The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Sven J Saupe - One of the best experts on this subject based on the ideXlab platform.
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Amyloid Prions in Fungi
Microbiology spectrum, 2017Co-Authors: Sven J Saupe, Daniel F. Jarosz, Heather L. TrueAbstract:Prions are infectious protein polymers that have been found to cause fatal diseases in mammals. Prions have also been identified in fungi (yeast and filamentous fungi), where they behave as cytoplasmic non-Mendelian genetic elements. Fungal Prions correspond in most cases to fibrillary β-sheet-rich protein aggregates termed amyloids. Fungal Prion models and, in particular, yeast Prions were instrumental in the description of fundamental aspects of Prion structure and propagation. These models established the “protein-only” nature of Prions, the physical basis of strain variation, and the role of a variety of chaperones in Prion propagation and amyloid aggregate handling. Yeast and Fungal Prions do not necessarily correspond to harmful entities but can have adaptive roles in these organisms.
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Diversity of Amyloid Motifs in NLR Signaling in Fungi.
Biomolecules, 2017Co-Authors: Antoine Loquet, Sven J SaupeAbstract:Amyloid folds not only represent the underlying cause of a large class of human diseases but also display a variety of functional roles both in prokaryote and eukaryote organisms. Among these roles is a recently-described activity in signal transduction cascades functioning in host defense and programmed cell death and involving Nod-like receptors (NLRs). In different Fungal species, Prion amyloid folds convey activation signals from a receptor protein to an effector domain by an amyloid templating and propagation mechanism. The discovery of these amyloid signaling motifs derives from the study of [Het-s], a Fungal Prion of the species Podospora anserina. These signaling pathways are typically composed of two basic components encoded by adjacent genes, the NLR receptor bearing an amyloid motif at the N-terminal end and a cell death execution protein with a HeLo pore-forming domain bearing a C-terminal amyloid motif. Activation of the NLR receptor allows for amyloid folding of the N-terminal amyloid motifs which then template trans-conformation of the homologous motif in the cell death execution protein. A variety of such motifs, which differ by their sequence signature, have been described in fungi. Among them, the PP-motif bears resemblance with the RHIM amyloid motif involved in the necroptosis pathway in mammals suggesting an evolutionary conservation of amyloid signaling from fungi to mammals.
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As a toxin dies a Prion comes to life: A tentative natural history of the [Het-s] Prion
Prion, 2015Co-Authors: Asen Daskalov, Sven J SaupeAbstract:A variety of signaling pathways, in particular with roles in cell fate and host defense, operate by a Prion-like mechanism consisting in the formation of open-ended oligomeric signaling complexes termed signalosomes. This mechanism emerges as a novel paradigm in signal transduction. Among the proteins forming such signaling complexes are the Nod-like receptors (NLR), involved in innate immunity. It now appears that the [Het-s] Fungal Prion derives from such a cell-fate defining signaling system controlled by a Fungal NLR. What was once considered as an isolated oddity turns out to be related to a conserved and widespread signaling mechanism. Herein, we recall the relation of the [Het-s] Prion to the signal transduction pathway controlled by the NWD2 Nod-like receptor, leading to activation of the HET-S pore-forming cell death execution protein. We explicit an evolutionary scenario in which formation of the [Het-s] Prion is the result of an exaptation process or how a loss-of-function mutation in a pore-fo...
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High natural prevalence of a Fungal Prion
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Alfons J. M. Debets, Henk J. P. Dalstra, M. Slakhorst, Bertha Koopmanschap, Rolf F. Hoekstra, Sven J SaupeAbstract:Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of Fungal Prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a Prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of Prion formation and the het-S allele encoding a protein variant that cannot form a Prion. We document here that het-s alleles, capable of Prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] Prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] Prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread Prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism.
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Characterization of the amyloid bacterial inclusion bodies of the HET-s Fungal Prion
Microbial Cell Factories, 2009Co-Authors: Raimon Sabaté, Sven J Saupe, Alba Espargaró, Salvador VenturaAbstract: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.
Beat H. Meier - One of the best experts on this subject based on the ideXlab platform.
