Necroptosis, a lytic form of programmed cell death, is responsible for the regulation of viral infections and occurs in response to cell or tissue damage. Dysregulation of necroptosis is also associated with several disease states, including inflammatory bowel disease and cancer. The formation of functional amyloid fibrils, containing Receptor Interacting Protein Kinase 3 (RIPK3) and other adapter proteins, is crucial to necroptosis. These fibrils are formed through interactions mediated by the RIP Homotypic Interaction Motifs (RHIMs) found within key proteins associated with necroptosis. Herpesviruses can manipulate the pathway through the expression of RHIM‑containing proteins that form hybrid, competing functional amyloid fibrils with the host RHIM‑containing proteins. Within the cell, multiple competing RHIM interactions are possible and the rate of assembly, or the stability of the resulting fibrils, may control the cellular outcome.
To determine the hierarchy of RHIM interactions, the rate at which different host and viral RHIM-containing proteins form amyloid assemblies, and the stability of different host and viral homomeric and heteromeric amyloid assemblies, have been studied. Kinetic assays have been performed with Thioflavin T, a small molecule that exhibits a characteristic increase in fluorescence when bound to amyloid fibrils. The stability of the homomeric and heteromeric RHIM amyloid assemblies have been probed using semi-denaturing agarose gel electrophoresis, revealing the degree of depolymerisation of the amyloid structures as a function of sodium dodecyl sulfate detergent concentration. Understanding the intricacies of the initiation and inhibition of the necroptosis pathways could allow the future manipulation of necroptosis for therapeutic purposes.