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Mechanistic Studies of MLKL Function via Optogenetic Control and Intracellular Trafficking

광유전학적 제어와 세포 내 수송을 통한 MLKL 기능의 작용기전 연구

  • 정다혜 (아주대학교 일반대학원)

초록/요약

Mixed lineage kinase domain-like protein （MLKL） functions as the terminal effector of necroptosis, an immunogenic form of cell death implicated in antitumor immunity. However, precise activation of MLKL remains challenging due to the complexity of upstream signaling networks and the limited understanding of tumor cell–intrinsic regulatory mechanisms. To address these challenges, I developed an optogenetically activatable necroptosis system that enables precise spatiotemporal control of MLKL activation independent of upstream regulatory pathways. This system, exemplified by optoMLKL, forms higher-order assemblies within membrane compartments without requiring RIPK3-mediated phosphorylation. Activation of optoMLKL promotes the release of immunogenic danger signals, including intracellular and classical damage- associated molecular patterns （iDAMPs and cDAMPs）, thereby enhancing innate immune responses. Furthermore, activation of optoMLKL in patient-derived pancreatic cancer organoids induces antitumor effects, supporting its potential as a controllable platform for immunogenic cell death. In addition to its utility as an experimental tool, I identify a previously unrecognized role of MLKL in the regulation of intracellular trafficking during necroptosis. Upon activation, MLKL translocates to the endoplasmic reticulum （ER） and induces condensation of Sec24A, a cargo- selective component of the coat protein complex II （COPII） vesicle machinery. This interaction is mediated through recognition of a cargo motif within the MLKL brace region, promoting liquid-like phase separation of Sec24A. As a consequence, COPII vesicle assembly is disrupted, leading to selective impairment of ER-to-Golgi trafficking. Notably, this process inhibits the maturation and surface presentation of programmed death-ligand 1 （PD-L1） by preventing its glycosylation and delivery to the plasma membrane. Reduced PD-L1 availability at the cell surface limits PD-1 engagement and attenuates adaptive immune checkpoint signaling. Collectively, this work establishes a dual framework for MLKL function in necroptosis, highlighting its role as both a programmable effector that can be precisely controlled using optogenetic approaches and a regulator of vesicular trafficking that modulates immune checkpoint regulation. These findings provide mechanistic insights into necroptosis-driven immune regulation and suggest new s trategies for enhancing antitumor immunity through controlled induction of immunogenic cell death. Keywords: Necroptosis, MLKL, Optogenetics, Sec24A, PD-L1

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목차

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

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