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Localized and Collective Motions in HET‐s(218‐289) Fibrils from Combined NMR Relaxation and MD Simulation
Angewandte Chemie (International ed. in English), 2019Co-Authors: Albert A. Smith, Matthias Ernst, Sereina Riniker, Beat H. MeierAbstract:Nuclear magnetic resonance (NMR) relaxation data and molecular dynamics (MD) simulations are combined to characterize the dynamics of the Fungal Prion HET-s(218-289) in its amyloid form. NMR data is analyzed with the dynamics detector method, which yields timescale-specific information. An analogous analysis is performed on MD trajectories. Because specific MD predictions can be verified as agreeing with the NMR data, MD was used for further interpretation of NMR results: for the different timescales, cross-correlation coefficients were derived to quantify the correlation of the motion between different residues. Short timescales are the result of very local motions, while longer timescales are found for longer-range correlated motion. Similar trends on ns- and μs-timescales suggest that μs motion in fibrils is the result of motion correlated over many fibril layers.
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Atomic-Resolution Three-Dimensional Structure of HET-s(218−289) Amyloid Fibrils by Solid-State NMR Spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Helene Van Melckebeke, Christian Wasmer, Adam Lange, Antoine Loquet, Anja Böckmann, Beat H. MeierAbstract:We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the Prion domain of the Fungal Prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218−289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.
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atomic resolution three dimensional structure of het s 218 289 amyloid fibrils by solid state nmr spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Helene Van Melckebeke, Christian Wasmer, Adam Lange, Antoine Loquet, Anja Böckmann, Beat H. MeierAbstract:We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the Prion domain of the Fungal Prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218−289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.
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The molecular organization of the Fungal Prion HET-s in its amyloid form.
Journal of molecular biology, 2009Co-Authors: Christian Wasmer, Roland Riek, Anne K. Schütz, Antoine Loquet, Carolin Buhtz, Jason Greenwald, Anja Böckmann, Beat H. MeierAbstract:Abstract The Prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the Prion and nonPrion forms of the protein. For HET-s, the atomic-resolution structure of the isolated Prion domain HET-s(218–289), consisting of a highly ordered triangular cross-β arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s Prion in which we compare their spectra with spectra from isolated C-terminal Prion domain fibrils and the crystalline N-terminal globular domain HET-s(1–227). The spectra reveal unequivocally that the highly ordered structure of the isolated Prion domain HET-s(218–289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT ( i nsensitive n uclei e nhanced by p olarization t ransfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-β core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing Prions despite their similar appearances at the microscopic level.
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Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218-289) are amyloids.
Angewandte Chemie (International ed. in English), 2009Co-Authors: Christian Wasmer, Bénédicte Coulary-salin, Sven J Saupe, Laura Benkemoun, Raimon Sabaté, Michel O. Steinmetz, Lei Wang, Roland Riek, Beat H. MeierAbstract:Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the Prion-forming domain from the Fungal Prion HET-s is the same as that previously determined for fibrils assembled in vitro, and show Prion infectivity. These results demonstrate that the IBs of HET-s(218-289) are amyloids.
Christian Wasmer - One of the best experts on this subject based on the ideXlab platform.
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Atomic-Resolution Three-Dimensional Structure of HET-s(218−289) Amyloid Fibrils by Solid-State NMR Spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Helene Van Melckebeke, Christian Wasmer, Adam Lange, Antoine Loquet, Anja Böckmann, Beat H. MeierAbstract:We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the Prion domain of the Fungal Prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218−289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.
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atomic resolution three dimensional structure of het s 218 289 amyloid fibrils by solid state nmr spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Helene Van Melckebeke, Christian Wasmer, Adam Lange, Antoine Loquet, Anja Böckmann, Beat H. MeierAbstract:We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the Prion domain of the Fungal Prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218−289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints.
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The molecular organization of the Fungal Prion HET-s in its amyloid form.
Journal of molecular biology, 2009Co-Authors: Christian Wasmer, Roland Riek, Anne K. Schütz, Antoine Loquet, Carolin Buhtz, Jason Greenwald, Anja Böckmann, Beat H. MeierAbstract:Abstract The Prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the Prion and nonPrion forms of the protein. For HET-s, the atomic-resolution structure of the isolated Prion domain HET-s(218–289), consisting of a highly ordered triangular cross-β arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s Prion in which we compare their spectra with spectra from isolated C-terminal Prion domain fibrils and the crystalline N-terminal globular domain HET-s(1–227). The spectra reveal unequivocally that the highly ordered structure of the isolated Prion domain HET-s(218–289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT ( i nsensitive n uclei e nhanced by p olarization t ransfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-β core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing Prions despite their similar appearances at the microscopic level.
