Anti-prion Systems Research Articles

Human proteins curing yeast prions.

Recognition that common human amyloidoses are prion diseases makes the use of the Saccharomyces cerevisiae prion model systems to screen for possible anti-prion components of increasing importance. [PSI+] and [URE3] are amyloid-based prions of Sup35p and Ure2p, respectively. Yeast has at least six anti-prion systems that together cure nearly all [PSI+] and [URE3] prions arising in their absence. We made a GAL-promoted bank of 14,913 human open reading frames in a yeast shuttle plasmid and isolated 20 genes whose expression cures [PSI+] or [URE3]. PRPF19 is an E3 ubiquitin ligase that cures [URE3] if its U-box is intact. DNAJA1 is a J protein that cures [PSI+] unless its interaction with Hsp70s is defective. Human Bag5 efficiently cures [URE3] and [PSI+]. Bag family proteins share a 110 to 130 residue "BAG domain"; Bag 1, 2, 3, 4, and 6 each have one BAG domain while Bag5 has five BAG domains. Two BAG domains are necessary for curing [PSI+], but one can suffice to cure [URE3]. Although most Bag proteins affect autophagy in mammalian cells, mutations blocking autophagy in yeast do not affect Bag5 curing of [PSI+] or [URE3]. Curing by Bag proteins depends on their interaction with Hsp70s, impairing their role, with Hsp104 and Sis1, in the amyloid filament cleavage necessary for prion propagation. Since Bag5 curing is reduced by overproduction of Sis1, we propose that Bag5 cures prions by blocking Sis1 access to Hsp70s in its role with Hsp104 in filament cleavage.

Soluble amyloid-β dimers are resistant to amyloid-β prion conversion in vivo suggesting antiprion properties.

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Anti-Prion Systems in Saccharomyces cerevisiae Turn an Avalanche of Prions into a Flurry.

Prions are infectious proteins, mostly having a self-propagating amyloid (filamentous protein polymer) structure consisting of an abnormal form of a normally soluble protein. These prions arise spontaneously in the cell without known reason, and their effects were generally considered to be fatal based on prion diseases in humans or mammals. However, the wide array of prion studies in yeast including filamentous fungi revealed that their effects can range widely, from lethal to very mild (even cryptic) or functional, depending on the nature of the prion protein and the specific prion variant (or strain) made by the same prion protein but with a different conformation. This prion biology is affected by an array of molecular chaperone systems, such as Hsp40, Hsp70, Hsp104, and combinations of them. In parallel with the systems required for prion propagation, yeast has multiple anti-prion systems, constantly working in the normal cell without overproduction of or a deficiency in any protein, which have negative effects on prions by blocking their formation, curing many prions after they arise, preventing prion infections, and reducing the cytotoxicity produced by prions. From the protectors of nascent polypeptides (Ssb1/2p, Zuo1p, and Ssz1p) to the protein sequesterase (Btn2p), the disaggregator (Hsp104), and the mysterious Cur1p, normal levels of each can cure the prion variants arising in its absence. The controllers of mRNA quality, nonsense-mediated mRNA decay proteins (Upf1, 2, 3), can cure newly formed prion variants by association with a prion-forming protein. The regulator of the inositol pyrophosphate metabolic pathway (Siw14p) cures certain prion variants by lowering the levels of certain organic compounds. Some of these proteins have other cellular functions (e.g., Btn2), while others produce an anti-prion effect through their primary role in the normal cell (e.g., ribosomal chaperones). Thus, these anti-prion actions are the innate defense strategy against prions. Here, we outline the anti-prion systems in yeast that produce innate immunity to prions by a multi-layered operation targeting each step of prion development.

Anti-Prion Systems Block Prion Transmission, Attenuate Prion Generation, Cure Most Prions as They Arise and Limit Prion-Induced Pathology in Saccharomyces cerevisiae.

Simple SummaryVirus and bacterial infections are opposed by their hosts at many levels. Similarly, we find that infectious proteins (prions) are severely restricted by an array of host systems, acting independently to prevent infection, generation, propagation and the ill effects of yeast prions. These ‘anti-prion systems’ work in normal cells without the overproduction or deficiency of any components. DNA repair systems reverse the effects of DNA damage, with only a rare lesion propagated as a mutation. Similarly, the combined effects of several anti-prion systems cure and block the generation of all but 1 in about 5000 prions arising. We expect that application of our approach to mammalian cells will detect analogous or even homologous systems that will be useful in devising therapy for human amyloidoses, most of which are prions.All variants of the yeast prions [PSI+] and [URE3] are detrimental to their hosts, as shown by the dramatic slowing of growth (or even lethality) of a majority, by the rare occurrence in wild isolates of even the mildest variants and by the absence of reproducible benefits of these prions. To deal with the prion problem, the host has evolved an array of anti-prion systems, acting in normal cells (without overproduction or deficiency of any component) to block prion transmission from other cells, to lower the rates of spontaneous prion generation, to cure most prions as they arise and to limit the damage caused by those variants that manage to elude these (necessarily) imperfect defenses. Here we review the properties of prion protein sequence polymorphisms Btn2, Cur1, Hsp104, Upf1,2,3, ribosome-associated chaperones, inositol polyphosphates, Sis1 and Lug1, which are responsible for these anti-prion effects. We recently showed that the combined action of ribosome-associated chaperones, nonsense-mediated decay factors and the Hsp104 disaggregase lower the frequency of [PSI+] appearance as much as 5000-fold. Moreover, while Btn2 and Cur1 are anti-prion factors against [URE3] and an unrelated artificial prion, they promote [PSI+] prion generation and propagation.

