Partial prion cross-seeding between fungal and mammalian amyloid signaling motifs

Abstract

In fungi, NLR-based signalosomes activate downstream membrane-targeting cell-death inducing proteins by a mechanism of amyloid templating. In the species Podospora anserina, two such signalosomes, NWD2/HET-S and FNT1/HELLF have been described as well as an analogous system, involving a distinct amyloid signaling motif termed PP in the species Chaetomium globosum. The PP-motif bears resemblance to the RHIM and RHIM-like motifs controlling necroptosis in mammals and innate immunity in Drosophila. We identified here, a third NLR signalosome in Podospora anserina comprising a PP-motif and organized as a two-gene cluster encoding a NLR and a HELL-domain cell-death execution protein termed HELLP. We show that the PP-motif region of HELLP forms a prions we term [π] and that [π] prions trigger the cell-death inducing activity of full length HELLP. We detect no prion cross-seeding between HET-S, HELLF and HELLP amyloid motifs. In addition, we find that akin to PP-motifs, RHIM motifs from human RIP1 and RIP3 kinases are able to form prions in Podospora, and that [π] and [Rhim] prions cross-seed. Our study shows Podospora display three independent amyloid signalosomes. Based on the described functional similarity between RHIM and PP it appears likely that these amyloid motifs constitute evolutionary related cell-death signaling modules.Author summaryAmyloids are fibrillar protein polymers assembled from cross β-sheets. Pathogenic amyloids are responsible for a range of neuronal and systemic protein misfolding diseases. In addition, functional amyloids fulfill a variety of biological functions in microbes and animals. In fungi, amyloids control prion-like signaling pathways with roles in host defense and immune programmed cell death. These signaling pathways involve a Nod-like receptor and a cell-death execution protein activated through amyloid templating. In P. anserina two such amyloid signaling systems were identified so far. Here we identified and characterized a third amyloid signaling system comprising a different amyloid motif termed PP. We showed a total absence of cross-induction between the three P. anserina systems allowing for the co-existence of three independent amyloid signaling cascades in the same organism. An evolutionary relationship between fungal amyloid motifs and mammalian RHIM amyloid motif involved in the necroptosis pathway was previously suggested. We validated this assumption by showing that the human RHIM motif could not only propagate as a prion in P. anserina but also functionally cross-interact with the fungal PP-motif prions. These results point to a long term evolutionary conservation of amyloid signaling mechanisms from fungi to mammals.

The expanding scope of amyloid signalling

“…We discuss here recent findings concerning the molecular mechanisms of NLR-associated amyloid signal transduction [29] and the evolutionary history of this class of functional amyloids [30,33]. We reflect on the origin of a signal-transducing paradigm, where signal transmission is achieved through 'structural templating' (or replication of a protein fold), from the early observation of non-Mendelian inheritance phenomena in P. anserina [1], through the discovery and characterization of the [Het-s] prion [35], to the very recent identification of signal-transducing amyloids in multicellu lar prokaryotes [33].…”

“…The structure of the necrosome -a heterotypic RHIM-based amyloid formed by the RHIM domains of the RIPK1 and RIPK3 kinaseshas recently been resolved [39]. Our recent discovery that mammalian RHIM motifs can propagate as prions and cross-seed with the fungal PP-based prion amyloids and BASS3 bacterial amyloids [30] indicates a conserved signalling specificity and suggests, in the frame of the amyloid templating paradigm, that a degree of structural similarity exists between these signalling amyloids. Further structural exploration between these signalling amyloids would be of great interest considering that the short amyloidogenic domains are not found in similar protein architectures, specifically in mammalian signalling compared to fungi and prokaryotes.…”

“…In addition to HELLF and HET-S, HELLP constitutes a third amyloid-activated cell-death execution protein of Podospora anserina. The hellp gene occurs in a twogene cluster immediately adjacent to a gene encoding an NLR termed PNT1 displaying an N-terminal PPmotif, a central nucleotide-binding and oligomerisation domain of the NB-ARC type and a C-terminal TPR domain (tetratricopeptide repeats) [30]. HELLP functions in all respects analogously to HET-S and HELLF (Figure 1).…”