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Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218-289) are amyloids.
Angewandte Chemie (International ed. in English), 2009Co-Authors: Christian Wasmer, Bénédicte Coulary-salin, Sven J Saupe, Laura Benkemoun, Raimon Sabaté, Michel O. Steinmetz, Lei Wang, Roland Riek, Beat H. MeierAbstract:Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the Prion-forming domain from the Fungal Prion HET-s is the same as that previously determined for fibrils assembled in vitro, and show Prion infectivity. These results demonstrate that the IBs of HET-s(218-289) are amyloids.
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Infectious and noninfectious amyloids of the HET-s(218-289) Prion have different NMR spectra.
Angewandte Chemie (International ed. in English), 2008Co-Authors: Christian Wasmer, Raimon Sabaté, Roland Riek, Adam Lange, Alice Soragni, Beat H. MeierAbstract:The molecular basis for Prion infectivity is not yet understood. The NMR spectra of noninfectious and infectious amyloids of the Prion-forming domain 218-289 of the Fungal Prion HET-s are clearly different (see picture) but are indicative for a cross- arrangement in both cases. The fibrils formed at pH 3 are not infectious because their molecular structure apparently differs substantially from that formed at physiological pH.
Gerald Stubbs - One of the best experts on this subject based on the ideXlab platform.
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Fiber Diffraction of the Prion-Forming Domain HET-s(218–289) Shows Dehydration-Induced Deformation of a Complex Amyloid Structure
Biochemistry, 2014Co-Authors: William Wan, Gerald StubbsAbstract:Amyloids are filamentous protein aggregates that can be formed by many different proteins and are associated with both disease and biological functions. The pathogenicities or biological functions of amyloids are determined by their particular molecular structures, making accurate structural models a requirement for understanding their biological effects. One potential factor that can affect amyloid structures is hydration. Previous studies of simple stacked β-sheet amyloids have suggested that dehydration does not impact structure, but other studies indicated dehydration-related structural changes of a putative water-filled nanotube. Our results show that dehydration significantly affects the molecular structure of the Fungal Prion-forming domain HET-s(218–289), which forms a β-solenoid with no internal solvent-accessible regions. The dehydration-related structural deformation of HET-s(218–289) indicates that water can play a significant role in complex amyloid structures, even when no obvious water-accessible cavities are present.
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Fungal Prion HET-s as a model for structural complexity and self-propagation in Prions.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: William Wan, Gerald StubbsAbstract:The highly ordered and reproducible structure of the Fungal Prion HET-s makes it an excellent model system for studying the inherent properties of Prions, self-propagating infectious proteins that have been implicated in a number of fatal diseases. In particular, the HET-s Prion-forming domain readily folds into a relatively complex two-rung β-solenoid amyloid. The faithful self-propagation of this fold involves a diverse array of inter- and intramolecular structural features. These features include a long flexible loop connecting the two rungs, buried polar residues, salt bridges, and asparagine ladders. We have used site-directed mutagenesis and X-ray fiber diffraction to probe the relative importance of these features for the formation of β-solenoid structure, as well as the cumulative effects of multiple mutations. Using fibrillization kinetics and chemical stability assays, we have determined the biophysical effects of our mutations on the assembly and stability of the Prion-forming domain. We have found that a diversity of structural features provides a level of redundancy that allows robust folding and stability even in the face of significant sequence alterations and suboptimal environmental conditions. Our findings provide fundamental insights into the structural interactions necessary for self-propagation. Propagation of Prion structure seems to require an obligatory level of complexity that may not be reproducible in short peptide models.
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Heterogeneous seeding of a Prion structure by a generic amyloid form of the Fungal Prion-forming domain HET-s(218-289)
The Journal of biological chemistry, 2013Co-Authors: William Wan, Wen Bian, Michele Mcdonald, Aleksandra Kijac, David E. Wemmer, Gerald StubbsAbstract:The Fungal Prion-forming domain HET-s(218–289) forms infectious amyloid fibrils at physiological pH that were shown by solid-state NMR to be assemblies of a two-rung β-solenoid structure. Under acidic conditions, HET-s(218–289) has been shown to form amyloid fibrils that have very low infectivity in vivo, but structural information about these fibrils has been very limited. We show by x-ray fiber diffraction that the HET-s(218–289) fibrils formed under acidic conditions have a stacked β-sheet architecture commonly found in short amyloidogenic peptides and denatured protein aggregates. At physiological pH, stacked β-sheet fibrils nucleate the formation of the infectious β-solenoid Prions in a process of heterogeneous seeding, but do so with kinetic profiles distinct from those of spontaneous or homogeneous (seeded with infectious β-solenoid fibrils) fibrillization. Several serial passages of stacked β-sheet-seeded solutions lead to fibrillization kinetics similar to homogeneously seeded solutions. Our results directly show that structural mutation can occur between substantially different amyloid architectures, lending credence to the suggestion that the processes of strain adaptation and crossing species barriers are facilitated by structural mutation.
