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Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission. [ABSTRACT FROM AUTHOR]

Misfolding of tau proteins into prions and their propagation along neural circuits are thought to result in neurodegeneration causing Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy, and other tauopathies. Little is known about the molecular processes mediating tau prion replication and spreading in different brain regions. Using transgenic (Tg) mice with a neuronal promoter driving expression of human mutant (P301S) tau, we found that tau prion formation and histopathologic deposition is largely restricted to the hindbrain. Unexpectedly, tau mRNA and protein levels did not differ between the forebrain and hindbrain, suggesting that other factors modulating the conversion of tau into a prion exist and are region specific. Using a cell-based prion propagation assay, we discovered that tau prion replication is suppressed by forebrain-derived inhibitors, one of which is sortilin, a lysosomal sorting receptor. We also show that sortilin expression is higher in the forebrain than the hindbrain across the life span of the Tg mice, suggesting that sortilin, at least in part, inhibits forebrain tau prion replication in vivo. Our findings provide evidence for selective vulnerability in mice resulting in highly regulated levels of tau prion propagation, thus affording a model for identification of additional molecules that could mitigate the levels of tau prions in human tauopathies.

[PSI+] and [URE3] are prions of Saccharomyces cerevisiae based on amyloids of Sup35p and Ure2p, respectively. In normal cells, antiprion systems block prion formation, cure many prions that arise, prevent infection by prions, and prevent toxicity of those prions that escape the other systems. The upf1Δ, ssz1Δ, and hsp104T160M single mutants each develop [PSI+] at 10- to 15-fold, but the triple mutant spontaneously generates [PSI+] at up to ∼5,000-fold the wild-type rate. Most such [PSI+] variants are cured by restoration of any one of the three defective antiprion systems, defining a previously unknown type of [PSI+] variant and proving that these three antiprion systems act independently. Generation of [PSI+] variants stable in wild-type cells is also increased in upf1Δ ssz1Δ hsp104T160M strains 25- to 500-fold. Btn2 and Cur1 each cure 90% of [URE3] prions generated in their absence, but we find that btn2Δ or cur1Δ diminishes the frequency of [PSI+] generation in an otherwise wild-type strain. Most [PSI+] isolates in a wild-type strain are destabilized on transfer to a btn2Δ or cur1Δ host. Single upf1Δ or hsp104T160M mutants show the effects of btn2Δ or cur1Δ but not upf1Δ ssz1Δ hsp104T160M or ssz1Δ hsp104T160M strains. The disparate action of Btn2 on [URE3] and [PSI+] may be a result of [PSI+]’s generally higher seed number and lower amyloid structural stability compared with [URE3]. Thus, prion generation is not a rare event, but the escape of a nascent prion from the surveillance by the antiprion systems is indeed rare. [ABSTRACT FROM AUTHOR]

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models. [ABSTRACT FROM AUTHOR]

Neurodegenerative disorders are frequently associated with ß-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. 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 ß-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 sheds light on key molecular features concerning an important class of pathogenic agents. [ABSTRACT FROM AUTHOR]

TIA1, a protein critical for eukaryotic stress response and stress granule formation, is structurally characterized in full-length form. TIA1 contains three RNA recognition motifs (RRMs) and a C-terminal low-complexity domain, sometimes referred to as a "prion-related domain" or associated with amyloid formation. Under mild conditions, full-length (fl) mouse TIA1 spontaneously oligomerizes to form a metastable colloid-like suspension. RRM2 and RRM3, known to be critical for function, are folded similarly in excised domains and this oligomeric form of apo fl TIA1, based on NMR chemical shifts. By contrast, the termini were not detected by NMR and are unlikely to be amyloid-like. We were able to assign the NMR shifts with the aid of previously assigned solution-state shifts for the RRM2,3 isolated domains and homology modeling. We present a micellar model of fl TIA1 wherein RRM2 and RRM3 are colocalized, ordered, hydrated, and available for nucleotide binding. At the same time, the termini are disordered and phase separated, reminiscent of stress granule substructure or nanoscale liquid droplets. [ABSTRACT FROM AUTHOR]

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs. [ABSTRACT FROM AUTHOR]

