Yeast prions [URE3] and [PSI+] are diseases

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises de novo in more than one in 10(6) yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild Saccharomyces strains, we found the [PIN+] prion in 11 strains, but the [URE3] and [PSI+] prions were uniformly absent. In contrast, the "selfish" 2mu DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [PSI+] in wild strains indicates that each prion has a net deleterious effect on its host.     

Interactions among prions and prion "strains" in yeast

Prions are "infectious" proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PSI(+)] prion form its function is compromised. When Rnq1 is aggregated in its [PIN(+)] prion form, it promotes the de novo appearance of [PSI(+)]. Heritable variants (strains) of [PSI(+)] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PIN(+)] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PSI(+)]. Unlike [PSI(+)] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PIN(+)] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PSI(+)], or different [PIN(+)] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PIN(+)] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PSI(+)] appearance, [PIN(+)] enhances the de novo appearance of [URE3]. Surprisingly, [PSI(+)] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.     

Amyloid of Rnq1p, the basis of the [PIN+] prion, has a parallel in-register beta-sheet structure

The [PIN(+)] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI(+)] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1Delta strains have no known phenotype except for inability to propagate the [PIN(+)] prion. We used solid-state NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153-405) labeled with Tyr-1-(13)C (14 residues), Leu-1-(13)C (7 residues), or Ala-3-(13)C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in beta-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were approximately 0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel beta-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3-(13)C indicate that the beta-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN(+)]'s defining characteristic.     

Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro

Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ+], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ+] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, alpha-synuclein, and synphilin do not. Furthermore, transthyretin and alpha-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states.     

Specificity of the J-protein Sis1 in the propagation of 3 yeast prions

Yeast prions, such as [PSI(+)], [RNQ(+)], and [URE3], are heritable elements formed by proteins capable of acquiring self-perpetuating conformations. Their propagation is dependent on fragmentation of the amyloid protein complexes formed to generate the additional seeds necessary for conversion of nascent soluble protein to the prion conformation. We report that, in addition to its known role in [RNQ(+)] propagation, Sis1, a J-protein cochaperone of Hsp70 Ssa, is also specifically required for propagation of [PSI(+)] and [URE3]. Whereas both [RNQ(+)] and [URE3] are cured rapidly upon SIS1 repression, [PSI(+)] loss is markedly slower. This disparity cannot be explained simply by differences in seed number, as [RNQ(+)] and [PSI(+)] are lost with similar kinetics upon inhibition of Hsp104, a remodeling protein required for propagation of all yeast prions. Rather, in the case of [PSI(+)], our results are consistent with the partial impairment, rather than the complete abolition, of fragmentation of prion complexes upon Sis1 depletion. We suggest that a common set of molecular chaperones, the J-protein Sis1, the Hsp70 Ssa, and the AAA+ ATPase Hsp104, act sequentially in the fragmentation of all yeast prions, but that the threshold of Sis1 activity required for each prion varies.     