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Degradation of Fungal Prion HET-s(218-289) Induces Formation of a Generic Amyloid Fold
Biophysical journal, 2012Co-Authors: William Wan, Ulrich Baxa, Holger Wille, Jan Stöhr, Stanley B. Prusiner, Gerald StubbsAbstract:The Prion-forming domain of the Fungal Prion protein HET-s, HET-s(218-289), is known from solid-state NMR studies to have a β-solenoidal structure; the β-solenoid has the cross-β structure characteristic of all amyloids, but is inherently more complex than the generic stacked β-sheets found in studies of small synthetic peptides. At low pH HET-s(218-289) has also been reported to form an alternative structure, which has not been characterized. We have confirmed by x-ray fiber diffraction that HET-s(218-289) adopts a β-solenoidal structure at neutral pH, and shown that at low pH, it forms either a β-solenoid or a stacked β-sheet structure, depending on the integrity of the protein and the conditions of fibrillization. The low pH stacked-sheet structure is usually formed only by proteolyzed HET-s(218-289), but intact HET-s(218-289) can form stacked sheets when seeded with proteolyzed stacked-sheet HET-s(218-289). The polymorphism of HET-s parallels the structural differences between the infectious brain-derived and the much less infectious recombinant mammalian Prion protein PrP. Taken together, these observations suggest that the functional or pathological forms of amyloid proteins are more complex than the simple generic stacked-sheet amyloids commonly formed by short peptides.
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Stacked Sheets and Solenoids: Implications of Polymorphic Amyloid Structures of the Fungal Prion HET-s
Biophysical Journal, 2012Co-Authors: William Wan, Holger Wille, Jan Stöhr, Wen Bian, Michele Mcdonald, Gerald StubbsAbstract:Prions are aberrantly folded infectious proteins that propagate by inducing the refolding of normal proteins. Prions generally form amyloids: chemically homogeneous, fibrillar protein aggregates. Amyloids are associated with many diseases including Alzheimer's, type II diabetes, and more specifically to Prions, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (“mad cow disease”). Despite this variety, all amyloids share a common cross-β structural motif, β-strands running perpendicular to a central fiber axis. Data from tissue extracted amyloids and studies of short amyloidogenic peptides have led some to hypothesize that all amyloids have a generic amyloid fold consisting of stacks of β-sheets. However, structural models of Alzheimer's related Aβ-amyloid and diabetes related IAPP amyloid indicate that amyloid structure is more diverse. Studies of brain-derived and recombinant Prion protein, PrP, show that the generic amyloid fold shows only marginal biological activity while a β-solenoid fold is highly pathogenic.We have looked at HET-s(218-289), the Prion forming domain of a functional Prion in the fungus Podospora anserina. It has been determined by solid state NMR that under physiological conditions, HET-s(218-289) fibrilizes into a β-solenoid fold. Others have shown that when fibrilized under low pH conditions, a non-functional polymorph is formed. We have determined by X-ray fiber diffraction that at low pH, proteolysis leads to the formation of stacked β-sheet amyloids. These amyloids can propagate the generic fold onto undegraded HET-s(218-289), though only under non-physiological conditions. These results indicate that the biological activity of HET-s(218-289) is intimately tied to its specific amyloid structure and that short amyloidogenic fragments may not adequately reproduce the interactions of larger Prion domains. Supported by NIH grants P01-AG002132 and T32-GM008320-21.
Raimon Sabaté - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the amyloid bacterial inclusion bodies of the HET-s Fungal Prion
Microbial Cell Factories, 2009Co-Authors: Raimon Sabaté, Sven J Saupe, Alba Espargaró, Salvador VenturaAbstract: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.
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Energy barriers for HET-s Prion forming domain amyloid formation.
