Prion Domain Research Articles

De novo design of synthetic prion domains

Prions are important disease agents and epigenetic regulatory elements. Prion formation involves the structural conversion of proteins from a soluble form into an insoluble amyloid form. In many cases, this structural conversion is driven by a glutamine/asparagine (Q/N)-rich prion-forming domain. However, our understanding of the sequence requirements for prion formation and propagation by Q/N-rich domains has been insufficient for accurate prion propensity prediction or prion domain design. By focusing exclusively on amino acid composition, we have developed a prion aggregation prediction algorithm (PAPA), specifically designed to predict prion propensity of Q/N-rich proteins. Here, we show not only that this algorithm is far more effective than traditional amyloid prediction algorithms at predicting prion propensity of Q/N-rich proteins, but remarkably, also that PAPA is capable of rationally designing protein domains that function as prions in vivo.

Molecular Analysis of Genetic Variation in Prions of Saccharomyces cerevisiae

Several proteins in the yeast Saccharomyces cerevisiae act as prions, forming infectious amyloids that are transmissible to other cells during division and outcrossed mating. The parallel in‐register β‐sheet structure of yeast prions such as [PSI+] and [RNQ+] provides a template for conversion of normally unstructured prion domains into a β‐sheet with folds at sites determined by the template on the ends of the filament. Prion propagation is highly sequence specific, and polymorphisms can disrupt templating and abrogate prion transmission. To investigate intra‐species barriers for prion transmission in yeast, we sequenced prion genes of 80 wild isolates from a diverse array of geographic locations and ecological niches. Molecular population genetics was used to quantify nucleotide diversity and examine the evolutionary basis for heterogeneity of prion alleles. An array of indels, SNPs and premature stop codons were detected in wild isolates. Many novel prion haplotypes were characterized, and several variants blocked prion propagation, resulting in barriers to transmission between strains. Almost half of the wild strains analyzed were heterozygous at one or more loci, suggesting that outcrossed mating occurs quite frequently in yeast. It is possible that the observed prion sequence heterogeneity is a consequence of natural selection against prion infections in wild yeast populations.

Investigating the Effect of the Oligopeptide Repeat Region on Prion Propagation

Prions are proteins that can adopt distinct conformations in vivo: a native form and a collection of misfolded or prion forms, which alter the activity of the protein. Remarkably, the prion forms self‐replicate their structure by assembling into ordered aggregates, which template the conversion of the native form into a like state and allow the associated phenotypes to persist. This process of self‐replication is dictated by the sequence of the prion protein. For example, the Sup35 prion in S. cerevisiae has a bipartite prion domain containing a poly‐glutamine‐rich element and five and a half imperfect repeats of a nonapeptide, which are both required for prion propagation. The polyglutamine region mediates templating, but the function of the repeats is currently unknown. Our studies suggest that the steady‐state size of prion aggregates decreases as repeat number increases, and these effects are exacerbated by the deletion of a newly identified sequence element proximal to the repeats. Intriguingly, changes in aggregate size in either direction decreases the abundance of heritable prion aggregates. Together, our studies suggest that the repeat and proximal regions fine‐tune the process of conformational self‐replication through a change in prion aggregate dynamics to create a protein‐based epigenetic element. Supported by NIH 1F31GM099383‐01.

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.

PSI+] Prion Transmission Barriers Protect Saccharomyces cerevisiae from Infection: Intraspecies 'Species Barriers'

[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.

Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins

Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences for synthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93-231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during the synthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.

RNA-Binding Proteins in Amyotrophic Lateral Sclerosis and Neurodegeneration

Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease, which is universally fatal. While the causes of this devastating disease are poorly understood, recent advances have implicated RNA-binding proteins (RBPs) that contain predicted prion domains as a major culprit. Specifically, mutations in the RBPs TDP-43 and FUS can cause ALS. Cytoplasmic mislocalization and inclusion formation are common pathological features of TDP-43 and FUS proteinopathies. Though these RBPs share striking pathological and structural similarities, considerable evidence suggests that the ALS-linked mutations in TDP-43 and FUS can cause disease by disparate mechanisms. In a recent study, Couthouis et al. screened for protein candidates that were also involved in RNA processing, contained a predicted prion domain, shared other phenotypic similarities with TDP-43 and FUS, and identified TAF15 as a putative ALS gene. Subsequent sequencing of ALS patients successfully identified ALS-linked mutations in TAF15 that were largely absent in control populations. This study underscores the important role that perturbations in RNA metabolism might play in neurodegeneration, and it raises the possibility that future studies will identify other RBPs with critical roles in neurodegenerative disease.