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.The yeast prion [URE3] is a self-propagating amyloid of Ure2p (1–4). Ure2p normally functions as a soluble regulator of nitrogen catabolism (5, 6), and its conversion to the aggregated amyloid prion form produces inappropriate derepression of many genes of nitrogen catabolism, resulting in slightly slowed growth (7). Many [URE3] isolates have a severe toxic effect, producing extremely slowed growth (8). A single protein sequence can assume any of many different heritable/infectious forms with different biological properties and, one infers, different protein conformations. The parallel in-register β-sheet architecture of the [URE3] prion amyloid (9) can explain the templating properties of this prion (10) and the ability of the Ure2p amyloid to act as a gene with multiple alleles (4).[PSI+] is similarly an amyloid prion of Sup35p, a subunit of the translation termination factor [reviewed by Liebman and Chernoff (11)], and [PIN+] is a prion of Rnq1p, a protein of unknown function (12–14).Eukaryotic cells deal with protein aggregates in several ways, including resolubilization, degradation, sequestration, and combinations of these processes. Several organelles and systems have been recognized to play a role in these processes, including vacuoles (the yeast equivalent of the mammalian lysosomes), the ubiquitin–proteasome systems, the autophagy system, and the various chaperones.In mammalian cells the aggresome is a centrosomal structure to which aggregates are brought in a microtubule-dependent process (15). A yeast site near the spindle pole body (the yeast centrosome) collects huntingtin/polyQ aggregates in a process that depends on microtubule function, suggesting that this is the yeast aggresome (16). Bmh1p, a 14-3-3 protein, was found associated with huntingtin in yeast aggresomes, and bmh1Δ prevented aggresome accumulation of huntingtin (16).Btn2p and Cur1p were found to cure the [URE3] prion when overproduced (17). These proteins are paralogs, members of the Hook family, consistent with their similar effects, but Btn2 localized to a site next to the nucleus, whereas Cur1 was localized largely within the nucleus (17). During the curing process, those [URE3] cells having both Ure2p-GFP aggregates and Btn2-RFP dots show striking colocalization (17–19). Partial colocalization of Btn2-RFP with Sup35NM-GFP in a [PSI+] strain or with the huntingtin-like Q103-GFP were also observed (17), but overexpression of Btn2p or Cur1p did not cure [PSI+]. Doubly deleted btn2Δ cur1Δ strains show an elevated seed number of [URE3] and partial resistance to prion curing by dimethyl sulfoxide, overexpression of Ssa1p, overexpression of Hsp104, and other agents (17). These results suggested that Btn2p and Cur1p expressed at their normal levels affect [URE3] propagation. It was suggested that at least Btn2p collects Ure2p amyloid aggregates at the Btn2 site near the nucleus, thereby decreasing the operative seed number and destabilizing the prion (17).Another study described two morphological sites of accumulation of nonspecific aggregates, one near the nucleus (JUNQ) and the other peripheral to the nucleus (IPOD), neither localized at the spindle pole body (20). Ubiquitinated proteins accumulated at the juxtanuclear site, whereas aggregates of overproduced “amyloidogenic” prion proteins Ure2p and Rnq1p, in strains not carrying prions, were reported to be moved to the peripheral site (20). Prion formation by Ure2p is induced 20- to 200-fold by overproduction of the prion protein (2), but still only a relatively small minority of cells become [URE3], although most cells may have aggregates of Ure2p while it is overproduced. The same is true of induction of [PSI+], a prion of Sup35p (2, 21, 22) and of [PIN+], a prion of Rnq1p (14). Thus, aggregates of overproduced Ure2p or Rnq1p in cells not carrying the corresponding prion will be largely nonprion aggregates, and there is no reason to assume that they are amyloid, even though those proteins are indeed “amyloidogenic.” The formation of both IPOD and JUNQ foci were inhibited by benomyl (20), a drug that depolymerizes microtubules.Specht et al. (23) found that Hsp42 was necessary for aggregate accumulation at the peripheral site, but not at the juxtanuclear site. They found that Hsp42 did not affect the localization of “amyloidogenic” aggregates of Rnq1p, but again, the cells were not carrying the corresponding prion, [PIN+], so these aggregates were likely nonamyloid. Specht et al. found that although benomyl inhibited formation of both peripheral and juxtanuclear aggregate foci, a benomyl-resistant tubulin2 mutant showed the same effect. In contrast, LatrunculinA, which depolymerizes the actin cytoskeleton, inhibited accumulation of aggregates at both sites, but a mutation in actin-1 that prevents LatrunculinA action prevented this action of the drug (23). Thus, the actin cytoskeleton is invovled in IPOD and JUNQ focus formation, distinguishing both from the aggresome. Overproduction of either Hsp26 or Hsp42 were reported to cure the [PSI+] prion (24), but hsp26Δ did not affect IPOD or JUNQ aggregate focus formation (23). Hsp42p associates with Btn2p in vivo (25).The prion-curing ability of overproduced Btn2p and Cur1p was confirmed by Malinovska et al. using an artificial prion (25). As in other studies cited above, this group overproduced the RFP-labeled prion domains of Ure2, Sup35, Rnq1, or Nrp1 in a strain carrying none of the corresponding prions. There was no significant colocalization with Btn2-GFP, from which it was inferred that Btn2p was not directly associating with prion amyloid in the curing process (25). As discussed above, only a tiny minority of cells overexpressing these prion domains form prions, although most cells show aggregates of the overproduced protein, so this inference is not justified. Indeed, Btn2p does colocalize with amyloid aggregates of Ure2p in a [URE3] strain early in the curing process (17–19). Btn2p is also partially colocalized with amyloid aggregates of Sup35p in a [PSI+] strain (17). Btn2p also colocalizes with nonamyloid aggregates of optineurin, PrP, Rnq1p, and Q103P (19). Malinovska et al. further showed that Btn2p and Cur1p each interact physically with the Hsp40 family member Sis1p, and that both Btn2p and Cur1p are drawn into the nucleus as a result of this association (25). It was suggested that overproduced Btn2p or Cur1p cure prions by sequestering the Sis1p that is needed for prion propagation (25).Here we show that normal levels of Btn2p and Cur1p can cure most [URE3] variants despite being 20–400 times less abundant in the cell than Sis1p. [URE3] variants cured by normal levels of Btn2 and Cur1 are much lower in copy number than those not cured under this condition, suggesting that sequestration of seeds is the mechanism of prion curing. Btn2p and Cur1p are thus elements of normal prion-curing systems, suggesting that yeast does not welcome [URE3] but has systems in place to rapidly eliminate it. We find that Hsp42 is important for curing by overproduction of Btn2p or Cur1p and that overproduction of Hsp42 itself cures [URE3] in a process requiring Cur1p, but not Btn2p.     