Antiprion systems in yeast cooperate to cure or prevent the generation of nearly all [PSI+] and [URE3] prions

[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.

Innate immunity to prions: anti-prion systems turn a tsunami of prions into a slow drip

The yeast prions (infectious proteins) [URE3] and [PSI+] are essentially non-functional (or even toxic) amyloid forms of Ure2p and Sup35p, whose normal function is in nitrogen catabolite repression and translation termination, respectively. Yeast has an array of systems working in normal cells that largely block infection with prions, block most prion formation, cure most nascent prions and mitigate the toxic effects of those prions that escape the first three types of systems. Here we review recent progress in defining these anti-prion systems, how they work and how they are regulated. Polymorphisms of the prion domains partially block infection with prions. Ribosome-associated chaperones ensure proper folding of nascent proteins, thus reducing [PSI+] prion formation and curing many [PSI+] variants that do form. Btn2p is a sequestering protein which gathers [URE3] amyloid filaments to one place in the cells so that the prion is often lost by progeny cells. Proteasome impairment produces massive overexpression of Btn2p and paralog Cur1p, resulting in [URE3] curing. Inversely, increased proteasome activity, by derepression of proteasome component gene transcription or by 60S ribosomal subunit gene mutation, prevents prion curing by Btn2p or Cur1p. The nonsense-mediated decay proteins (Upf1,2,3) cure many nascent [PSI+] variants by associating with Sup35p directly. Normal levels of the disaggregating chaperone Hsp104 can also cure many [PSI+] prion variants. By keeping the cellular levels of certain inositol polyphosphates / pyrophosphates low, Siw14p cures certain [PSI+] variants. It is hoped that exploration of the yeast innate immunity to prions will lead to discovery of similar systems in humans.

Mutable yeast prion variants are stabilized by a defective Hsp104 chaperone.

Gorkovskiy et al. observed that many [PSI+ ] prion isolates, obtained in yeast with the mutant Hsp104T160M chaperone, propagate poorly in wild-type cells and suggested that Hsp104 is part of the cellular anti-prion system, curing many nascent [PSI+ ] variants. Here, we argue that the concept may require reassessment. We induced [PSI+ ] variants in both the wild-type and the mutant background. Three new variants were isolated in the T160M background. They exhibited lower thermostability, possessed novel structural features, and were inherently mutable, changing to well-characterized VH, VK, and VL variants in wild-type cells. In contrast, VH, VK, and VL of the wild-type background, could not change freely and were lost in the mutant, due to insufficient chaperone activity. Thus, mutant Hsp104 can impose as much restriction against emerging prion variants as the wild-type protein. Such restriction conserved the transmutable variants in the T160M background, since new structures mis-templated from them could not gain a foothold. We further demonstrate excess Hsp104T160M or Hsp104∆2-147 can eliminate nearly all of the [PSI+ ] variants in their native background. This finding contradicts the generally held belief that Hsp104-induced [PSI+ ] curing requires its N-terminal domain, and may help settling the current contention regarding how excess Hsp104 cures [PSI+ ].

Mutations Outside the Ure2 Amyloid-Forming Region Disrupt [URE3] Prion Propagation and Alter Interactions with Protein Quality Control Factors.

The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise de novo While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation in vitro Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion's amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.

How Do Yeast Cells Contend with Prions?

Infectious proteins (prions) include an array of human (mammalian) and yeast amyloid diseases in which a protein or peptide forms a linear β-sheet-rich filament, at least one functional amyloid prion, and two functional infectious proteins unrelated to amyloid. In Saccharomyces cerevisiae, at least eight anti-prion systems deal with pathogenic amyloid yeast prions by (1) blocking their generation (Ssb1,2, Ssz1, Zuo1), (2) curing most variants as they arise (Btn2, Cur1, Hsp104, Upf1,2,3, Siw14), and (3) limiting the pathogenicity of variants that do arise and propagate (Sis1, Lug1). Known mechanisms include facilitating proper folding of the prion protein (Ssb1,2, Ssz1, Zuo1), producing highly asymmetric segregation of prion filaments in mitosis (Btn2, Hsp104), competing with the amyloid filaments for prion protein monomers (Upf1,2,3), and regulation of levels of inositol polyphosphates (Siw14). It is hoped that the discovery of yeast anti-prion systems and elucidation of their mechanisms will facilitate finding analogous or homologous systems in humans, whose manipulation may be useful in treatment.