The yeast prion [PSI+] is a self-propagating amyloid of the translation termination factor, Sup35p. For known pathogenic prions, such as [PSI+], a single protein can form an array of different amyloid structures (prion variants) each stably inherited and with differing biological properties. The ribosome-associated chaperones, Ssb1/2p (Hsp70s), and RAC (Zuo1p (Hsp40) and Ssz1p (Hsp70)), enhance de novo protein folding by protecting nascent polypeptide chains from misfolding and maintain translational fidelity by involvement in translation termination. Ssb1/2p and RAC chaperones were previously found to inhibit [PSI+] prion generation. We find that most [PSI+] variants arising in the absence of each chaperone were cured by restoring normal levels of that protein. [PSI+] variants hypersensitive to Ssb1/2p have distinguishable biological properties from those hypersensitive to Zuo1p or Ssz1p. The elevated [PSI+] generation frequency in each deletion strain is not due to an altered [PIN+], another prion that primes [PSI+] generation. [PSI+] prion generation/propagation may be inhibited by Ssb1/2/RAC chaperones by ensuring proper folding of nascent Sup35p, thus preventing its joining amyloid fibers. Alternatively, the effect of RAC/Ssb mutations on translation termination and the absence of an effect on the [URE3] prion suggest an effect on the mature Sup35p such that it does not readily join amyloid filaments. Ssz1p is degraded in zuo1Δ [psi-] cells, but not if the cells carry any of several [PSI+] variants. Our results imply that prions arise more frequently than had been thought but the cell has evolved exquisite antiprion systems that rapidly eliminate most variants. [ABSTRACT FROM AUTHOR]

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence. [ABSTRACT FROM AUTHOR]

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE. [ABSTRACT FROM AUTHOR]

Electrochemical reduction of CO2 to multicarbon products is a significant challenge, especially for molecular complexes. We report here CO2 reduction to multicarbon products based on a Ru(II) polypyridyl carbene complex that is immobilized on an N-doped porous carbon (RuPC/NPC) electrode. The catalyst utilizes the synergistic effects of the Ru(II) polypyridyl carbene complex and the NPC interface to steer CO2 reduction toward C2 production at low overpotentials. In 0.5 M KHCO3/CO2 aqueous solutions, Faradaic efficiencies of 31.0 to 38.4% have been obtained for C2 production at -0.87 to -1.07 V (vs. normal hydrogen electrode) with 21.0 to 27.5% for ethanol and 7.1 to 12.5% for acetate. Syngas is also produced with adjustable H2/CO mole ratios of 2.0 to 2.9. The RuPC/NPC electrocatalyst maintains its activity during 3-h CO2-reduction periods. [ABSTRACT FROM AUTHOR]

The yeast prion [PSI+] is a self-propagating amyloid of Sup35p with a folded in-register parallel ß-sheet architecture. In a genetic screen for antiprion genes, using the yeast knockout collection, UPF1/NAM7 and UPF3, encoding nonsense-mediated mRNA decay (NMD) factors, were frequently detected. Almost all [PSI+] variants arising in the absence of Upf proteins were eliminated by restored normal levels of these proteins, and [PSI+] arises more frequently in upf mutants. Upf1p, complexedwith Upf2p and Upf3p, is a multifunctional protein with helicase, ATP-binding and RNA-binding activities promoting efficient translation termination and degradation of mRNAs with premature nonsense codons. We find that the curing ability of Upf proteins is uncorrelated with these previously reported functions but does depend on their interaction with Sup35p and formation of the Upf1p--Upf2p--Upf3p complex (i.e., the Upf complex). Indeed, Sup35p amyloid formation in vitro is inhibited by substoichiometric Upf1p. Inhibition of [PSI+] prion generation and propagation by Upf proteins may be due to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers. Alternatively, the association of the Upf complex with amyloid filaments may block the addition of new monomers. Our results suggest that maintenance of normal protein--protein interactions prevents prion formation and can even reverse the process. [ABSTRACT FROM AUTHOR]

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype. [ABSTRACT FROM AUTHOR]

The yeast prions [PSI+] and [URE3] are folded in-register parallel β-sheet amyloids of Sup35p and Ure2p, respectively. In a screen for antiprion systems curing [PSI+] without protein overproduction, we detected Siw14p as an antiprion element. An array of genetic tests confirmed that many variants of [PSI+] arising in the absence of Siw14p are cured by restoring normal levels of the protein. Siw14p is a pyrophosphatase specifically cleaving the β phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5), suggesting that increased levels of this or some other inositol polyphosphate favors [PSI+] propagation. In support of this notion, we found that nearly all variants of [PSI+] isolated in a WT strain were lost upon loss of ARG82, which encodes inositol polyphosphate multikinase. Inactivation of the Arg82p kinase by D131A and K133A mutations (preserving Arg82p's nonkinase transcription regulation functions) resulted the loss of its ability to support [PSI+] propagation. The loss of [PSI+] in arg82Δ is independent of Hsp104's antiprion activity. [PSI+] variants requiring Arg82p could propagate in ipk1Δ (IP5 kinase), kcs1Δ (IP6 5-kinase), vip1Δ (IP6 1-kinase), ddp1Δ (inositol pyrophosphatase), or kcs1Δ vip1Δ mutants but not in ipk1Δ kcs1Δ or ddp1Δ kcs1Δ double mutants. Thus, nearly all [PSI+] prion variants require inositol poly-/pyrophosphates for their propagation, and at least IP6 or 5PP-IP4 can support [PSI+] propagation. [ABSTRACT FROM AUTHOR]

