Biology subjects

Daskalov, A.

Publications and source records attributed to Daskalov, A..

4 recordsLinked to original sources

bioRxiv2021

Fungal gasdermin-like proteins are controlled by proteolytic cleavage

Gasdermins are a family of pore-forming proteins controlling an inflammatory cell death reaction in the mammalian immune system. The pore-forming ability of the gasdermin proteins is released by proteolytic cleavage with the removal of their inhibitory C-terminal domain. Recently, gasdermin-like proteins have been discovered in fungi and characterized as cell death-inducing toxins in the context of conspecific non-self discrimination (allorecognition). Although functional analogies have been established between mammalian and fungal gasdermins, the molecular pathways regulating gasdermin activity in fungi remain largely unknown. Here, we characterize a gasdermin-based cell death reaction, controlled by the het-Q allorecognition genes in the filamentous fungus Podospora anserina. We show that the cytotoxic activity of the HET-Q1 gasdermin is controlled by proteolysis. HET-Q1 loses a [~]5 kDa C-terminal fragment during the cell death reaction in presence of a subtilisin-like serine protease, termed HET-Q2. Mutational analyses and successful reconstitution of the cell death reaction in a heterologous host (Saccharomyces cerevisiae) suggest that HET-Q2 directly cleaves HET-Q1 to induce cell death. By analysing the genomic landscape of het-Q1 homologs in fungi, we uncovered that the vast majority of the gasdermin genes are clustered with protease-encoding genes. These HET-Q2-like proteins carry either subtilisin-like or caspase-related proteases, which in some cases correspond to the N-terminal effector domain of NOD-like receptor proteins (NLRs). This study thus reveals the proteolytic regulation of gasdermins in fungi and establishes evolutionary parallels between fungal and mammalian gasdermin-dependent cell death pathways. SignificanceThe recent discovery of gasdermin-like proteins in fungi have brought to light that this family of pore-forming proteins controls cell death in two of the major eukaryotic kingdoms, fungi and mammals. Yet, the regulation of cytotoxicity of the fungal gasdermins and their molecular pathways remain uncharacterized. Here, we describe the regulation through proteolytic cleavage of the fungal gasdermin HET-Q1 and uncover that majority of fungal gasdermins are genomically clustered with protease-encoding genes. Some of these genes encode proteins with caspase-related domains and/or are members of a family of immune receptors in mammals and plants. Overall, this work contributes towards our understanding of the evolution of gasdermin-dependent cell death, enlightening multiple evolutionary parallels between signaling pathways in mammals and fungi.

genetics↗

bioRxiv2020

Prion cross-seeding between fungal and mammalian amyloid signaling motifs

In filamentous 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. An analogous system, involving a distinct amyloid signaling motif termed PP was also identified in the genome of the species Chaetomium globosum and studied using heterologous expression in Podospora anserina. The PP-motif bears resemblance to the RHIM and RHIM-like motifs controlling necroptosis in mammals and innate immunity in flies. 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 prion we term [{pi}] and that [{pi}] 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 [{pi}] and [Rhim] prions partially cross-seed. Our study shows that Podospora anserina displays three independent cell-death inducing 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. ImportanceAmyloids are {beta}-sheet-rich protein polymers that can be pathological or display a variety of biological roles. In filamentous fungi, specific immune receptors activate programmed cell-death execution proteins through a process of amyloid templating akin to prion propagation. Among these fungal amyloid signaling sequences, the PP-motif stands out because it shows similarity to RHIM, an amyloid sequence controlling necroptotic cell-death in mammals. We characterized an amyloid signaling system comprising a PP-motif in the model species Podospora anserina thus bringing to three the number of independent amyloid signaling cell death pathways described in that species. We then show that human RHIM motifs not only propagate as prions in P. anserina but also partially cross-seed with fungal PP-prions. These results indicate that in addition to show sequence similarity, PP and RHIM-motif are at least partially functionally related, supporting a model of long-term evolutionary conservation of amyloid signaling mechanisms from fungi to mammals.

microbiology↗

bioRxiv2020

Structural and molecular basis of cross-seeding barriers in amyloids

Neurodegenerative disorders are frequently associated with β-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behaviour and distinct patho-phenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical β-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and shed new light on key molecular features concerning an important class of pathogenic agents.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Identification of NLR-associated amyloid signaling motifs in filamentous bacteria

NLRs (Nod-like receptors) are intracellular receptors regulating immunity, symbiosis, non-self recognition and programmed cell death in animals, plants and fungi. Several fungal NLRs employ amyloid signaling motifs to activate downstream cell-death inducing proteins. Herein, we identify in Archaea and Bacteria, short sequence motifs that occur in the same genomic context as fungal amyloid signaling motifs. We identify 10 families of bacterial amyloid signaling sequences (we term BASS), one of which (BASS3) is related to mammalian RHIM and fungal PP amyloid motifs. We find that BASS motifs occur specifically in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of these motifs and find that they behave as prion forming domains when expressed in a fungal model. All tested bacterial motifs also formed fibrils in vitro. We analyze by solid-state NMR and X-ray diffraction, the amyloid state of a protein from Streptomyces coelicolor bearing the most common BASS1 motif and find that it forms highly ordered non-polymorphic amyloid fibrils. This work expands the paradigm of amyloid signaling to prokaryotes and underlies its relation to multicellularity.

microbiology↗