Poster Presentation 50th Lorne Proteins Conference 2025

When Necroptosis meets Protein Liquid-liquid Phase Separation (#109)

Tianchen Li 1 , Brayden Williams 1 , Margie Sunde 1 2 , Yi Shen 1 2
  1. The University of Sydney, Darlington, NSW, Australia
  2. Sydney Nano, The University of Sydney Nano Institute, Sydney, NSW, Australia

Necroptosis is a programmable cell death mechanism that plays an important role in removing infectious cells and preventing cell proliferation. In TNF or viral-induced necroptosis, the assembly of RIPK3 into amyloid fibrils, with its subsequent phosphorylation, is critical to trigger downstream cell death mechanisms. 1 Multiple upstream proteins, such as TRIF 2, RIPK1 3, and ZBP1 4, have been identified to co-assemble with RIPK3 through RHIM domains to concentrate RIPK3 molecules, regulate the kinetics, and manipulate the necroptosis. However, the initial stage of RIPK3 fibrillation remains unclear. It is of particular interest how these functional proteins can meet in the intracellular environment in the presence of numerous other biomolecules.

In recent decades, protein condensates have been identified as membrane-less organelles responsible for partitioning intracellular components and organizing biological reactions. 5 They can serve as reaction crucibles to recruit aggregation-prone protein molecules and either suppress or facilitate their self-assembly into amyloids. 6 However, as metastable liquid droplets, protein condensates can also solidify into fibrillar structures. Such aberrant protein phase transitions are closely related to neurodegenerative diseases. 7,8 Moreover, there is an increasing interest in the connection between necroptosis and protein liquid-liquid phase separation. 9,10

Here, we show that the RHIM domain of RIPK3 from murine and humans can form liquid-like condensates in the presence of a molecular crowder in vitro. These condensates can further solidify into gel or fibrillar structures in the presence of external stimuli. We also found that during the co-assembly of ZBP1 and RIPK3, the RHIM domains of ZBP1 and RIPK3 can form a double-layer pearl necklace-like structure. We speculate that this is initiated by protein liquid-liquid phase separation followed by rapid liquid-to-solid transitions. These results imply that the formation of liquid-like condensates can be a potential early-stage mechanism to manipulate necroptosis-mediated immunity.

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