In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal "cellular" prion protein (PrPC) is transformed into amyloid aggregates representing the "scrapie form" of the protein (PrPSc). Continued research on this system is of keen interest, since new information on the physiological function of PrPC in healthy organisms is emerging, as well as new data on the mechanism of the transformation of PrPC to PrPSc. In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of PrPC. The focus of the data analysis was on an 11-residue polypeptide segment in mouse PrPC(121-231) that includes the β2-α2 loop of residues 167-170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse PrPC and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on PrPC, providing quantitative insights into the nature of the structural transition-related function of the β2-α2 loop. [ABSTRACT FROM AUTHOR]

Overproduction or deficiency of many chaperones and other cellular components cure the yeast prions [PSI+] (formed by Sup35p) or [URE3] (based on Ure2p). However, at normal expression levels, Btn2p and Cur1p eliminate most newly arising [URE3] variants but do not cure [PSI+], even after overexpression. Deficiency or overproduction of Hsp104 cures the [PSI+] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [PSI+], but cannot cure it by overproduction, thus separating filament cleavage from curing activities. Here we show that most [PSI+] variants arising spontaneously in an hsp104(T160M) strain are cured by restoration of just normal levels of the WT Hsp104. Both strong and weak [PSI+] variants are among those cured by this process. This normal-level Hsp104 curing is promoted by Sti1p, Hsp90, and Sis1p, proteins previously implicated in the Hsp104 overproduction curing of [PSI+]. The [PSI+] prion arises in hsp104(T160M) cells at more than 10-fold the frequency in WT cells. The curing activity of Hsp104 thus constitutes an antiprion system, culling many variants of the [PSI+] prion at normal Hsp104 levels. [ABSTRACT FROM AUTHOR]

Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles. [ABSTRACT FROM AUTHOR]

Electron tomography is an increasingly powerfulmethod to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions. [ABSTRACT FROM AUTHOR]

Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid- controlled fungal programmed cell death and mammalian necroptosis. [ABSTRACT FROM AUTHOR]

Yeasts contain various protein-based genetic elements, termed prions, that result from the structural conversion of proteins into self-propagating amyloid forms. Most yeast prion proteins contain glutamine/asparagine (Q/N)-rich prion domains that drive prion activity. Here, we explore two mechanisms by which new prion domains could evolve. First, it has been proposed that mutation and natural selection will tend to result in proteins with aggregation propensities just low enough to function under physiological conditions and thus that a small number of mutations are often sufficient to cause aggregation. We hypothesized that if the ability to form prion aggregates was a sufficiently generic feature of Q/N-rich domains, many nonprion Q/N-rich domains might similarly have aggregation propensities on the edge of prion formation. Indeed, we tested four yeast Q/N-rich domains that had no detectable aggregation activity; in each case, a small number of rationally designed mutations were sufficient to cause the proteins to aggregate and, for two of the domains, to create prion activity. Second, oligopeptide repeats are found in multiple prion proteins, and expansion of these repeats increases prion activity. However, it is unclear whether the effects of repeat expansion are unique to these specific sequences or are a generic result of adding additional aggregation-prone segments into a protein domain. We found that within nonprion Q/N-rich domains, repeating aggregation-prone segments in tandem was sufficient to create prion activity. Duplication of DNA elements is a common source of genetic variation and may provide a simple mechanism to rapidly evolve prion activity. [ABSTRACT FROM AUTHOR]

Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin-proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome. [ABSTRACT FROM AUTHOR]

