Structure and assembly properties of the yeast prion Ure2p

The non-Mendelian phenotype [URE3] is due to a transmissible conformational change of the protein Ure2. The infectious protein form of Ure2p has lost its function and gained the capacity to transform the active form of the protein into an inactive form. The molecular basis of this conversion process is unknown. There are however indications that the conformational changes at the origin of the propagation of the inactive form of Ure2p in yeast cells are similar to those at the origin of the transition of PrPC into the scrapie-associated PrPSc form of the protein. To better understand the nature of the conformational changes at the origin of prion propagation, we have purified, characterized biochemically, examined the assembly properties and solved the crystal structure of Ure2p. Our data are presented below and a number of conclusions dealing with the molecular basis of the conversion of soluble Ure2p into its amyloid-forming state are derived.     

Small angle X-ray scattering study of the yeast prion Ure2p

The GdmCl-induced equilibrium unfolding and dissociation of the dimeric yeast prion protein Ure2, and its prion domain deletion mutants Delta 15-42Ure2 and 90Ure2, was studied by small angle X-ray scattering (SAXS) using synchrotron radiation and by chemical cross-linking with dithiobis(succinimidyl propionate) (DTSP). The native state is globular and predominantly dimeric prior to the onset of unfolding. R(g) values of 32 and 45A were obtained for the native and 5M GdmCl denatured states of Delta 15-42Ure2, respectively; the corresponding values for 90Ure2 were 2-3A lower. SAXS suggests residual structure in the 4M GdmCl denatured state and chemical cross-linking detects persistence of dimeric structure under these conditions. Hexamers consisting of globular subunits could be detected by SAXS at high protein concentration under partially denaturing conditions. The increased tendency of partially folded states to form small oligomers points to a mechanism for prion formation.     

An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain

Production of the pathogenic prion isoform PrP^sc-like molecules is thought to be useful forunderstanding the mysterious mechanism of conformational conversion process of prion diseases andproving the “protein-only“ hypothesis. In this report, an engineered PrP^sc-like conformation was producedfrom a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD).Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD,spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-likecharacteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increasedfluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopyexperiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high contentB-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit moresoluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrP^se and will be helpful for further understanding the mechanism of conformational conversion process.     

Pressure denaturation of the yeast prion protein Ure2

Denaturation of the Saccharomyces cerevisiae prion protein Ure2 was investigated using hydrostatic pressure. Pressures of up to 600 MPa caused only limited perturbation of the structure of the 40-kDa dimeric protein. However, nondenaturing concentrations of GdmCl in combination with high pressure resulted in complete unfolding of Ure2 as judged by intrinsic fluorescence. The free energy of unfolding measured by pressure denaturation or by GdmCl denaturation is the same, indicating that pressure does not induce dimer dissociation or population of intermediates in 2 M GdmCl. Pressure-induced changes in 5 M GdmCl suggest residual structure in the denatured state. Cold denaturation under pressure at 200 MPa showed that unfolding begins below -5 degrees C and Ure2 is more susceptible to cold denaturation at low ionic strength. Results obtained using two related protein constructs, which lack all or part of the N-terminal prion domain, were very similar.     

Characterization of oligomeric species in the fibrillization pathway of the yeast prion Ure2p

The [URE3] prion of Saccharomyces cerevisiae shares many features with mammalian prions and poly-glutamine related disorders and has become a model for studying amyloid diseases. The development of the [URE3] phenotype is thought to be caused by a structural switch in the Ure2p protein. In [URE3] cells, Ure2p is found predominantly in an aggregated state, while it is a soluble dimer in wild-type cells. In vitro, Ure2p forms fibrils with amyloid-like properties. Several studies suggest that the N-terminal domain of Ure2p is essential for prion formation. In this work, we investigated the fibril formation of Ure2p by isolating soluble oligomeric species, which are generated during fibrillization, and characterized them with respect to size and structure. Our data support the critical role of the N-terminal domain for fibril formation, as we observed fibrils in the presence of 5 M guanidinium chloride, conditions at which the C-terminal domain is completely unfolded. Based on fluorescence measurements, we conclude that the structure of the C-terminal domain is very similar in dimeric and fibrillar Ure2p. When studying the time course of fibrillization, we detected the formation of small, soluble oligomeric species during the early stages of the process. Their remarkable resistance against denaturants, their increased content of beta-structure, and their ability to 'seed' Ure2p fibrillization suggest that conversion to the amyloid-like conformation has already occurred. Thus, they likely represent critical intermediates in the fibrillization pathway of Ure2p.     

