Application of cyclic peptides as novel probes for amyloid-forming proteins

Abstract

Amyloid fibrils are protein polymers that are distinguished by their unique, cross β structure, which is formed irrespective of the component protein sequence. Amyloid fibrils have traditionally been associated with disease, for example, the microtubule associated protein, tau, which is implicated in multiple neurodegenerative conditions including Alzheimer’s disease and chronic traumatic encephalopathy (CTE), collectively termed tauopathies.

In contrast to pathogenic amyloid, some amyloid forming proteins have a functional role, where fibrillar structures are required to maintain and regulate some biological process within organisms. Necroptosis is a form of programmed, lytic cell death that requires the inhibition of caspase 8 and involves the formation of an amyloid signaling platform known as the necrosome. This necrosome structure is a multi protein complex that is largely comprised of Receptor Interacting Protein Kinase 3 (RIPK3). The RIPK3 C terminal region contains an ~18 residue sequence which drives amyloid formation called the RIP Homotypic Interaction Motif (RHIM). At the N terminal, RIPK3 contains a kinase domain for RIPK3 autophosphorylation and for phosphorylation of downstream effector protein Mixed Lineage Kinase domain Like protein (MLKL), which subsequently lyses the cell.

A significant amount of research has been directed towards developing probes and inhibitors for both functional and pathological amyloid, often through the use of small molecule screening or antibody development. The potential of cyclic peptides as reporters or modulators of amyloidogenic activity has not yet been fully explored. The work in this thesis presents efforts towards the identifying and characterising three series of cyclic peptides against both the kinase domain and the amyloid forming region of RIPK3 for use in studies of necroptosis. This thesis also presents work on screening cyclic peptides against tau amyloid to act as markers or inhibitors of tau in CTE.