Chaperone-dependent amyloid assembly protects cells from prion toxicity

Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed.     

A prion of yeast metacaspase homolog (Mca1p) detected by a genetic screen

Saccharomyces cerevisiae can be infected with four amyloid-based prions: [URE3], [PSI⁺], [PIN⁺], and [SWI⁺], due to self-propagating aggregation of Ure2p, Sup35p, Rnq1p and Swi1p, 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 Mca1 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 Mca1p based on genetic, cytological and biochemical tests.     

The [URE3] prion is an aggregated form of Ure2p that can be cured by overexpression of Ure2p fragments

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p^1–65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p–green fluorescent protein (GFP) or a Ure2p^1–65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p^1–65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] “crystal” of Ure2p poisons its propagation.     

The prion model for [URE3] of yeast: Spontaneous generation and requirements for propagation

The genetic properties of the non-Mendelian element, [URE3], suggest that it is a prion (infectious protein) form of Ure2p, a mediator of nitrogen regulation in Saccharomyces cerevisiae. Into a ure2Δ strain (necessarily lacking [URE3]), we introduced a plasmid overproducing Ure2p. This induced the frequent “spontaneous generation” of [URE3], with properties identical to the original [URE3]. Altering the translational frame only in the prion-inducing domain of URE2 shows that it is Ure2 protein (and not URE2 RNA) that induces appearance of [URE3]. The proteinase K-resistance of Ure2p is unique to [URE3] strains and is not seen in nitrogen regulation of normal strains. The prion-inducing domain of Ure2p (residues 1–65) can propagate [URE3] in the absence of the C-terminal part of the molecule. In contrast, the C-terminal part of Ure2p cannot be converted to the prion (inactive) form without the prion-inducing domain covalently attached. These experiments support the prion model for [URE3] and extend our understanding of its propagation.     

