MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Mixed-lineage kinase domain-like protein (MLKL) is a key mediator of tumor necrosis factor-alpha (TNF-α)-induced necroptosis.
• The precise mechanism by which MLKL triggers necroptosis remains incompletely understood.

Purpose of the Study:

• To elucidate the structural assembly of MLKL during necroptosis.
• To determine the role of MLKL polymerization in the induction of cell death.

Main Methods:

• Formation of SDS-resistant, disulfide bond-dependent MLKL polymers was analyzed during necroptosis.
• MLKL polymer size, proteinase K resistance, and ultrastructure were assessed using biochemical assays and electron microscopy.
• Amyloid-like fiber formation by the MLKL N-terminal domain was investigated using Congo red binding assays.
• The impact of MLKL polymerization mutants on necroptosis induction was evaluated.
• The effect of necrosulfonamide on MLKL polymerization and cell death was examined.

Main Results:

• MLKL forms large ( > 2 million Da), SDS-resistant, disulfide bond-dependent polymers during necroptosis, independent of RIPK1/RIPK3.
• These polymers exhibit fiber-like structures (5 nm diameter) and are resistant to proteinase K digestion.
• The N-terminal domain of MLKL self-assembles into amyloid-like fibers, suggesting a structural basis for polymerization.
• Mutants impaired in polymer formation showed reduced necroptosis induction efficiency.
• Necrosulfonamide treatment conjugated MLKL at Cys86, inhibited polymer formation, and blocked necroptosis.

Conclusions:

• Disulfide bond-dependent, amyloid-like polymerization of MLKL is a critical step in necroptosis.
• MLKL polymers are both necessary and sufficient for inducing necroptosis.
• Targeting MLKL polymerization represents a potential therapeutic strategy for controlling necroptosis.