The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies

The degraded, misfolded C terminus of TAR DNA-binding protein-43 is associated with a wide spectrum of neurodegenerative diseases, particularly frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. However, the precise mechanism of pathological cleavage of the TAR DNA-binding protein-43 remains unknown. Here we show that the TAR DNA-binding protein-43 C-terminal protein physically interacts with itself or with the cellular-folded yeast prion domain of Sup35 forming dynamic aggregates. This prion-like nature governs known cellular functions of the TAR DNA-binding protein-43, including subcellular localisation and exon skipping of the cystic fibrosis transmembrane conductance regulator. Significantly, mutants with a failure to engage in prion-like interactions are processed into an ~24-kDa C-terminal fragment of the TAR DNA-binding protein-43. The estimated cleavage site of degraded TAR DNA-binding protein-43 fragments corresponds to the pathological cleavage site identified in patients with the TAR DNA-binding protein-43 proteinopathies. Consistently, epigallocatechin gallate constrains prion-like interactions, attenuating pathological-like degradation. Thus, the native folding of TAR DNA-binding protein-43 C terminus acts as a guardian of pathogenesis, which is directly associated with loss-of-function.

Stacked Sheets and Solenoids: Implications of Polymorphic Amyloid Structures of the Fungal Prion HET-s

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.

Temperature Dependence of the Aggregation Kinetics of Sup35 and Ure2p Yeast Prions

Fungal prions are protein-based genetic elements. Sup35 and Ure2p constitute the best-characterized prion proteins in the yeast Saccharomyces cerevisiae. No high-resolution molecular models of the amyloid conformations adopted by the prion domains of these proteins are available yet. A quantitative description of the kinetics and thermodynamics of their self-assembly processes might provide clues on the nature of the structural changes originating their heritable and transmissible phenotypes. Here we study the temperature dependence of Sup35 and Ure2p amyloid fibril nucleation and elongation reactions at physiological pH. Both processes follow the Arrhenius law, allowing calculation of their associated thermodynamic activation parameters. Although the Gibbs energies (ΔG*) for the nucleation and elongation of both prions are similar, the enthalpic and entropic contributions to these two processes are dramatically different. In addition, the structural properties of the two types of prion fibrils exhibit different dependence on the polymerization temperature. Overall, we show here that the amyloidogenic pathways of Sup35 and Ure2p prions diverge significantly.

Dual Conformation of H2H3 Domain of Prion Protein in Mammalian Cells

The concept of prion is applied to protein modules that share the ability to switch between at least two conformational states and transmit one of these through intermolecular interaction and change of conformation. Although much progress has been achieved through the understanding of prions from organisms such as Saccharomyces cerevisiae, Podospora anserina, or Aplysia californica, the criteria that qualify a protein module as a prion are still unclear. In addition, the functionality of known prion domains fails to provide clues to understand the first identified prion, the mammalian infectious prion protein, PrP. To address these issues, we generated mammalian cellular models of expression of the C-terminal two helices of PrP, H2 and H3, which have been hypothesized, among other models, to hold the replication and conversion properties of the infectious PrP. We found that the H2H3 domain is an independent folding unit that undergoes glycosylations and glycosylphosphatidylinositol anchoring similar to full-length PrP. Surprisingly, in some conditions the normally folded H2H3 was able to systematically go through a conversion process and generate insoluble proteinase K-resistant aggregates. This structural switch involves the assembly of amyloid structures that bind thioflavin S and oligomers that are reactive to A11 antibody, which specifically detects protein oligomers from neurological disorders. Overall, we show that H2H3 is a conformational switch in a cellular context and is thus suggested to be a candidate for the conversion domain of PrP.

