Eligible Student Poster 49th Lorne Conference on Protein Structure and Function 2024

Assessing the viability of RAPIDly discovered cyclic peptides as specific binders to the RIPK3 RHIM functional amyloid (#119)

Jessica A Buchanan 1 , Chi L Pham 1 , Toby Passioura 2 , Huy T Nguyen 3 , Kat Harrison 3 , Cheree Fitzgibbon 4 , Olivia Lavidis 1 , Richard Payne 3 , James Murphy 4 , Meghan Steain 1 , Margie Sunde 1 2
  1. School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia
  2. Sydney Analytical, The University of Sydney, Sydney, NSW, Australia
  3. School of Chemistry, The University of Sydney, Sydney, NSW, Australia
  4. Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia

Organisms use multiple forms of programmed cell death to protect against invading pathogens. Necroptosis is a highly immunogenic form of lytic cell death. Dysregulation of necroptosis has been implicated in multiple disease states, including inflammatory bowel disease and ischaemic injury after stroke. Central to necroptosis is a functional amyloid complex referred to as the necrosome, which is comprised largely of the protein RIPK3. The necrosome forms when the ~18‑residue RIP Homotypic Interaction Motif (RHIM) within RIPK3 adopts an amyloid cross-β structure and recruits additional RIPK3 monomers into a fibrillar form, whilst the N‑terminal kinase domain of RIPK3 remains exposed and available for phosphorylation. Functional amyloid formation by RIPK3 results in autophosphorylation of this kinase and subsequent phosphorylation of downstream effector protein, MLKL, leading to lytic cell death.

There are currently no small molecules available that selectively bind to the amyloidogenic RHIM region of RIPK3. Here, we have aimed to identify cyclic peptides that bind with high specificity to the RIPK3 RHIM region or to the RIPK3 kinase domain. The two RIPK3 regions were used as targets for screening against a cyclic peptide library in a selection known as random non-standard peptide integrated discovery (RaPID). Novel cyclic peptide binders to the RIPK3 RHIM amyloid fibril interface have the potential to be used as diagnostic tools to detect RIPK3 functional amyloid formation. Peptides that bind with high specificity to the RIPK3 kinase domain may allow future investigations of the distinct actions and interfaces of these two regions of the protein. Further characterisation has revealed that RIPK3 RHIM‑binding peptides independently form assemblies that display amyloid‑like properties. Investigation of peptide:RIPK3 RHIM binding and peptide:RIPK3 kinase domain binding is underway using multiple techniques, including fluorescent colocalization studies, transmission electron microscopy and immunofluorescence assays with cells that have undergone necroptosis.