Prion Variants of Yeast are Numerous, Mutable, and Segregate on Growth, Affecting Prion Pathogenesis, Transmission Barriers, and Sensitivity to Anti-Prion Systems.

The known amyloid-based prions of Saccharomyces cerevisiae each have multiple heritable forms, called “prion variants” or “prion strains”. These variants, all based on the same prion protein sequence, differ in their biological properties and their detailed amyloid structures, although each of the few examined to date have an in-register parallel folded β sheet architecture. Here, we review the range of biological properties of yeast prion variants, factors affecting their generation and propagation, the interaction of prion variants with each other, the mutability of prions, and their segregation during mitotic growth. After early differentiation between strong and weak stable and unstable variants, the parameters distinguishing the variants has dramatically increased, only occasionally correlating with the strong/weak paradigm. A sensitivity to inter- and intraspecies barriers, anti-prion systems, and chaperone deficiencies or excesses and other factors all have dramatic selective effects on prion variants. Recent studies of anti-prion systems, which cure prions in wild strains, have revealed an enormous array of new variants, normally eliminated as they arise and so not previously studied. This work suggests that defects in the anti-prion systems, analogous to immune deficiencies, may be at the root of some human amyloidoses.

Hermes Transposon Mutagenesis Shows [URE3] Prion Pathology Prevented by a Ubiquitin-Targeting Protein: Evidence for Carbon/Nitrogen Assimilation Cross Talk and a Second Function for Ure2p in Saccharomyces cerevisiae.

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism. While most "variants" of the [URE3] prion are toxic, mild variants that only slightly slow growth are more widely studied. The existence of several antiprion systems suggests that some components may be protecting cells from potential detrimental effects of mild [URE3] variants. Our extensive Hermes transposon mutagenesis showed that disruption of YLR352W dramatically slows the growth of [URE3-1] strains. Ylr352wp is an F-box protein, directing selection of substrates for ubiquitination by a "cullin"-containing E3 ligase. For efficient ubiquitylation, cullin-dependent E3 ubiquitin ligases must be NEDDylated, modified by a ubiquitin-related peptide called NEDD8 (Rub1p in yeast). Indeed, we find that disruption of NEDDylation-related genes RUB1, ULA1, UBA3, and UBC12 is also counterselected in our screen. We find that like ylr352wΔ [URE3] strains, ylr352wΔ ure2Δ strains do not grow on nonfermentable carbon sources. Overexpression of Hap4p, a transcription factor stimulating expression of mitochondrial proteins, or mutation of GLN1, encoding glutamine synthetase, allows growth of ylr352w∆ [URE3] strains on glycerol media. Supplying proline as a nitrogen source shuts off the nitrogen catabolite repression (NCR) function of Ure2p, but does not slow growth of ylr352wΔ strains, suggesting a distinct function of Ure2p in carbon catabolism. Also, gln1 mutations impair NCR, but actually relieve the growth defect of ylr352wΔ [URE3] and ylr352wΔ ure2Δ strains, again showing that loss of NCR is not producing the growth defect and suggesting that Ure2p has another function. YLR352W largely protects cells from the deleterious effects of otherwise mild [URE3] variants or of a ure2 mutation (the latter a rarer event), and we name it LUG1 (lets [URE3]/ure2 grow).

Anti-Prion Systems in Yeast and Inositol Polyphosphates.

The amyloid-based yeast prions are folded in-register parallel β-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties resulting from different self-propagating amyloid conformations. Yeast has several anti-prion systems, acting in normal cells (without protein overexpression or deficiency). Some anti-prion proteins partially block prion formation (Ssb1,2p, ribosome-associated Hsp70s); others cure a large portion of prion variants that arise [Btn2p, Cur1p, Hsp104 (a disaggregase), Siw14p, and Upf1,2,3p, nonsense-mediated decay proteins], and others prevent prion-induced pathology (Sis1p, essential cytoplasmic Hsp40). Study of the anti-prion activity of Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5), led to the discovery that inositol polyphosphates, signal transduction molecules, are involved in [PSI+] prion propagation. Either inositol hexakisphosphate or 5PP-IP4 (or 5PP-IP5) can supply a function that is needed by nearly all [PSI+] variants. Because yeast prions are informative models for mammalian prion diseases and other amyloidoses, detailed examination of the anti-prion systems, some of which have close mammalian homologues, will be important for the development of therapeutic measures.

Prion propagation and inositol polyphosphates.

