Amyloids are filamentous polymers of aberrantly folded proteins distinguished by their cross-beta structures. Accumulation of amyloid is associated with approximately 20 human diseases, including Alzheimer's, type-2 diabetes, and rheumatoid arthritis. Amyloids fall into two broad categories: infectious and non-infectious. Infectious amyloids are called prions. We started studying yeast prion structures in 1998, focusing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain, which performs its regulatory function, remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. With this model, it envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin, and growing support for models of this kind is appearing in the scientific literature. Ongoing work is aimed at testing and refining this model; investigating fibril polymorphism; and relating amyloids to native conformations. In FY15 we focused on the following projects. (1) Parkinson disease (PD) is a chronic and progressive neurodegenerative disease affecting motor function. PD is characterized by dopaminergic neuronal cell death and by the presence of Lewy bodies. Amyloid fibrils of alfa-synuclein (aS) are the main component of Lewy bodies, and previous research suggests that its fibrillation is part of the disease pathology. Normally, the 140 aa-long protein has a membrane remodeling function, which we have also researched, as reported previously in project report AR027015-19. aS is alpha-helical when associated with a membrane and a random coil in solution. In fibril formation, the protein polymerizes into a cross-beta structure. Despite their high clinical relevance, structural information on aS-containing amyloid fibrils has been elusive and such information is the goal of this project. Recombinant aS was expressed in E. coli, purified and assembled into fibrils, which were observed by cryo-EM in our laboratory and by dark-field STEM at the Brookhaven STEM facility. The resulting cryo-EM images revealed that aS fibrils are polymorphic (as in previous reports). Our analysis focused on a twisting fibril with an axial repeat length of 77 nm between crossovers. These fibrils have an average diameter of 8.6 nm. Reconstructing their cross-section showed them to consist of two asymmetrically associated protofibrils, with each protofibril subdividing into two protofilaments. Mass-per-length measurements made from the STEM data gave a unimodal distribution with a mean density equivalent to two subunits per 0.47 nm axial rise, i.e. one subunit per protofibril. The STEM images showed two thread-like densities running along each fibril that we interpret as metal ions. Similar threads were observed after doping metal-free fibrils with copper. We find that multiple - but not all - fibril morphotypes have these axially stacked metal coordination sites. These observations support the idea that metal binding promotes fibrillation and hence Lewy body formation in PD. A paper reporting these observations was published earlier this year. (2) Necroptosis-signaling molecules TRIF and HSV-1 R1 form amyloid fibrils. Receptor-interacting protein 1 (RIP1) and homolog receptor-interacting protein 3 (RIP3) are serine-threonine kinases associated with the mediation of cell death and inflammation signaling. RIP1 and RIP3 activate necroptosis by forming amyloid fibrils of their RIP-Homotypic Interaction Motifs (RHIMs), which creates a linear array of RIP kinase domains in high local concentration. During active HSV-1 infection, the HSV-1 ICP6 protein, R1, inhibits necroptosis by interacting with RIP1 thereby blocking RIP3 activation. Pathogen-sensing receptors TLR3 or TLR4 are activated by the binding of dsRNA in the endosome or LPS at the cell surface, respectively. TRIF recruits RIP1 to activate inflammatory cytokines via a MyD88-dependent pathway. TLR3 also signals apoptosis via caspase-8. If caspase-8 is inhibited, then TRIF recruits RIP3 independent of RIP1, triggering necroptosis via a MyD88-independent pathway. TRIF and R1 have RHIMs, so we hypothesized that these motifs formed amyloid fibrils. Using negative stain TEM and Congo Red staining, we found that synthetic peptides of the TRIF and R1 RHIMs form fibrils, and that they are amyloids. Under the conditions used, RIP1 required higher concentration to form amyloid. We have also observed that TRIF can seed RIP3 to form amyloid complexes. Under the same conditions this was not observed for RIP1. The assembly of signal transduction complexes is crucial to cell survival and inflammation-related signaling. RIP1 and RIP3 have been shown to form amyloid complexes that signal necroptosis. We have shown that the RHIM peptides of both TRIF and HSV R1 form amyloid fibril-type complexes similar to those of the RIP kinases. The TRIF RHIM nucleates RIP3 amyloid fibrils, suggesting that formation of a functional amyloid is the mechanism by which the TLR3-associated necroptosis pathway occurs.
