Alzheimer's, prion diseases and other neurodegenerative disorders are associated with insoluble protein fibres called amyloid fibrils. Gathering structural information about these has proved difficult, but three groups now report success with contrasting approaches to the problem. The cover shows the atomic structure of the ‘spine’ of an amyloid-like fibril formed by the yeast prion protein Sup35. To obtain this structure, Nelson et al. performed X-ray crystallography on amyloid microcrystals. Krishnan and Lindquist used a raft of techniques to study folding of the amyloid core. And Ritter et al. determined the infectious conformation of HET-s prion of a filamentous fungus. Christopher Dobson ponders on what this flood of structural data says about prion and amyloid formation in sickness and in health. Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins1,2. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast3,4,5, Podospora anserina6 and mammals7 can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218–289. Amyloid fibrils of HET-s(218–289) are necessary and sufficient for the induction and propagation of prion infectivity6. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218–289). This approach revealed four β-strands constituted by two pseudo-repeat sequences, each forming a β-strand-turn-β-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.

SUMMARY: Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.

The [Het-s] infectious element of the filamentous fungus Podospora anserina is a prion. We have recently reported that recombinant HET-s protein aggregates in vitro into amyloid fibers. In vivo , the protein aggregates specifically in the [Het-s] prion strains. Here, we show that biolistic introduction of aggregated recombinant HET-s protein into fungal cells induces emergence of the [Het-s] prion with a high frequency. Thus, we demonstrate that prion infectivity can be created de novo , in vitro from recombinant protein in this system. Although the amyloid filaments formed from HET-s could transmit [Het-s] efficiently, neither the soluble form of the protein nor amorphous aggregates would do so. In addition, we have found that ( i ) [Het-s] infectivity correlates with the ability to convert HET-s to amyloids in vitro , ( ii ) [Het-s] infectivity is resistant to proteinase K digestion, and ( iii ) HET-s aggregates formed in vivo in [Het-s] strains have the ability to convert the recombinant protein to aggregates. Together, our data designate the HET-s amyloids as the molecular basis of [Het-s] prion propagation.

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The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the β-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins. Filamentous fungi have the potential for genetically distinct individuals to fuse, resulting in a cell with multiple nuclei known as a heterokaryon. This fusion event is controlled by genetic variants that determine the compatibility of the individuals, such that the fusion of incompatible genotypes triggers a cell death reaction in the heterokaryon. We have investigated the molecular mechanism of toxicity in the HET-S/HET-s incompatibility system in the fungus P. anserina. HET-s is an infectious yet non-toxic protein (prion) whose interaction with the almost identical protein HET-S has been shown to re-localize HET-S to the cell periphery, an event that is associated with the death of heterokaryons that simultaneously contain both proteins. We find that the HET-s prion converts soluble HET-S into a protein that binds to and destabilizes lipid membranes. Furthermore, we identify a potential transmembrane helix that is normally buried within the soluble fold of HET-S and show that its presence is associated with toxicity. We conclude that upon interaction with a HET-s prion, the HET-S globular domain partially unfolds, exposing a previously buried transmembrane segment that targets HET-S to the membrane. Once there, it further oligomerizes into a structure that causes a loss of membrane integrity, reminiscent of the mode of action of pore-forming toxins.

Significance: Programmed cell death plays a central role in host defense in plants, animals, and fungi, but the extent to which cell death modalities are evolutionarily related and mechanistically similar in different kingdoms is unclear. The involvement of prion-like mechanisms in host defense and cell death cascades has been reported in animals and fungi. In fungi, a cell death-inducing pore-forming protein termed HET-S is activated by amyloid templating. Here we characterize a protein termed HELLP, which behaves analogously to HET-S as a membrane-targeting cell death-inducing protein regulated by amyloid templating. We find that HELLP is homologous to MLKL, the protein involved in execution of necroptotic cell death in mammals, thus revealing transkingdom conservation of amyloid-regulated programmed cell death.