Necroptosis is an immunogenic form of programmed, lytic cell death. Dysregulation of necroptosis is implicated in certain inflammatory conditions, including ischaemic injury after stroke. 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-terminal of RIPK3, which forms the amyloid cross‑β core of these functional amyloid fibrils. The N‑terminal kinase domain of RIPK3 remains available for autophosphorylation on the surface of the fibrils and subsequently phosphorylates the downstream effector protein, MLKL, leading to lytic cell death.
There is currently limited information available about the formation or location of the necrosome during necroptosis. No small molecules that are selective for the RIPK3 amyloid structure have been identified, and no antibodies suitable for immunofluorescence have been developed. This limits the investigation of the role and fate of the necrosome in cells. Cyclic peptides display relatively large interaction interfaces with their targets and may confer desirable selectivity towards large structures such as amyloid fibrils. The C-terminal region of RIPK3, containing the RHIM, was targeted using an mRNA-display cyclic peptide screening system termed RaPID. This identified several novel cyclic peptides that bind to RIPK3. The binding of these peptides to the RIPK3 RHIM amyloid has been characterised in vitro, and the peptides have now been successfully applied in immunofluorescence assays of programmed cell death. Analysis of these assays has shown that fluorescently labelled peptides demonstrate specific detection of the RIPK3 RHIM‑based necrosome amyloid structure within cells following induction of necroptosis.