Expansion of a poly-glutamine (polyQ) repeat in a group of functionally unrelated proteins is the cause of several inherited neurodegenerative disorders, including Huntington's disease. The polyQ length-dependent aggregation and toxicity of these disease proteins can be reproduced in Saccharomyces cerevisiae. This system allowed us to screen for genes that when overexpressed reduce the toxic effects of an N-terminal fragment of mutant huntingtin with 103 Q. Surprisingly, among the identified suppressors were three proteins with Q-rich, prion-like domains (PrDs): glycine threonine serine repeat protein (Gts1p), nuclear polyadenylated RNA-binding protein 3, and minichromosome maintenance protein 1. Overexpression of the PrD of Gts1p, containing an imperfect 28 residue glutamine-alanine repeat, was sufficient for suppression of toxicity. Association with this discontinuous polyQ domain did not prevent 103Q aggregation, but altered the physical properties of the aggregates, most likely early in the assembly pathway, as reflected in their increased SDS solubility. Molecular simulations suggested that Gts1p arrests the aggregation of polyQ molecules at the level of nonfibrillar species, acting as a cap that destabilizes intermediates on path to form large fibrils. Quantitative proteomic analysis of polyQ interactors showed that expression of Gts1p reduced the interaction between polyQ and other prion-like proteins, and enhanced the association of molecular chaperones with the aggregates. These findings demonstrate that short, Q-rich peptides are able to shield the interactive surfaces of toxic forms of polyQ proteins and direct them into nontoxic aggregates. [ABSTRACT FROM AUTHOR]

[ PSI + ] and [URE3] are prions of Saccharomyces cerevisiae based on amyloids of Sup35p and Ure2p, respectively. In normal cells, antiprion systems block prion formation, cure many prions that arise, prevent infection by prions, and prevent toxicity of those prions that escape the other systems. The upf1Δ , ssz1Δ , and hsp104 T160M single mutants each develop [ PSI + ] at 10- to 15-fold, but the triple mutant spontaneously generates [ PSI + ] at up to ∼5,000-fold the wild-type rate. Most such [ PSI + ] variants are cured by restoration of any one of the three defective antiprion systems, defining a previously unknown type of [ PSI + ] variant and proving that these three antiprion systems act independently. Generation of [ PSI + ] variants stable in wild-type cells is also increased in upf1Δ ssz1Δ hsp104 T160M strains 25- to 500-fold. Btn2 and Cur1 each cure 90% of [URE3] prions generated in their absence, but we find that btn2Δ or cur1Δ diminishes the frequency of [ PSI + ] generation in an otherwise wild-type strain. Most [ PSI + ] isolates in a wild-type strain are destabilized on transfer to a btn2Δ or cur1Δ host. Single upf1Δ or hsp104 T160M mutants show the effects of btn2Δ or cur1Δ but not upf1Δ ssz1Δ hsp104 ]'s generally higher seed number and lower amyloid structural stability compared with [URE3]. Thus, prion generation is not a rare event, but the escape of a nascent prion from the surveillance by the antiprion systems is indeed rare.T160M or ssz1Δ hsp104 T160M strains. The disparate action of Btn2 on [URE3] and [ PSI + ] may be a result of [ PSI + ]'s generally higher seed number and lower amyloid structural stability compared with [URE3]. Thus, prion generation is not a rare event, but the escape of a nascent prion from the surveillance by the antiprion systems is indeed rare.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction. [ABSTRACT FROM AUTHOR]

The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based. sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain. which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the Nterminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular β-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain β-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage. [ABSTRACT FROM AUTHOR]

The formation of extracellular amyloid plaques is a common pathobiochemical event underlying several debilitating human conditions, including Alzheimer's disease (AD). Considerable evidence implies that AD damage arises primarily from small oligomeric amyloid forms of Aβ peptide, but the precise mechanism of pathogenicity remains to be established. Using a cell culture system that reproducibly leads to the formation of Alzheimer's Aβ amyloid plaques, we show here that the formation of a single amyloid plaque represents a template-dependent process that critically involves the presence of endocytosisor phagocytosis-competent cells. Internalized Aβ peptide becomes sorted to multivesicular bodies where fibrils grow out, thus penetrating the vesicular membrane. Upon plaque formation, cells undergo cell death and intracellular amyloid structures become released into the extracellular space. These data imply a mechanism where the pathogenic activity of Aβ is attributed, at least in part, to intracellular aggregates. [ABSTRACT FROM AUTHOR]

