Mechanistic Studies of MLKL Function via Optogenetic Control and Intracellular Trafficking = 광유전학적 제어와 세포 내 수송을 통한 MLKL 기능의 작용기전 연구

목차 (Table of Contents)

• INTRODUCTION 1
• PART Ⅰ. Optogenetic Activation of MLKL as a Standlone Module for Necroptosis 4
• I. INTRODUCTION 5
• II. RESULTS 8
• A. Design and functional characterization of optoRIPK3 8
• B. OptoRIPK3 activates the necroptotic signaling cascade. 11
• C. RHIM-independent optoRIPK3 oligomerization induce necroptosis 16
• D. OptoMLKL activation induces RIPK3-independent necroptosis 20
• E. MLKL hotspot formation is required for light-induced necroptosis 26
• F. Phosphorylation-independent MLKL oligomerization is sufficient to induces necroptosis 31
• G. Optogenetic modulation of MLKL increased immunognenicity through the release of both iDAMPs and cDAMPs 35
• H. Application of optoMLKL in patient-derived pancreatic cancer organoid 40
• III. DISCUSSION 45
• IV. MATERIAL & METHODS 48
• A. Antibodies and reagents 48
• B. Cell culture 48
• C. Plasmid and cloning 49
• D. Plasmid transfection and lentiviral transduction 49
• E. Live-cell imaging and photoactivation 50
• F. Cell cytotoxicity and viability assay 50
• G. Reducing and non-reducing western blot analysis 51
• H. BN-PAGE analysis 51
• I. Quantitative real-time PCR 51
• J. Immunocytochemistry 52
• K. Silver staining 52
• L. Measurement of Extracellular ATP assay 53
• M. Measurement of HMGB1 secretion (CM concentration) 53
• N. THP-1 migration assay 53
• O. Generation of murine bone marrow DCs, and DMs 54
• P. Phagocytosis assay 54
• Q. Organoid culture 54
• R. Light stimulation and ICC of organoids 55
• S. Quantification and statistical analysis 55
• PART II. Active MLKL restricts PD-L1 trafficking via Sec24A condensation 61
• I. INTRODUCTION 62
• II. RESULTS 65
• A. Activated MLKL translocate to the ER 65
• B. MLKL activation promotes association with COPII cargo adaptor Sec24A 74
• C. Activated MLKL associates with Sec24A in translocation-dependent manner 77
• D. MLKL activation induces Sec24A puncta formation in the perinuclear region 83
• E. Activated MLKL engages Sec24A via an exposed cargo-recognition motif 91
• F. Liquid like phage separation of Sec24A by active MLKL 95
• G. MLKL-induced Sec24A condensation impairs COPII vesicle formation 102
• H. Necroptosis signaling induces redistribution of PD-L1 104
• I. MLKL activation induces ER retention of PD-L1 and impairs its surface trafficking 110
• J. ER-retained PD-L1 fails to undergo sequential N-glycosylation along the ER-Golgi pathway 117
• K. MLKL-induced PD-L1 retention occurs independently of canonical ER stress signaling 126
• L. Active MLKL impairs PD-L1 surface trafficking and PD-1 engagement 129
• M. Necroptotic competence enhances anti-tumor immunity and ICB responsiveness 133
• III. DISCUSSION 139
• IV. MATERIAL & METHODS 143
• A. Antibodies and reagents 143
• B. RNA-seq data analysis of public ICB cohort 144
• C. Cell culture 145
• D. Treatment conditions 145
• E. Plasmid construction and transfection 146
• F. Lentivirus production and in vitro viral transduction 147
• G. Immunofluorescence and microscopy 147
• H. Proximity Ligation Assay (PLA) 148
• I. Live cell imaging 148
• J. Cell death assay 148
• K. In vitro COPII vesicle reconstitution assay. 149
• L. RUSH assay 150
• M. Western blot analysis 150
• N. Glycosidase digestion 151
• O. Immunoprecipitation and lectin blotting 151
• P. Flow cytometry 151
• Q. Quantitative real-time PCR 152
• R. BioID proximity labeling and mass spectrometry 152
• S. Functional annotation and enrichment analysis 152
• T. Image quantification and statistical analysis 153
• REFERENCE 155