Structural characterization of the fibrillar form of the yeast Saccharomyces cerevisiae prion Ure2p

The protein Ure2 from the yeast Saccharomyces cerevisiae has prion properties. It assembles in vitro into long, straight, insoluble fibrils that are similar to amyloids in that they bind Congo Red and show green-yellow birefringence and have an increased resistance to proteolysis. We recently showed that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta-core. A model for fibril formation, where assembly is driven by non-native inter- and/or intramolecular interaction between Ure2p monomers following subtle conformational changes was proposed [Bousset et al. (2002) EMBO J. 21, 2903-2911]. An alternative model for the assembly of Ure2p into fibrils where assembly is driven by the stacking of 40-70 N-terminal amino acid residues of Ure2p into a central beta-core running along the fibrils from which the C-terminal domains protrude was proposed [Baxa et al. (2003) J. Biol. Chem. 278, 43717-43727]. We show here that Ure2p fibril congophilia and the associated yellow-green birefringence in polarized light are not indicative that the fibrils are of amyloid nature. We map the structures of the fibrillar and soluble forms of Ure2p using limited proteolysis and identify the reaction products by microsequencing and mass spectrometry. Finally, we demonstrate that the C-terminal domain of Ure2p is tightly involved in the fibrillar scaffold using a sedimentation assay and a variant Ure2p where a highly specific cleavage site between the N- and C-terminal domains of the protein was engineered. Our results are inconsistent with the cross-beta-core model and support the model for Ure2p assembly driven by subtle conformational changes and underline the influence of the natural context of the N-terminal domain on the assembly of Ure2p.     

The yeast prion protein Ure2: structure, function and folding

The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.     

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

Assembly of the yeast prion Ure2p into protein fibrils. Thermodynamic and kinetic characterization

The [URE3] phenotype in Saccharomyces cerevisiae propagates by a prion mechanism, involving the aggregation of the normally soluble and highly helical protein Ure2. Previous data have shown that the protein spontaneously forms in vitro long, straight, insoluble fibrils at neutral pH that are similar to amyloids in that they bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis. These fibrils are not amyloids as they are devoid of a cross-beta core. Here we further document the mechanism of assembly of Ure2p into fibrils. The critical concentration for Ure2p assembly is measured, and the minimal size of the nuclei that are the precursors of Ure2p fibrils is determined. Our data indicate that the assembly process is irreversible. As a consequence, the critical concentration is very low. By analyzing the elongation rates of preformed fibrils and combining the results with single-fiber imaging experiments of a variant Ure2p labeled by fluorescent dyes, we reveal the polarity of the fibrils and differences in the elongation rates at their ends. These results bring novel insight in the process of Ure2p assembly into fibrils and the mechanism of propagation of yeast prions.     

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.     

Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.     

The yeast prion Ure2p retains its native alpha-helical conformation upon assembly into protein fibrils in vitro

The yeast inheritable phenotype [URE3] is thought to result from conformational changes in the normally soluble and highly helical protein Ure2p. In vitro, the protein spontaneously forms long, straight, insoluble protein fibrils at neutral pH. Here we show that fibrils of intact Ure2p assembled in vitro do not possess the cross beta-structure of amyloid, but instead are formed by the polymerization of native-like helical subunits that retain the ability to bind substrate analogues. We further show that dissociation of the normally dimeric protein to its constituent monomers is a prerequisite for assembly into fibrils. By analysing the nature of early assembly intermediates, as well as fully assembled Ure2p fibrils using atomic force microscopy, and combining the results with experiments that probe the fidelity of the native fold in protein fibrils, we present a model for fibril formation, based on assembly of native-like monomers, driven by interactions between the N-terminal glutamine and asparagine-rich region and the C-terminal functional domain. The results provide a rationale for the effect of mutagenesis on prion formation and new insights into the mechanism by which this, and possibly other inheritable factors, can be propagated.     

The prion domain of yeast Ure2p induces autocatalytic formation of amyloid fibers by a recombinant fusion protein

The Ure2 protein from Saccharomyces cerevisiae has been proposed to undergo a prion-like autocatalytic conformational change, which leads to inactivation of the protein, thereby generating the [URE3] phenotype. The first 65 amino acids, which are dispensable for the cellular function of Ure2p in nitrogen metabolism, are necessary and sufficient for [URE3] (Masison & Wickner, 1995), leading to designation of this domain as the Ure2 prion domain (UPD). We expressed both UPD and Ure2 as glutathione-S-transferase (GST) fusion proteins in Escherichia coli and observed both to be initially soluble. Upon cleavage of GST-UPD by thrombin, the released UPD formed ordered fibrils that displayed amyloid-like characteristics, such as Congo red dye binding and green-gold birefringence. The fibrils exhibited high beta-sheet content by Fourier transform infrared spectroscopy. Fiber formation proceeded in an autocatalytic manner. In contrast, the released, full-length Ure2p formed mostly amorphous aggregates; a small amount polymerized into fibrils of uniform size and morphology. Aggregation of Ure2p could be seeded by UPD fibrils. Our results provide biochemical support for the proposal that the [URE3] state is caused by a self-propagating inactive form of Ure2p. We also found that the uncleaved GST-UPD fusion protein could polymerize into amyloid fibrils by a strictly autocatalytic mechanism, forcing the GST moiety of the protein to adopt a new, beta-sheet-rich conformation. The findings on the GST-UPD fusion protein indicate that the ability of the prion domain to mediate a prion-like conversion process is not specific for or limited to the Ure2p.     