We performed mass-per-length (MPL) measurements and electron cryomicroscopy (cryo-EM) with 3D reconstruction on an Aβ(1-42) amyloid fibril morphology formed under physiological pH conditions. The data show that the examined Aβ(1-42) fibril morphology has only one protofilament, although two protofilaments were observed with a previously studied Aβ(1-40) fibril. The latter fibril was resolved at 8 Å resolution showing pairs of β-sheets at the cores of the two protofilaments making up a fibril. Detailed comparison of the Aβ(1-42) and Aβ(1-40) fibril structures reveals that they share an axial twofold symmetry and a similar protofilaent structure. Furthermore, the MPL data indicate that the protofilaments of the examined Aβ(1-40) and Aβ(1-42) fibrils have the same number of Aβ molecules per crossβ repeat. Based on this data and the previously studied Aβ(1-40) fibril structure, we describe a model for the arrangement of peptides within the Aβ(1-42) fibril. [ABSTRACT FROM AUTHOR]

Despite the importance of the aberrant polymerization of Aβ in the early pathogenic cascade of Alzheimer's disease, little is known about the induction of Aβ aggregation in vivo. Here we show that induction of cerebral β-amyloidosis can be achieved in many different brain areas of APP23 transgenic mice through the injection of dilute Aβ-containing brain extracts. Once the amyloidogenic process has been exogenously induced, the nature of the induced Aβ-deposition is determined by the brain region of the host. Because these observations are reminiscent of a prion-like mechanism, we then investigated whether cerebral β-amyloidosis also can be induced by peripheral and systemic inoculations or by the intracerebral implantation of stainless steel wires previously coated with minute amounts of Aβ-containing brain extract. Results reveal that oral, intravenous, intraocular, and intranasal inoculations yielded no detectable induction of cerebral β-amyloidosis in APP23 transgenic mice. In contrast, transmission of cerebral β-amyloidosis through the Aβ-contaminated steel wires was demonstrated. Notably, plasma sterilization, but not boiling of the wires before implantation, prevented the induction of β-amyloidosis. Our results suggest that minute amounts of Aβ-containing brain material in direct contact with the CNS can induce cerebral β-amyloidosis, but that systemic cellular mechanisms of prion uptake and transport to the CNS may not apply to Aβ. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae can be infected with four amyloid-based prions: (URE3], [PSI+], [PIN+J, and [SWI+], due to self-propagating aggregation of Ure2p, Sup35p, Rnqlp and Swilp, respectively. We searched for new prions of yeast by fusing random segments of yeast DNA to SUP35MC, encoding the Sup35 protein lacking its own prion domain, selecting clones in which Sup35MC function was impaired. Three different clones contained parts of the Q/N-rich amino-terminal domain of Mca1p/Yca1p with the Sup35 part of the fusion protein partially inactive. This inactivity was dominant, segregated 4:0 in meiosis, and was efficiently transferred by cytoplasmic mixing. The inactivity was cured by overexpression of Hsp104, but the prion could arise again in the cured strain (reversible curing). Overproduction of the Mcal N-terminal domain induced the de novo appearance of the prion form of the fusion. The prion state, which we name [MCA], was transmitted to the chromosomally encoded Mcalp based on genetic, cytological and biochemical tests. [ABSTRACT FROM AUTHOR]

Most transmissible spongiform encephalopathies arise either spontaneously or by infection. Mutations of PRNP, which encodes the prion protein, PrP, segregate with phenotypically similar diseases. Here we report that moderate overexpression in transgenic mice of mPrP(170N,174T), a mouse PrP with two point mutations that subtly affect the structure of its globular domain, causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease was reproduced with 100% attack rate by intracerebral inoculation of brain homogenate to tga20 mice overexpressing WT PrP, and from the latter to WT mice, but not to PrP-deficient mice. Upon successive transmissions, the incubation periods decreased and PrP became more protease-resistant, indicating the presence of a strain barrier that was gradually overcome by repeated passaging. This shows that expression of a subtly altered prion protein, with known 3D structure, efficiently generates a prion disease. [ABSTRACT FROM AUTHOR]

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimer's disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of β-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Aβ amyloid fibrils from disaggregated Aβ peptide as well as from specific Aβ oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Aβ protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation. [ABSTRACT FROM AUTHOR]

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate. [ABSTRACT FROM AUTHOR]

