Biophysical investigation of the amyloidogenic potential of human and viral necroptosis-associated proteins

Abstract

Functional amyloids form a growing list of proteins that can adopt a fibrillar amyloid structure for functional purposes. In cell death signalling via necroptosis, the RIP kinases 1 (RIPK1) and 3 (RIPK3) interact to form hybrid amyloid assemblies via a protein-protein interaction motif, termed RIP-homotypic interaction motif (RHIM). RHIM sequences are ~18 residue long and contain a highly conserved tetrad sequence (V/I)-Q-(V/I/L/C)-G. The cytosolic nucleic acid sensor Z-DNA binding protein 1 (ZBP1) can also drive necroptosis via interaction with RIPK3, upon sensing of viral nucleic acid. Unlike other RHIM-containing proteins, ZBP1 contains three putative RHIM sequences. Although the first RHIM, termed RHIM-A, has been suggested to be most important for RHIM mediated interactions, the role of the multiple ZBP1 RHIMs has not been probed extensively. I have introduced tetra-alanine mutations into the tetrad sequences in the multiple RHIMs of ZBP1 in order to identify their role in functional amyloid assembly. Biophysical characterisation of the recombinantly produced mutant ZBP1 proteins reveals that mutation of the tetrad sequence of the ZBP1 RHIMs does not completely abrogate amyloid formation, although differences in the extent of amyloid formation are seen upon binding to the amyloid-specific dye Thioflavin T. Importantly, mutation of RHIM-A but not RHIM-B nor C dramatically alters the morphology of ZBP1 amyloid as visualised by transmission electron microscopy. RHIM-A mutants also form smaller amyloid assemblies than the wild type protein and other mutants and these can be easily depolymerised by detergent. Surprisingly, the RHIM-B and C mutant constructs display an increased propensity to form large assemblies that are resistant to depolymerisation, even more-so than the wild type protein. Altogether amyloid formation by ZBP1 RHIM in vitro echoes its cellular function where RHIM-A is vital for necroptosis-related protein-protein interactions, while RHIM-B tempers the activity of RHIM-A. I have also utilised the multiple ZBP1 RHIM mutant proteins to investigate a series of putative new amyloid reporter dyes and have outlined potential binders to the ZBP1 amyloid scaffold. In the second part of this thesis, I have investigated the viral RHIM containing protein ICP6, encoded by herpes simplex virus 1 (HSV-1). Necroptosis is important for antiviral host defence but some viruses, including HSV 1, have evolved adaptations against this form of cell death. Investigation of recombinantly produced ICP6 RHIM reveals that it can form amyloid structures in vitro. This aligns with other work from the Sunde laboratory, which has shown that murine cytomegalovirus (MCMV) and varicella-zoster virus (VZV) encode the RHIM containing proteins M45 and ORF-20, respectively, which form amyloid structures in vitro and interact with host-RHIM containing proteins RIPK3, and ZBP1. Importantly, ICP6 RHIM amyloid is sensitive to a reducing milieu, a property not associated with other proteins of the RHIM family. Unlike M45 and ORF20, ICP6 does not form large homomeric amyloid assemblies, rather preferentially forms heteromeric assemblies with host RHIM proteins. Moreover, tetra alanine mutation of the ICP6 RHIM tetrad inhibits amyloid formation and disrupts its interaction with host-RHIM proteins. Characterisation of ICP6 RHIM revealed that an intra-molecular disulfide bond between Cys75 and Cys79 confers distinct amyloid characteristics not seen with previously characterised viral proteins M45 and ORF20. Formation of virus-host mixed amyloid may prevent host RHIM proteins from interacting and activating the downstream necroptosis cascade.