A G-protein γ subunit mimic is a general antagonist of prion propagation in Saccharomyces cerevisiae

The Gpg1 protein is a Gγ subunit mimic implicated in the G-protein glucose-signaling pathway in Saccharomyces cerevisiae, and its function is largely unknown. Here we report that Gpg1 blocks the maintenance of [PSI⁺], an aggregated prion form of the translation termination factor Sup35. Although the GPG1 gene is normally not expressed, over-expression of GPG1 inhibits propagation of not only [PSI⁺] but also [PIN⁺], [URE3] prions, and the toxic polyglutamine aggregate in S. cerevisiae. Over-expression of Gpg1 does not affect expression and activity of Hsp104, a protein-remodeling factor required for prion propagation, showing that Gpg1 does not target Hsp104 directly. Nevertheless, prion elimination by Gpg1 is weakened by over-expression of Hsp104. Importantly, Gpg1 protein is prone to self-aggregate and transiently colocalized with Sup35NM-prion aggregates when expressed in [PSI⁺] cells. Genetic selection and characterization of loss-of-activity gpg1 mutations revealed that multiple mutations on the hydrophobic one-side surface of predicted α-helices of the Gpg1 protein hampered the activity. Prion elimination by Gpg1 is unaffected in the gpa2Δ and gpb1Δ strains lacking the supposed physiological G-protein partners of Gpg1. These findings suggest a general inhibitory interaction of the Gpg1 protein with other transmissible and nontransmissible amyloids, resulting in prion elimination. Assuming the ability of Gpg1 to form G-protein heterotrimeric complexes, Gpg1 is likely to play a versatile function of reversing the prion state and modulating the G-protein signaling pathway.     

Primary sequence independence for prion formation

Many proteins can adopt self-propagating beta-sheet-rich structures, termed amyloid fibrils. The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are infectious amyloid forms of the proteins Ure2p and Sup35p, respectively. Ure2p forms prions primarily as a result of its sequence composition, as versions of Ure2p with the prion domain amino acids shuffled are still able to form prions. Here we show that prion induction by both Ure2p and Ure2-21p, one of the scrambled versions of Ure2p, is clearly dependent on the length of the inducing fragment. For Ure2-21p, no single sequence is found in all of the inducing fragments, highlighting the sequence independence of prion formation. Furthermore, the sequence of the Sup35p prion domain can also be randomized without blocking prion formation. Indeed, a single shuffled sequence could give rise to several prion variants. These results suggest that [PSI+] formation is driven primarily by the amino acid composition of the Sup35p prion domain, and that the Sup35p oligopeptide repeats are not required for prion maintenance.     

A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1Delta strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2(Val19) increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.     

Hsp40/JDP Requirements for the Propagation of Synthetic Yeast Prions

Yeast prions are protein-based transmissible elements, most of which are amyloids. The chaperone protein network in yeast is inexorably linked to the spreading of prions during cell division by fragmentation of amyloid prion aggregates. Specifically, the core “prion fragmentation machinery” includes the proteins Hsp104, Hsp70 and the Hsp40/J-domain protein (JDP) Sis1. Numerous novel amyloid-forming proteins have been created and examined in the yeast system and occasionally these amyloids are also capable of continuous Hsp104-dependent propagation in cell populations, forming synthetic prions. However, additional chaperone requirements, if any, have not been determined. Here, we report the first instances of a JDP-Hsp70 system requirement for the propagation of synthetic prions. We utilized constructs from a system of engineered prions with prion-forming domains (PrDs) consisting of a polyQ stretch interrupted by a single heterologous amino acid interspersed every fifth residue. These “polyQX” PrDs are fused to the MC domains of Sup35, creating chimeric proteins of which a subset forms synthetic prions in yeast. For four of these prions, we show that SIS1 repression causes prion loss in a manner consistent with Sis1′s known role in prion fragmentation. PolyQX prions were sensitive to Sis1 expression levels to differing degrees, congruent with the variability observed among native prions. Our results expand the scope known Sis1 functionality, demonstrating that Sis1 acts on amyloids broadly, rather than through specific protein–protein interactions with individual yeast prion-forming proteins.     

Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c^−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c^−/− mice.     

Locating folds of the in-register parallel β-sheet of the Sup35p prion domain infectious amyloid

The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae. The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1-¹³C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.Prions are infectious proteins, mostly self-propagating amyloids. Amyloid is a filamentous polymer, rich in β-sheet structure, in which the β-strands run perpendicular to the long axis of the filament and the hydrogen bonds joining β-strands to make a sheet are along the long axis of the filament. In mammals, prions are uniformly lethal diseases, caused by amyloid formation of the PrP protein. In yeast and fungi, prions are not uniformly fatal and have widely varying effects (reviewed in ref. 1). Perhaps the most remarkable feature of prions is that they have strains or variants, distinct self-propagating forms of the same protein, analogous to alleles of a gene, each relatively stably propagated. The existence of different self-propagating prion variants implies an array of self-propagating structures, each based on the same protein sequence. Because each prion variant is self-propagating, and each variant represents a different amyloid structure/conformation, there must be some mechanism by which the protein can template its conformation. This mechanism must operate for each of the many conformations that are possible for a given prion.An amino acid residue in a β-sheet can have interactions in three dimensions (Fig. 1A): (a) along the peptide chain; (b) with the residues it faces within the β-sheet but perpendicular to the peptide chain, including the residues to which its main chain N–H and >C = O are hydrogen bonded; and (c) with residues located in the direction perpendicular to the β-sheet, possibly another β-sheet, interacting with the distal part of the side chain. The distance between residues having the relation (b), between adjacent β-strands in the β-sheet, is generally 0.47–0.48 nm. The distance between adjacent sheets, as in (c), is about 1.0 nm.Open in a separate windowFig. 1.Amyloid structure. (A) An amino acid residue in amyloid has interactions in three directions: (a) along the peptide chain, (b) between aligned residues in the β-sheet along the fibril axis but perpendicular to the peptide chain (∼0.48 nm distant), and (c) with residues in an adjacent β-sheet (∼1.0 nm distant). Based on a model of Aβ amyloid (4). (B) Types of β-sheet. The dots represent a single ¹³C-labeled atom in each protein molecule. The expected separation is ∼0.48 nm for in-register parallel, ∼0.94 nm for antiparallel, and >0.9 nm for β-helix. (C) Amyloid fibers of Ile-1-¹³C–labeled Sup35NM with mutations Y13I, I152L, I220L, and I239L.Given that the filament is a regular/repeating structure, the molecules in a filament can have any of several architectures (Fig. 1B). Most β-sheets in soluble enzymes are antiparallel, with adjacent β-strands running in opposite directions (Fig. 1B). Labeling a single atom in each molecule and measuring the distance between nearest neighbors will usually produce a distance of >0.9 nm (the distance between strands in a β-sheet is ∼0.47 nm) for an antiparallel β-sheet. A parallel β-sheet can be in-register or out-of-register. In-register means that each residue in one molecule is aligned with the same residue in the neighboring molecule, producing lines of identical residues along the long axis of the filament. Labeling a single atom in each molecule will give a distance between labels of ∼0.48 nm if the filament has the parallel in-register β-sheet architecture (Fig. 1B). For an out-of-register β-sheet, the distance will be greater, depending on the register shift (Fig. 1B). In a β-helix, the distance between single labels will be >0.9 nm if each molecule constitutes two turns of the helix, and even greater if each molecule comprises more than two turns (Fig. 1B).The first demonstration of an in-register parallel amyloid structure was by Benzinger et al. studying the 10–35 fragment of the Aβ peptide, and established the approach for proving this architecture (2). The peptide was synthesized with a single ¹³C atom as the carbonyl carbon of one residue. Amyloid was assembled from each such synthetic peptide, and the distance between labels (necessarily in different molecules because there was only one label per molecule) was measured by solid-state NMR using dipolar recoupling. The distance between labeled carbonyl carbons was ∼0.5 nm regardless of which residue was labeled, establishing the in-register parallel architecture for this peptide.The full-size Aβ is a peptide of 40–42 residues. Tycko’s group used a similar approach to show that amyloid of Aβ40 has an in-register parallel β-sheet architecture, and long range intramolecular cross-peaks showed that the sheet was folded along