The [PSI+] prion is a folded in-register parallel β-sheet amyloid (filamentous polymer) of Sup35p, a subunit of the translation termination factor. Our searches for anti-prion systems led to our finding that certain soluble inositol polyphosphates (IPs) are important for the propagation of the [PSI+] prion. The IPs affect a wide range of processes, including mRNA export, telomere length, phosphate and polyphosphate metabolism, energy regulation, transcription and translation. We found that 5-diphosphoinositol tetra(or penta)kisphosphate or inositol hexakisphosphate could support [PSI+] prion propagation, and 1-diphosphoinositol pentakisphosphate appears to inhibit the process.

PSI+] prion propagation is controlled by inositol polyphosphates

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.

Hsp104 disaggregase at normal levels cures many [PSI+] prion variants in a process promoted by Sti1p, Hsp90, and Sis1p

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.

Yeast and Fungal Prions

Yeast and fungal prions are infectious proteins, most being self-propagating amyloids of normally soluble proteins. Their effects range from a very mild detriment to lethal, with specific effects dependent on the prion protein and the specific prion variant ("prion strain"). The prion amyloids of Sup35p, Ure2p, and Rnq1p are in-register, parallel, folded β-sheets, an architecture that naturally suggests a mechanism by which a protein can template its conformation, just as DNA or RNA templates its sequence. Prion propagation is critically affected by an array of chaperone systems, most notably the Hsp104/Hsp70/Hsp40 combination, which is responsible for generating new prion seeds from old filaments. The Btn2/Cur1 antiprion system cures most [URE3] prions that develop, and the Ssb antiprion system blocks [PSI+] generation.

Yeast prions: proteins templating conformation and an anti-prion system.

Most yeast prions (infectious proteins) are amyloids, linear β-sheet-rich polymers of a single protein with the β-strands perpendicular to the long axis of the filament. A single prion protein can form any of many different prion variants, differing in structure and biological properties, but with the same amino acid sequence. The folded in-register parallel β-sheet architecture we have shown for several yeast prions explains how a given prion variant can be propagated stably, how a protein can template its conformation, just as DNA can template its sequence. We have found an anti-prion system that sequesters prion seeds, preventing their even distribution to daughter cells. This system involves Btn2p, Cur1p, and Hsp42, and cures most of the [URE3] variants arising. Btn2p is weakly homologous to mammalian HOOK proteins, which include the aggresome-active HOOK2. While mammalian prions (infectious proteins) are rare, many common human amyloid diseases, such as Alzheimer disease, Parkinson disease, and others, have prion aspects, including infectivity in some cases. Most yeast prions are infectious amyloids, filamentous polymers of a single protein. Work on yeast prions was the first to establish the protein-only (prion) hypothesis, and, now, work on the structure of yeast prion infectious amyloid explains how a prion protein can template its conformation, just as DNA can template its sequence. The recent discovery of a cellular anti-prion system that cures most arising prions of the yeast Ure2 protein offers a possible direction to look for treatments of amyloidoses. The yeast non-chromosomal genetic elements [URE3] and [PSI+] were discovered to be prions of Ure2p and Sup35p, respectively, based on their unusual genetic properties [1], and were demonstrated to be based on amyloids of these proteins [2–4]. There are now at least eight amyloid-based prions of Saccharomyces cerevisiae (reviewed in [5,6]) and one of Podospora anserina [7]. In addition to the PrP-related mammalian prion diseases, recent work indicates that Alzheimer disease and other common human amyloidoses have prion-like aspects, including infectivity (reviewed by [8]). This broadens the importance of detailed studies of yeast prions.

Normal levels of the antiprion proteins Btn2 and Cur1 cure most newly formed [URE3] prion variants.

[URE3] is an amyloid prion of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. Overproduction of Btn2p, involved in late endosome to Golgi protein transport, or its paralog Cur1p, cures [URE3]. Btn2p, in curing, is colocalized with Ure2p in a single locus, suggesting sequestration of Ure2p amyloid filaments. We find that most [URE3] variants generated in a btn2 cur1 double mutant are cured by restoring normal levels of Btn2p and Cur1p, with both proteins needed for efficient curing. The [URE3] variants cured by normal levels of Btn2p and Cur1p all have low seed number, again suggesting a seed sequestration mechanism. Hsp42 overproduction also cures [URE3], and Hsp42p aids Btn2 overproduction curing. Cur1p is needed for Hsp42 overproduction curing of [URE3], but neither Btn2p nor Cur1p is needed for overproduction curing by the other. Although hsp42Δ strains stably propagate [URE3-1], hsp26Δ destabilizes this prion. Thus, Btn2p and Cur1p are antiprion system components at their normal levels, acting with Hsp42. Btn2p is related in sequence to human Hook proteins, involved in aggresome formation and other transport activities.