The complexity and implications of yeast prion domains

Prions are infectious proteins with altered conformations converted from otherwise normal host proteins. While there is only one known mammalian prion protein, PrP, a handful of prion proteins have been identified in the yeast Saccharomyces cerevisiae. Yeast prion proteins usually have a defined region called prion domain (PrD) essential for prion properties, which are typically rich in glutamine (Q) and asparagine (N). Despite sharing several common features, individual yeast PrDs are generally intricate and divergent in their compositional characteristics, which potentially implicates their prion phenotypes, such as prion-mediated transcriptional regulations.

A Small, Glutamine-Free Domain Propagates the [SWI+] Prion in Budding Yeast

Yeast prions are self-propagating protein conformations that transmit heritable phenotypes in an epigenetic manner. The recently identified yeast prion [SWI(+)] is an alternative conformation of Swi1, a component of the evolutionarily conserved SWI/SNF chromatin-remodeling complex. Formation of the [SWI(+)] prion results in a partial loss-of-function phenotype for Swi1. The amino-terminal region of Swi1 is dispensable for its normal function but is required for [SWI(+)] formation and propagation; however, the precise prion domain (PrD) of Swi1 has not been elucidated. Here, we define the minimal Swi1 PrD as the first 37 amino acids of the protein. This region is extremely asparagine rich but, unexpectedly, contains no glutamine residues. This unusually small prion domain is sufficient for aggregation, propagation, and transmission of the [SWI(+)] prion. Because of its unusual size and composition, the Swi1 prion domain defined here has important implications for describing and identifying novel prions.

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.

InVivo and In Vitro Analyses of Toxic Mutants of HET-s: FTIR Antiparallel Signature Correlates with Amyloid Toxicity

The folding and interactions of amyloid proteins are at the heart of the debate as to how these proteins may or may not become toxic to their host. Although little is known about this issue, the structure seems to be clearly involved with effects on molecular events. To understand how an amyloid may be toxic, we previously generated a yeast toxic amyloid (mutant 8) from the nontoxic HET-s (218–289) prion domain of Podospora anserina. Here, we performed a comprehensive structure–toxicity study by mutating individually each of the 10 mutations found in mutant 8. The study of the library of new mutants generated allowed us to establish a clear link between Fourier transform infrared antiparallel signature and amyloid toxicity. All of the mutants that form parallel β-sheets are not toxic. Double mutations may be sufficient to shift a parallel structure to antiparallel amyloids, which are toxic to yeast. Our findings also suggest that the toxicity of antiparallel structured mutants may be linked to interaction with membranes.

PSI+] Maintenance Is Dependent on the Composition, Not Primary Sequence, of the Oligopeptide Repeat Domain

[PSI+], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI+] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI+] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI+] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI+]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI+] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.

Amyloid of the Candida albicans Ure2p Prion Domain Is Infectious and Has an In-Register Parallel β-Sheet Structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.

Prion-Forming Ability of Ure2 of Yeasts Is Not Evolutionarily Conserved

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.

The Core of Ure2p Prion Fibrils Is Formed by the N-Terminal Segment in a Parallel Cross-β Structure: Evidence from Solid-State NMR

Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly 15N–13C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical 13C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1–89 of Ure2p. Thus, the N-terminal segment, or “prion domain” (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of 13C–13C magnetic dipole–dipole couplings among 13C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.

Suicidal [ PSI + ] is a lethal yeast prion

[PSI(+)] is a prion of the essential translation termination factor Sup35p. Although mammalian prion infections are uniformly fatal, commonly studied [PSI(+)] variants do not impair growth, leading to suggestions that [PSI(+)] may protect against stress conditions. We report here that over half of [PSI(+)] variants are sick or lethal. These "killer [PSI(+)]s" are compatible with cell growth only when also expressing minimal Sup35C, lacking the N-terminal prion domain. The severe detriment of killer [PSI(+)] results in rapid selection of nonkiller [PSI(+)] variants or loss of the prion. We also report variants of [URE3], a prion of the nitrogen regulation protein Ure2p, that grow much slower than ure2Δ cells. Our findings give a more realistic picture of the impact of the prion change than does focus on "mild" prion variants.