Title Page
Contents
Chapter Ⅰ. Background 14
1. Autophagy 15
2. Neurodegenerative diseases and protein aggregates 20
3. Stress granules (SGs) 24
Chapter Ⅱ. Studies on autophagic degradation of TDP-25 aggregates using newly developed selective autophagosome probes and deconjugases [Published on Autophagy, 2022] 27
1. Introduction 28
2. Materials and methods 32
1) Molecular cloning 32
2) Cell culture, transfection, confocal microscopy, and drug treatment 35
3) Quantitative analysis of A/C ratios 36
4) Spot number analysis 37
5) Western blot 37
6) Lentivirus production 38
7) Immunocytochemistry 39
3. Results 40
1) Identification of LIR motifs that bind to autophagic membrane-anchored mATG8 in hexa mATG8-knockout HeLa (HKO) cells 40
2) Development of probes that selectively monitor membrane-anchored mATG8 proteins and enzymes that selectively deconjugate mATG8–PE in autophagic membranes 47
3) Membrane-anchored GABARAP subfamily proteins regulate autophagic degradation of TDP-25 aggregates 58
4. Discussion 65
Chapter Ⅲ. Nonmuscle myosin IIB regulates TDP-25 degradation via parkin-mediated mitophagy [Published on Cell death and differentiation, 2020] 69
1. Introduction 70
2. Materials and methods 73
1) Molecular cloning 73
2) Cell culture, transfection and drug treatment 74
3) Mitochondrial membrane potential measurement 75
4) Urea fractionation and mitochondrial fractionation 76
5) Western blot 76
6) Immumocytochemistry 77
7) Electron Microscopic Analysis 78
8) Reverse transcription and quantitative real-time PCR 78
3. Results 80
1) TDP-43 C-terminal fragment (TDP-25) is colocalized to Tom20-positive abnormal and damaged mitochondria 80
2) Toxic TDP-25 recruits Parkin into damaged mitochondria and is degraded by mitophagy 88
3) Myosin IIB, an actin-based motor protein, is localized to TDP-25-positive abnormal mitochondria 93
4) Myosin IIB inhibition impairs mitophagy-mediated degradation of TDP-25 or Tom20 and reduces viability in cells expressing Myc-TDP-25 100
4. Discussion 105
Chapter Ⅳ. NS1-BP regulates stress granule dynamics and autophagy by p62 109
1. Introduction 110
2. Materials and methods 113
1) Molecular cloning 113
2) Generation of NS1-BP KO HeLa cell line using CRISPR/Cas9 system 116
3) Cell culture, transfection, drug treatment 116
4) Western blot 117
5) Immunocytochemistry and confocal microscopy 117
6) Silver staining and LC-MS/MS analysis 118
7) Co-immunoprecipitation 118
8) Proximity ligation assay (PLA) 119
9) In vivo p62 ubiquitination IP 119
10) FRAP analysis 120
3. Results 121
1) NS1-BP selectively binds to GABARAP, GABARAP-L1 or p62 as autophagy components 121
2) NS1-BP is a general stress granule component 125
3) NS1-BP regulates SGs number, size and dynamics 131
4) NS1-BP is associated with autophagy receptor protein, p62 upon oxidative stress 138
5) NS1-BP regulates protein stability of p62 via inhibition of ubiquitination of p62 143
6) Reduced levels of p62 contribute to SGs number and size in NS1-BP KO HeLa cells 150
4. Discussion 153
References 156
Abbreviation 174
Abstract 176
Abstract in Korean 179
Table 1. Primer sequences used for the experiments. 34
Table 2. Quantifications the binding property between each LC3/GABARAP family and LIR motifs using an A/C ratio assay. 44
Table 3. List of oligonucleotides and their sequences for sub-cloning. 74
Table 4. List of primer sequences for quantitative real-time PCR 79
Table 5. List of primers and their sequences for sub-cloning. 114
Figure 1. Autophagy pathway regulated by various ATG proteins 19
Figure 2. Protein aggregates causing of neurodegenerative disease 23
Figure 3. Stress granules formation upon various stress conditions in cells 26
Figure 4. Development of new tool to detect the binding affinities of LIR motifs for membrane-anchored mATG8... 43
Figure 5. Identification of selective mATG8-binding LIR motifs. 46
Figure 6. Schematic model of RavZ-based selective autophagic membrane-anchored mATG8 proteins monitoring probe and deconjugase. 49
Figure 7. gProbe-X selectively targets mATG8-containing autophagosomes. 53
Figure 8. Autophagic membrane-anchored mATG8 deconjugases selectively deconjugate mATG8 proteins. 55
Figure 9. Selective autophagic membrane-anchored mATG8 deconjugases insufficient to increase autophagy receptor protein p62 and NDP52 levels. 57
Figure 10. Cellular localization of TDP-25 aggregates with gProbe-X. 59
Figure 11. Regulation of TDP-25-positive aggregates by selective mATG8 deconjugases. 63
Figure 12. GABARAP subfamily proteins regulate TDP-25-positive aggregates. 64
Figure 13. Myc-TDP-43 is localized to G3BP1-positive stress granules but not Myc-TDP-25. 82
Figure 14. Myc-TDP-25 is localized to mitochondria in mouse cortical neurons. 83
Figure 15. TDP-25 is localized to mitochondrial proteins. 84
Figure 16. Glycine-rich domain on TDP-25 is required for mitochondrial localization. 85
Figure 17. TDP-25 induces damaged mitochondria. 87
Figure 18. Myc-TDP-25 recruits Parkin in mouse cortical neurons. 90
Figure 19. Autophagy proteins GABARAPL1, LC3B, phospho-p62 recruited to Myc-TDP-25 aggregates. 91
Figure 20. Myc-TDP25 is removed by Parkin-mediated mitophagy. 92
Figure 21. Myosin II mRNA expression level in human iPSC-derived neurons. 95
Figure 22. Myosin IIB colocalizes TDP-25 aggregates. 97
Figure 23. TDP-25 recruited to mitochondria by Myosin IIB. 99
Figure 24. Myosin IIB facilitates TDP-25-mediated mitophagy. 102
Figure 25. Myosin II inhibition caused mitochondrial accumulation in neurons expressing Myc-TDP-25. 103
Figure 26. Blebbistatin affects DNA fragmentation in Myc-TDP-25 expressing cells. 104
Figure 27. NS1-BP interacts with GABARAP and GABARAPL1 in a LIR dependent manner. 123
Figure 28. NS1-BP protein interacts with GABARAP and GABARAPL1. 124
Figure 29. NS1-BP interactome analysis. 127
Figure 30. NS1-BP interacts with stress granule components upon oxidative stress conditions. 129
Figure 31. NS1-BP colocalized to TIA-1-positive stress granules. 130
Figure 32. Generation of NS1-BP KO HeLa cells using CRISPR/Cas9 system. 133
Figure 33. NS1-BP regulates stress granules number, size and dynamics. 135
Figure 34. Stress granules number and size were regulated by knockdown of NS1-BP in mouse cortical neurons. 136
Figure 35. Stress granule dynamics was decreased in NS1-BP KO HeLa cells. 137
Figure 36. NS1-BP interacts with p62. 141
Figure 37. NS1-BP dose not interacts with p62 upon thapsigargin-induced ER stress conditions. 142
Figure 38. p62 protein level was significantly decreased in NS1-BP KO HeLa cells and their ubiquitination was regulated by NS1-BP. 146
Figure 39. P62 ubiquitination sites and ubiquitination regulation site by NS1-BP. 149
Figure 40. P62 regulates number and size of stress granules. 152