The structural characterization of amyloid fibers is one of the most investigated areas in structural biology. The structural motif, denoted as steric zipper, recently discovered for the peptide GNNQQNY [Nelson. R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., Grothe, R. & Eisenberg, D. (2005) Nature 435, 773-7781, is expected to exert strong influence in this field. To obtain further insights into the features of this unique structural motif, we report several molecular dynamics simulations of various GNNQQNY aggregates. Our analyses show that even pairs of β-sheets composed of a limited number of β-strands are stable in the 20-ns time interval considered, which suggests that: steric zipper interactions at a β-sheet-β-sheet interface strongly contribute to the stability of these aggregates. Moreover, although the basic features of side chain-side chain interactions are preserved in the simulation, the backbone structure undergoes significant variations. Upon equilibration, a significant twist of the β-strands that compose the β-sheets is observed. These results demonstrate that the occurrence of steric zipper interactions is compatible with flat and twisted β-sheets. Molecular dynamics simulations carried out on two pairs of β-sheets, separated in the crystal state by a hydrated interface, lead to interesting results. The two pairs of sheets, while twisting, associate through stable peptide-peptide interactions. These findings provide insight into the mechanism that leads to the formation of higher aggregates. On these bases, it is possible to reconcile the crystallographic and the EM data on the size of the basic GNNQQNY fiber unit. [ABSTRACT FROM AUTHOR]

We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid-β peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings high-light an important structural motif in a wide variety of normal and pathological processes. [ABSTRACT FROM AUTHOR]

Yeast strains can reversibly interconvert between (PSI+ and [psi-] states. The (PSI+] state is caused by a prion form of the translation termination factor eRF3. The [PSI+] state causes read-through at stop codons and can lead to phenotypic variation, although the molecular mechanisms causing those phenotypic changes remain unknown. We identify an interaction between (PSI+]-induced phenotypic variation and defects in nonstop mRNA decay. Nonstop mRNA decay is triggered when a ribosome reaches the 3' end of the transcript. In contrast, we observed little interaction between [PSI+]-induced phenotypic variation and defects in nonsense- mediated decay, which lead to suppression of premature stop codons. These results suggest that at least some of the phenotypic effects of (PSI+] may be due to read-through of "normal" stop codons, thereby producing extended proteins. Moreover, these observations suggest that nonstop mRNA decay may limit (PSI+]- induced phenotypic variation. Such a process would allow periodic sampling of the 3' UTR, which can diverge rapidly, for novel and beneficial protein extensions. [ABSTRACT FROM AUTHOR]

Secondary, or amyloid protein A (AA), amyloidosis is a complication of chronic inflammatory diseases, both infectious and noninfectious. AA constitutes the insoluble fibrils, which are deposited in different organs, and is a major N-terminal part of the acute phase protein serum AL It is not known why only some patients with chronic inflammation develop AA amyloidosis. Nucleation is a widely accepted mechanism in amyloidogenesis. Preformed amyloid-like fibrils act as nuclei in amyloid fibril formation in vitro, and AA amyloid fibrils and synthetic amyloid-like fibrils also may serve as seed for fibril formation in vivo. In addition to amyloid fibrils, there is a variety of similar nonmammalian protein fibrils with β-pleated structure in nature. We studied three such naturally occurring protein fibrils: silk from Bombyx mori Sup35 from Saccharomyces cerevisiae, and curli from Escherichia coli. Our results show that these protein fibrils exert amyloid-accelerating properties in the murine experimental AA amyloidosis, suggesting that such environment factors may be important risk factors in amyloidogenesis. [ABSTRACT FROM AUTHOR]

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs., Competing Interests: The authors declare no competing interest.

Overproduction or deficiency of many chaperones and other cellular components cure the yeast prions [ PSI + ] (formed by Sup35p) or [ URE3 ] (based on Ure2p). However, at normal expression levels, Btn2p and Cur1p eliminate most newly arising [ URE3 ] variants but do not cure [ PSI ] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [ + ], even after overexpression. Deficiency or overproduction of Hsp104 cures the [ PSI + ] prion. Hsp104 deficiency curing is a result of failure to cleave the Sup35p amyloid filaments to make new seeds, whereas Hsp104 overproduction curing occurs by a different mechanism. Hsp104(T160M) can propagate [ PSI + ], but cannot cure it by overproduction, thus separating filament cleavage from curing activities. Here we show that most [ PSI ] prion arises in + ] variants arising spontaneously in an hsp104(T160M) ] prion at normal Hsp104 levels.PSI + ] variants are among those cured by this process. This normal-level Hsp104 curing is promoted by Sti1p, Hsp90, and Sis1p, proteins previously implicated in the Hsp104 overproduction curing of [ PSI + ]. The [ PSI + ] prion arises in hsp104(T160M) cells at more than 10-fold the frequency in WT cells. The curing activity of Hsp104 thus constitutes an antiprion system, culling many variants of the [ PSI + ] prion at normal Hsp104 levels., Competing Interests: The authors declare no conflict of interest.