The genome sequence of Podospora anserina, a classic model fungus

The genome sequence of the coprophilous fungus Podospora anserina shows both typical ascomycete features and how this species is adapted to its specialized environment

Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied

WD-repeat instability and diversification of the Podospora anserina hnwd non-self recognition gene family

<p>Abstract</p> <p>Background</p> <p>Genes involved in non-self recognition and host defence are typically capable of rapid diversification and exploit specialized genetic mechanism to that end. Fungi display a non-self recognition phenomenon termed heterokaryon incompatibility that operates when cells of unlike genotype fuse and leads to the cell death of the fusion cell. In the fungus <it>Podospora anserina</it>, three genes controlling this allorecognition process <it>het-d, het-e </it>and <it>het-r </it>are paralogs belonging to the same <it>hnwd </it>gene family. HNWD proteins are STAND proteins (signal transduction NTPase with multiple domains) that display a WD-repeat domain controlling recognition specificity. Based on genomic sequence analysis of different <it>P. anserina </it>isolates, it was established that repeat regions of all members of the gene family are extremely polymorphic and undergoing concerted evolution arguing for frequent recombination within and between family members.</p> <p>Results</p> <p>Herein, we directly analyzed the genetic instability and diversification of this allorecognition gene family. We have constituted a collection of 143 spontaneous mutants of the <it>het-R </it>(<it>HNWD2</it>) and <it>het-E </it>(<it>hnwd5</it>) genes with altered recognition specificities. The vast majority of the mutants present rearrangements in the repeat arrays with deletions, duplications and other modifications as well as creation of novel repeat unit variants.</p> <p>Conclusions</p> <p>We investigate the extreme genetic instability of these genes and provide a direct illustration of the diversification strategy of this eukaryotic allorecognition gene family.</p

Characterization of the amyloid bacterial inclusion bodies of the HET-s fungal prion

The formation of amyloid aggregates is related to the onset of a number of human diseases. Recent studies provide compelling evidence for the existence of related fibrillar structures in bacterial inclusion bodies (IBs). Bacteria might thus provide a biologically relevant and tuneable system to study amyloid aggregation and how to interfere with it. Particularly suited for such studies are protein models for which structural information is available in both IBs and amyloid states. The only high-resolution structure of an infectious amyloid state reported to date is that of the HET-s prion forming domain (PFD). Importantly, recent solid-state NMR data indicates that the structure of HET-s PFD in IBs closely resembles that of the infectious fibrils. Here we present an exhaustive conformational characterization of HET-s IBs in order to establish the aggregation of this prion in bacteria as a consistent cellular model in which the effect of autologous or heterologous protein quality machineries and/or anti-aggregational and anti-prionic drugs can be further studied

Theme and variations: evolutionary diversification of the HET-s functional amyloid motif

International audienceIn mammals and fungi, Nod-like receptors (NLR) activate downstream cell death execution proteins by a prion-like mechanism. In Podospora anserina, the NWD2 NLR activates the HET-S Helo-domain pore-forming protein by converting its prion-forming domain into a characteristic β-solenoid amyloid fold. The amyloid forming region of HET-S/s comprises two repetitions of a 21 amino acid motif. Herein, we systematically analyze the sequences of C-terminal regions of fungal HeLo and HeLo-like domain proteins to identify HET-s-related amyloid motifs (HRAM). We now identify four novel HRAM subfamilies in addition to the canonical HET-S/s subfamily. These novel motifs share the pseudo-repeat structure of HET-S/s and a specific pattern of distribution of hydrophobic and polar residues. Sequence co-variance analyses predict parallel in-register β-stacking of the two repeats and residue-residue interactions compatible with the β-solenoid fold. As described for HET-S, most genes encoding the HeLo proteins are adjacent to genes encoding NLRs also displaying HRAMs. The motifs of the NLRs are similar to those of their cognate HeLo-domain protein, indicating concerted evolution between repeats. This study shows that HET-s-related amyloid motifs are more common than anticipated and that they have diversified into discrete subfamilies that apparently share a common overall fold. Amyloids are self-templating protein polymers assembled by stacking of β-strands 1. Amyloid formation is often associated with disease, in particular neurodegenerative age-related pathologies such as Alzheimer's or Parkinson's diseases or other conditions like type 2 diabetes 2,3. Amyloid formation has also been linked to cancer with a proposed role of p53 amyloids in tumorogenesis 4. Yet, amyloids also fulfill a range of different functions inside and outside cells 5–7. Such so-called functional amyloids are involved in hormone and toxin storage and release, formation of cell surface structures in microorganisms and scaffolding of pigment synthesis. A role in the regulation of cell states is also described in yeast, where prion amyloids behave as epigenetic regulatory switches 8. Recently, it has also been reported that functional amyloids play a role in cell fate controlling sig-naling cascades both in mammals and fungi. In mammals, the RIP1 and RIP3 kinases which control the necroptosis cell death pathway form an amyloid-like complex assembled via short conserved motifs termed RHIM (for RIP homotypic interaction motifs)