the long axis of the filaments (refs. 3 and 4, reviewed in ref. 5). Moreover, filament mass per length data implied that these folded sheets were associated to form filaments with either two or three molecules per layer (6, 7). Different filament formation conditions can also produce minor differences in the conformation of the monomer (8–10), but seeding can fix the amyloid form (6). Notably, seeding recombinant Aβ peptide with brain material of different Alzheimer’s disease patients produced different amyloid forms, with the same form produced by seeds from different parts of the brain of a single patient (11). This suggests that there may be different forms of Alzheimer’s disease based on different self-propagating amyloid variants, a notion now supported by animal studies (12).Most other pathologic human amyloids have been found to have the in-register parallel β-sheet architecture. Amylin/IAPP (type 2 diabetes), alpha-synuclein (Parkinson’s disease), and β2-microglobulin (dialysis-induced amyloidosis) all follow this pattern (13, 14). An 11-residue peptide from transthyretin also forms an in-register parallel β-sheet structure (15), but full-length transthyretin (senile systemic amyloidosis) breaks the rule in that amyloid formed in vitro does not have this architecture (16, 17).The [Het-s] prion of Podospora anserina is responsible for a heterokaryon incompatibility reaction, a phenomenon widespread in filamentous fungi that limits cell–cell fusion to the closest relatives to block the spread of fungal viruses (18, 19). Infectious amyloid formed by the prion domain of the HET-s prion protein has a β-helix structure, with two helical turns per molecule and three β-sheets forming the sides of the coil (20, 21). There is only a single prion variant of [Het-s] and the amyloid forms an unique structure in vitro with very narrow peaks in 2D solid-state NMR experiments, enabling complete assignments and structural determination (20, 21).The yeast prions [URE3], [PSI+], and [PIN+] (22, 23) are amyloids of Ure2p, Sup35p, and Rnq1p, respectively, with a restricted region (the prion domain) of each responsible for both the prion properties and the amyloid structure (24–33). Amyloid of the Ure2p prion domain (residues 1–89) was shown by solid-state NMR to be in-register parallel using preparations with [¹³C]carbonyl labeling of either the two Leu or the four Val residues or [¹³C]methyl labeling of the single Ala residue (34). These residues are scattered through the prion domain, and the result with the single Ala residue implies that the nearest neighbor distance measured was an intermolecular distance. This result was confirmed by solid-state NMR studies of full-length Ure2p labeled with Ile (three residues in the prion domain) (35) and by electron spin resonance (36).Sup35p consists of a Q/N-rich N-terminal domain (N, residues 1–123), a highly charged middle domain (M, residues 124–253), and the C-terminal domain essential for translation termination (C, residues 254–685). The Sup35p prion domain includes the N domain (residues 1–123) and an unknown part of the M domain, depending on how “prion domain” is defined. Residues 1–61 fused to GFP are sufficient, in amyloid form, to transmit several different prion variants by protein transformation (31), but some variants require up to residue 137 (37) and residues in the M domain are part of the barrier to [PSI+] prion transmission between wild strains of Saccharomyces cerevisiae (38). H–D exchange experiments indicate stable structures in the M domain in the amyloid of some [PSI+] variants (39). Of the eight Leu residues (seven of which are in M), solution NMR suggests that only four are fully unstructured (39). In agreement, solid-state NMR experiments suggested that four of the eight Leu residues are in parallel in-register structures (40).We previously showed that amyloid of Sup35NM having all Tyr carbonyl carbons labeled with ¹³C showed signal decay rates in dipolar recoupling experiments indicative of nearest neighbor ¹³C at a distance of ∼0.5 nm (40, 41). Dilution of this Tyr-labeled sample with unlabeled Sup35NM and then formation of amyloid resulted in a dramatic increase in the nearest neighbor distance, exactly as predicted if the nearest neighbor were on another molecule. We inferred that the Sup35NM amyloid has an in-register parallel architecture, and data with Sup35NM amyloid labeled with Phe-1-¹³C, Leu-1-¹³C or Ala-3-¹³C were consistent with this conclusion. However, one could imagine that although the nearest neighbor of a given Tyr was on another molecule, it was not the same Tyr. There are 20 Tyr residues in Sup35N (and none in Sup35M), making this a real possibility. We therefore sought a definitive proof of the in-register parallel architecture of Sup35NM. In addition, there continues to be a view that Sup35NM amyloid has a β-helix architecture (42–45), and a more rigorous test was therefore important to resolve this central issue.     

