallel modification pathways. In this study, to address this point, I selected ubiquitin and NEDD8 out of UBLs and analyzed whether and how they control DNA damage response and proteasomal proteolytic pathway. In the first study, I systematically screened 211 human E3 ubiquitin ligases (hE3s) related to DDR and identified 11 PARP1-dependent hE3s. Among them, drought-induced 19 (Di19) zinc-finger E3 ubiquitin ligases (DIZELs) translocate to DNA lesions in a PAR-dependent manner to generate unanchored K11 ubiquitin chains (UnK11Ubs). Notably, the conserved C2H2(I) domain of DIZELs RNF114 and RNF166 generates UnK11Ubs and interaction with PAR through C2H2(II) domain accelerates accumulation of UnK11Ubs. I also found that UnK11Ubs directly control PAR-seeded liquid demixing and enhance DOT1L-mediated H3K79 di-methylation on damaged chromatin, thereby facilitating recruitment of 53BP1 for DNA repair process. These findings demonstrate that DIZELs as a PAR-binding hE3s generate UnK11Ubs that control the PARP1-coupled DDR in an ATM-independent manner. In addition, accumulated evidence showed that crosstalk between NEDD8 and ubiquitin is important for the prompt elimination of misfolded proteins, which tends to form aggregates, through ubiquitin-proteasome system (UPS) or autophagy. However, it remains unclear how NEDD8 drives UPS or autophagy-mediated clearance of protein aggregates in responses to diverse stress conditions. Thus, in the second study, I screened NEDD8 binding proteins using a protein microarray system and identified HDAC6 as a direct NEDD8-binding protein. By systematic approach, I found that ubiquitin stress leads to accumulation of cytosolic NEDDylated protein aggregates, which form aggresome-like bodies (ALBs) in the perinuclear region. Intriguingly, HDAC6 colocalizes with stress-induced ALBs, and inhibition of HDAC6 activity suppresses ALBs formation, but not stress-induced NEDDylation, suggesting that HDAC6 carries NEDDylated proteins to generate ALBs. Then, I monitored the ALBs-associated proteostasis network and found that p62 directly controls ALBs formation as an acceptor of cytosolic NEDDylated aggregates. Interestingly, I also observed that ALBs are highly condensed in chloroquine-treated cells with impaired autophagic flux, indicating that ALBs rely on autophagy. Taken together, these data propose that NEDD8, HDAC6, and p62 are involved in the management of proteotoxic stress by forming cytosolic ALBs-coupled to the aggresome-autophagy flux . Collectively, all results in this study indicate functional roles of ubiquitin regulating DNA damage response to maintain genomic stability and of NEDD8 controlling proteostasis to protect cells from cellular stress. | - |
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