The yeast prion protein Ure2: insights into the mechanism of amyloid formation

Ure2, a regulator of nitrogen metabolism, is the protein determinant of the [URE3] prion state in Saccharomyces cerevisiae. Upon conversion into the prion form, Ure2 undergoes a heritable conformational change to an amyloid-like aggregated state and loses its regulatory function. A number of molecular chaperones have been found to affect the prion properties of Ure2. The studies carried out in our laboratory have been aimed at elucidating the structure of Ure2 fibrils, the mechanism of amyloid formation and the effect of chaperones on the fibril formation of Ure2.     

Hierarchical organization in the amyloid core of yeast prion protein Ure2

Formation of amyloid fibrils is involved in a range of fatal human disorders including Alzheimer, Parkinson, and prion diseases. Yeast prions, despite differences in sequence from their mammalian counterparts, share similar features with mammalian prions including infectivity, prion strain phenomenon, and species barrier and thus are good model systems for human prion diseases. Yeast prions normally have long prion domains that presumably form multiple β strands in the fibril, and structural knowledge about the yeast prion fibrils has been limited. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structures of amyloid fibrils of Ure2 prion domain. We show that 15 spin-labeled Ure2 mutants, with spin labels at every 5th residue from position 5 to position 75, show a single-line or nearly single-line feature in their EPR spectra as a result of strong spin exchange interactions. These results suggest that a parallel in-register β structure exists at these spin-labeled positions. More interestingly, we also show that residues in the segment 30-65 have stronger spin exchange interactions, higher local stability, and lower solvent accessibility than segments 5-25 and 70-75, suggesting different local environment at these segments. We propose a hierarchical organization in the amyloid core of Ure2, with the segment 30-65 forming an inner core and the segments 5-25 and 70-75 forming an outer core. The hierarchical organization in the amyloid core may be a structural origin for polymorphism in fibrils and prion strains.     

Equilibrium folding properties of the yeast prion protein determinant Ure2.

The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD). Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4. A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein.     

Conservation of the prion properties of Ure2p through evolution

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.     

Globular and pre-fibrillar prion aggregates are toxic to neuronal cells and perturb their electrophysiology

Prion diseases are characterised at autopsy by neuronal loss and accumulation of amorphous protein aggregates and/or amyloid fibrils in the brains of humans and animals. These protein deposits result from the conversion of the cellular, mainly alpha-helical prion protein (PrP(C)) to the beta-sheet-rich isoform (PrP(Sc)). Although the pathogenic mechanism of prion diseases is not fully understood, it appears that protein aggregation is itself neurotoxic and not the product of cell death. The precise nature of the neurotoxic species and mechanism of cell death are yet to be determined, although recent studies with other amyloidogenic proteins suggest that ordered pre-fibrillar or oligomeric forms may be responsible for cellular dysfunction. In this study we have refolded recombinant prion protein (rPrP) to two distinct forms rich in beta-sheet structure with an intact disulphide bond. Here we report on the structural properties of globular aggregates and pre-fibrils of rPrP and show that both states are toxic to neuronal cells in culture. We show that exogenous rPrP aggregates are internalised by neuronal cells and found in the cytoplasm. We also measured the changes in electrophysiological properties of cultured neuronal cells on exposure to exogenous prion aggregates and discuss the implications of these findings.     

The mechanisms of [URE3] prion elimination demonstrate that large aggregates of Ure2p are dead-end products

The yeast prion [URE3] is a self-propagating inactive form (the propagon) of the Ure2 protein. Ure2p is composed of two domains: residues 1-93--the prion-forming domain (PFD)--and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression. Guanidine hydrochloride, and the overproduction of Ure2p 1-65 or Ure2-GFP have been shown to induce the elimination of [URE3]. We demonstrate here, two different curing mechanisms: the inhibition of [URE3] replication by guanidine hydrochloride and its destruction by Ure2p aggregation. Such aggregation is observed if PFD or Ure2-GFP are overproduced and in heterozygous URE2/URE2-GFP, [URE3] diploids. We found that the GFP foci associated with the presence of the prion were dead-end products, the propagons remaining soluble. Surprisingly, [URE3] propagated via the Ure2-GFP fusion protein alone is resistant to these two curing mechanisms and cannot promote the formation of foci. The relationship between aggregation, prion and Hsp104 gives rise to a model in which the propagon is in equilibrium with larger aggregates and functional protein.