Poster Presentation 51st Lorne Proteins Conference 2026

De novo cyclic peptides allow visualisation of the monomeric and functional amyloid conformations of the kinase RIPK3 (#411)

Jessica A Buchanan 1 , Olivia Lavidis 1 , Nick Shields 1 , Kat Harrison 2 , Huy T Nguyen 2 , Toby Passioura 3 , Richard Payne 2 , Megan Steain 1 , Margie Sunde 1 3
  1. School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia
  2. School of Chemistry, The University of Sydney, Sydney, NSW, Australia
  3. Sydney Analytical, The University of Sydney, Sydney, NSW, Australia

Necroptosis is an immunogenic form of programmed, lytic cell death, which acts as a secondary mechanism for cell clearance following infection or inflammation. However, dysregulation of necroptosis is implicated in certain inflammatory conditions, including ischaemic injury after stroke and inflammatory bowel disease. Central to necroptosis is a functional amyloid complex designated the “necrosome”, which is comprised largely of the protein RIPK3. Necrosome formation occurs through intermolecular assembly driven by the 18‑residue RIP Homotypic Interaction Motif (RHIM) found towards the C-terminus of RIPK3, which forms the cross‑β amyloid core of this complex. The N‑terminal RIPK3 kinase domain remains available for autophosphorylation upon necrosome formation, subsequently phosphorylating the downstream effector protein, MLKL, leading to lytic cell death.

There is a dearth of information available on the role and the fate of the necrosome within cells. These investigations have been limited by a lack of effective human RIPK3 reporters, with current small molecules displaying poor selectively, and antibodies found unsuitable for immunofluorescence. Cyclic peptides are highly stable and offer large interaction interfaces, which can confer greater selectivity of their target proteins. Subsequently, a mRNA-display-based cyclic peptide screening technique (RaPID) was used to identify several novel cyclic peptides against RIPK3. Peptides were fluorescently labelled, and peptide binding to RIPK3 was assessed in vitro through fluorescence colocalization and microscale thermophoresis. Peptides have been successfully applied in fixed cells following necroptotic cell death, with results suggesting that peptides can bind to both the necrosome amyloid structure and to monomeric RIPK3 dispersed throughout the cytoplasm. Ongoing experimental work has utilised peptides in live cell assays, with preliminary imaging data showing that peptides can permeate live cells without causing toxicity. This offers great potential for live cell imaging with these peptides to monitor RIPK3 movement and necrosome formation during necroptosis.