Strain-specific sequences required for yeast [PSI+] prion propagation

Amyloid polymorphism underlies the prion strain phenomenon where a single protein polypeptide adopts different chain-folding patterns to form self-propagating cross-β structures. Three strains of the yeast prion [PSI], namely [VH], [VK], and [VL], have been previously characterized and are amyloid conformers of the yeast translation termination factor Sup35. Here we define specific sequences of the Sup35 protein that are necessary for in vivo propagation of each of these prion strains. By sequential substitution of residues 5–55 of Sup35 by proline and insertion of glycine at alternate sites in this segment, specific mutations have been identified that interfere selectively with the propagation of each of the three prion strains in yeast: the [VH] strain requires amino acid residues 7–21; [VK] requires residues 9–37; and [VL] requires residues 5 to at least 52. Minimal polypeptide segments capable of encoding prion conformations were defined by assembly of recombinant Sup35 fragments on purified prion nuclei to form amyloid fibers in vitro, whose infectivity was assayed in yeast. For the [VK] and [VL] strains, the minimal fragments approximately coincide with the strain-specific sequences defined by mutations of the N-terminal portion of the intact Sup35 (1–685); and for the [VH] strain, a longer Sup (1–53) fragment is required. Polymorphic structures of other amyloids might similarly involve different stretches of polypeptides to form cross-β amyloid cores with distinct molecular recognition surfaces.     