The FEBS journal, 2009Co-Authors: Raimon Sabaté, Sven J Saupe, Alba Espargaró, Virginia Castillo, Salvador VenturaAbstract:The Prion-forming domain comprising residues 218-289 of the Fungal Prion HET-s forms infectious amyloid fibrils at physiological pH. Because a high-resolution molecular model for the structure of these fibrils exists, it constitutes an attractive system with which to study the mechanism of amyloid assembly. Understanding aggregation under specific conditions requires a quantitative knowledge of the kinetics and thermodynamics of the self-assembly process. We report here the study of the temperature and agitation dependence of the HET-s(218-289) fibril nucleation (kn) and elongation (ke) rate constants at physiological pH. Over our temperature and agitation range, kn and ke increased 30-fold and three-fold, respectively. Both processes followed the Arrhenius law, allowing calculation of the thermodynamic activation parameters associated with them. The data confirm the nucleation reaction as the rate-limiting step of amyloid fibril formation. The formation of the nucleus appears to depend mainly on enthalpic factors, whereas both enthalpic and entropic effects contribute similarly to the energy barrier to fibril elongation. A kinetic model is proposed in which nucleation depends on the presence of an initially collapsed, but poorly structured, HET-s(218-289) state and in which the fibril tip models the conformation of the incoming monomers without substantial disorganization of its structure during the elongation process.
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Solid-state NMR spectroscopy reveals that E. coli inclusion bodies of HET-s(218-289) are amyloids.
Angewandte Chemie (International ed. in English), 2009Co-Authors: Christian Wasmer, Bénédicte Coulary-salin, Sven J Saupe, Laura Benkemoun, Raimon Sabaté, Michel O. Steinmetz, Lei Wang, Roland Riek, Beat H. MeierAbstract:Protein deposition frequently occurs as inclusion bodies (IBs) during heterologous protein expression in E. coli. The structure of these E. coli IBs of the Prion-forming domain from the Fungal Prion HET-s is the same as that previously determined for fibrils assembled in vitro, and show Prion infectivity. These results demonstrate that the IBs of HET-s(218-289) are amyloids.
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Infectious and noninfectious amyloids of the HET-s(218-289) Prion have different NMR spectra.
Angewandte Chemie (International ed. in English), 2008Co-Authors: Christian Wasmer, Raimon Sabaté, Roland Riek, Adam Lange, Alice Soragni, Beat H. MeierAbstract:The molecular basis for Prion infectivity is not yet understood. The NMR spectra of noninfectious and infectious amyloids of the Prion-forming domain 218-289 of the Fungal Prion HET-s are clearly different (see picture) but are indicative for a cross- arrangement in both cases. The fibrils formed at pH 3 are not infectious because their molecular structure apparently differs substantially from that formed at physiological pH.
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Role of Hsp104 in the propagation and inheritance of the [Het-s] Prion.
Molecular biology of the cell, 2007Co-Authors: Laurent Malato, Raimon Sabaté, Laura Benkemoun, Suzana Dos Reis, Sven J SaupeAbstract:The chaperones of the ClpB/HSP100 family play a central role in thermotolerance in bacteria, plants, and fungi by ensuring solubilization of heat-induced protein aggregates. In addition in yeast, Hsp104 was found to be required for Prion propagation. Herein, we analyze the role of Podospora anserina Hsp104 (PaHsp104) in the formation and propagation of the [Het-s] Prion. We show that DeltaPaHsp104 strains propagate [Het-s], making [Het-s] the first native Fungal Prion to be propagated in the absence of Hsp104. Nevertheless, we found that [Het-s]-propagon numbers, propagation rate, and spontaneous emergence are reduced in a DeltaPaHsp104 background. In addition, inactivation of PaHsp104 leads to severe meiotic instability of [Het-s] and abolishes its meiotic drive activity. Finally, we show that DeltaPaHSP104 strains are less susceptible than wild type to infection by exogenous recombinant HET-s(218-289) Prion amyloids. Like [URE3] and [PIN(+)] in yeast but unlike [PSI(+)], [Het-s] is not cured by constitutive PaHsp104 overexpression. The observed effects of PaHsp104 inactivation are consistent with the described role of Hsp104 in Prion aggregate shearing in yeast. However, Hsp104-dependency appears less stringent in P. anserina than in yeast; presumably because in Podospora Prion propagation occurs in a syncitium.