On the Binding of Congo Red to Amyloid Fibrils

Amyloids are characterized by their capacity to bind Congo red (CR), one of the most used amyloid‐specific dyes. The structural features of CR binding were unknown for years, mainly because of the lack of amyloid structures solved at high resolution. In the last few years, solid‐state NMR spectroscopy enabled the determination of the structural features of amyloids, such as the HET‐s prion forming domain (HET‐s PFD), which also has recently been used to determine the amyloid-CR interface at atomic resolution. Herein, we combine spectroscopic data with molecular docking, molecular dynamics, and excitonic quantum/molecular mechanics calculations to examine and rationalize CR binding to amyloids. In contrast to a previous assumption on the binding mode, our results suggest that CR binding to the HET‐s PFD involves a cooperative process entailing the formation of a complex with 1:1 stoichiometry. This provides a molecular basis to explain the bathochromic shift in the maximal absorbance wavelength when CR is bound to amyloids

The [Het-s] Prion, an Amyloid Fold as a Cell Death Activation Trigger

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Signal Transduction by a Fungal NOD-Like Receptor Based on Propagation of a Prion Amyloid Fold

In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s β-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S pore-forming domain. The gene immediately adjacent to het-S encodes NWD2, a Nod-like receptor (NLR) with an N-terminal motif similar to the elementary repeat unit of the β-solenoid fold. NLRs are immune receptors controlling cell death and host defense processes in animals, plants and fungi. We have proposed that, analogously to [Het-s], NWD2 can activate the HET-S pore-forming protein by converting its prion-forming region into the β-solenoid fold. Here, we analyze the ability of NWD2 to induce formation of the β-solenoid prion fold. We show that artificial NWD2 variants induce formation of the [Het-s] prion, specifically in presence of their cognate ligands. The N-terminal motif is responsible for this prion induction, and mutations predicted to affect the β-solenoid fold abolish templating activity. In vitro, the N-terminal motif assembles into infectious prion amyloids that display a structure resembling the β-solenoid fold. In vivo, the assembled form of the NWD2 N-terminal region activates the HET-S pore-forming protein. This study documenting the role of the β-solenoid fold in fungal NLR function further highlights the general importance of amyloid and prion-like signaling in immunity-related cell fate pathways

Protein folding activity of ribosomal rna is a selective target of two unrelated antiprion drugs

Background: 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases.Methodology/Principal Findings: Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome.Conclusion/Significance: 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity

Genesis of a Fungal Non-Self Recognition Repertoire

Conspecific allorecognition, the ability for an organism to discriminate its own cells from those of another individual of the same species, has been developed by many organisms. Allorecognition specificities are determined by highly polymorphic genes. The processes by which this extreme polymorphism is generated remain largely unknown. Fungi are able to form heterokaryons by fusion of somatic cells, and somatic non self-recognition is controlled by heterokaryon incompatibility loci (het loci). Herein, we have analyzed the evolutionary features of the het-d and het-e fungal allorecognition genes. In these het genes, allorecognition specificity is determined by a polymorphic WD-repeat domain. We found that het-d and het-e belong to a large gene family with 10 members that all share the WD-repeat domain and show that repeats of all members of the family undergo concerted evolution. It follows that repeat units are constantly exchanged both within and between members of the gene family. As a consequence, high mutation supply in the repeat domain is ensured due to the high total copy number of repeats. We then show that in each repeat four residues located at the protein/protein interaction surface of the WD-repeat domain are under positive diversifying selection. Diversification of het-d and het-e is thus ensured by high mutation supply, followed by reshuffling of the repeats and positive selection for favourable variants. We also propose that RIP, a fungal specific hypermutation process acting specifically on repeated sequences might further enhance mutation supply. The combination of these evolutionary mechanisms constitutes an original process for generating extensive polymorphism at loci that require rapid diversification