Propagation of prions causing synucleinopathies in cultured cells

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing self-propagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.James Parkinson first described a progressive deterioration of the nervous system in 1817 and called it “shaking palsy” (1). Almost one century later, Friederich Heinrich Lewy described the neuropathological hallmark now known as Lewy bodies (LBs) (2). Progress toward discerning the etiology of Parkinson’s disease (PD) was achieved 85 years later when the first of several studies identified mutations in or multiplications of the gene encoding α-synuclein, SNCA, in inherited cases of PD (3–5). These studies were corroborated by immunostaining for α-synuclein in brain sections from PD patients (6) and subsequently from dementia with Lewy bodies (DLB) cases (7, 8), which found that LBs are surrounded by a halo of α-synuclein polymers.Along with point mutations in SNCA (3), and duplication and triplication of the gene (4, 5) as causes of inherited PD, meta-analysis of genome-wide association studies (9) have identified common variations in SNCA as a risk factor for sporadic PD cases. Combined, these data strongly support an etiological role for α-synuclein in the pathogenesis of both the inherited and sporadic forms of PD.In 1998, brain sections from cases classified as multiple system atrophy (MSA) were analyzed for α-synuclein. Although no LBs were found, abundant immunostaining in the cytoplasm of glial cells was identified (8, 10, 11). A decade earlier, these large immunopositive deposits of α-synuclein were called glial cytoplasmic inclusions (GCIs) based on silver staining (12); they are primarily found in oligodendrocytes but have been occasionally observed in astrocytes and neurons. Limited ultrastructural studies performed on GCIs suggest that they are collections of poorly organized bundles of α-synuclein fibrils (8).In addition to the accumulation of α-synuclein into LBs in PD and GCIs in MSA, depigmentation of the substantia nigra pars compacta is a hallmark of both PD and the majority of MSA cases (13). This loss of dopaminergic neurons results in diminished input to the basal ganglia that is reflected in the motor deficits exhibited by patients. In the 1990s, fetal tissue transplants into the substantia nigra of PD patients were performed in an attempt to counteract the effects of dopamine loss. Strikingly, upon autopsy of patients that survived at least 10 years posttransplant, LBs were found in the grafted fetal tissue. Because these grafts were no more than 16 years old, the findings argued for host-to-graft transmission of LBs (14, 15). The results of these transplant studies offered evidence supporting the hypothesis that PD is a prion disease, characterized by a misfolded protein that self-propagates and gives rise to progressive neurodegeneration (16, 17). Additional support for this hypothesis came from studies on the spread of α-synuclein deposits from the substantia nigra to other regions of the CNS in PD patients (18).Even more convincing support for α-synuclein prions came from animal studies demonstrating the transmissibility of an experimental synucleinopathy. The first report used transgenic (Tg) mice expressing human α-synuclein containing the A53T mutation found in familial PD; the mice were designated TgM83 (19). Homozygous mice (TgM83^+/+) were found to develop spontaneous motor deficits along with increased amounts of insoluble phosphorylated α-synuclein throughout the brain between 8–16 months of age. Ten years later, Mougenot et al. (20) intracerebrally inoculated brain homogenates from sick TgM83^+/+ mice into ∼2-months-old TgM83^+/+ mice and found a substantial reduction in the survival time with incubation periods of ∼130 days. Similar observations were reported from two other groups using either homozygous TgM83^+/+ (21) or hemizygous TgM83^+/− (22) mice.Although our initial attempts to transmit PD to TgM83^+/− mice failed (23), the transmission of MSA to the same mouse line was the first demonstration of α-synuclein prions in human brain (22). The TgM83^+/− mice, which differ from their homozygous counterparts by not developing spontaneous disease, exhibited progressive CNS dysfunction ∼120 days following intrathalamic inoculation of brain homogenates from two MSA patients. Inoculation of brain fractions enriched for LBs from PD patients into wild-type (WT) mice and macaque monkeys induced aberrant α-synuclein deposits, but neither species developed neurological disease (24). In a similar approach, inoculation of WT mice with the insoluble protein fraction isolated from DLB patients also induced phosphorylated α-synuclein pathology after 15 months, but it failed to induce neurological disease characteristic of DLB (25).Because α-synuclein prions from MSA patients were transmissible to TgM83^+/− mice, we asked whether a more rapid cell-based bioassay could be developed to characterize the MSA prions. With the cell bioassay for progressive supranuclear palsy (PSP) in mind (26, 27), we began by constructing WT and mutant α-synuclein cDNAs fused to yellow fluorescent protein (YFP) (28–30) and expressed these in human embryonic kidney (HEK) cells. By testing the cells with full-length recombinant mutant human α-syn140*A53T fibrils, we induced aggregate formation in HEK cells expressing WT and mutant human SNCA transgenes. To expand these findings beyond synthetic prions and to examine natural prions, we report here that phosphotungstic acid (PTA) (31) can be used to selectively precipitate α-synuclein from MSA patients. Screening PTA-precipitated brain homogenate with our cellular bioassay, we detected MSA prions in all six of the cases examined. By measuring the distribution of prions in the substantia nigra, basal ganglia, cerebellum, and temporal gyrus, we found evidence to suggest that at least three different strains of α-synuclein prions may give rise to MSA. We also found that after enrichment by PTA precipitation, ∼6 million α-synuclein molecules comprised an infectious unit of MSA prions in cell culture. Importantly, we transmitted neurodegenerative disease to TgM83^+/− mice using PTA-precipitated brain homogenate from an MSA patient, confirming that the aggregate isolation methods used successfully purify prions from patient samples.