RESUMEN
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.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Priones/genética , Priones/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Mutación , Amiloide/genética , Amiloide/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Proteínas Fúngicas/metabolismo , Mamíferos/metabolismo , Factores de Empalme de ARN/genética , Proteínas Nucleares/metabolismo , Enzimas Reparadoras del ADN/genéticaRESUMEN
[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 â¼5,000-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.
Asunto(s)
Amiloidosis , Priones , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas Amiloidogénicas , Proteínas de Choque Térmico/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Factores de Terminación de Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Resistencia a la Enfermedad/inmunología , Susceptibilidad a Enfermedades , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Enfermedades por Prión/etiología , Priones/inmunología , Amiloide/química , Amiloide/inmunología , Amiloide/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/inmunología , Proteínas Amiloidogénicas/metabolismo , Animales , Autofagia , Susceptibilidad a Enfermedades/inmunología , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Chaperonas Moleculares/metabolismo , Mutación , Degradación de ARNm Mediada por Codón sin Sentido , Enfermedades por Prión/metabolismo , Priones/química , Priones/genética , Priones/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Ribosomas/metabolismoRESUMEN
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, complexed with 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.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido/genética , Priones/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Codón sin Sentido , Regulación Fúngica de la Expresión Génica , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Priones/genética , Biosíntesis de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Yeast prions have become important models for the study of the basic mechanisms underlying human amyloid diseases. Yeast prions are pathogenic (unlike the [Het-s] prion of Podospora anserina), and most are amyloid-based with the same in-register parallel ß-sheet architecture as most of the disease-causing human amyloids studied. Normal yeast cells eliminate the large majority of prion variants arising, and several anti-prion/anti-amyloid systems that eliminate them have been identified. It is likely that mammalian cells also have anti-amyloid systems, which may be useful in the same way humoral, cellular, and innate immune systems are used to treat or prevent bacterial and viral infections.
Asunto(s)
Priones/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , HumanosRESUMEN
The yeast prions [PSI+] and [URE3] are folded in-register parallel ß-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 ß 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.
Asunto(s)
Inositol/metabolismo , Polifosfatos/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Priones/genética , Biosíntesis de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/metabolismo , Priones/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Infectious proteins (prions) include an array of human (mammalian) and yeast amyloid diseases in which a protein or peptide forms a linear ß-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.
Asunto(s)
Priones/genética , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Animales , Evolución Molecular , Genes Fúngicos , Variación Genética , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Priones/antagonistas & inhibidores , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
I retrace my path from math to medicine to biochemistry to yeast genetics, my focus on infectious diseases of yeast and finally prions. My discovery of yeast prions relied on my particular focus on the logical relations of non-chromosomal genetic elements and the chromosomal genes involved in their propagation and expression. Pursuing an understanding of yeast prions involved structural biology based on genetics, solid-state NMR, population genetics and more genetics.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Priones/química , Priones/metabolismo , Pliegue de Proteína , Levaduras/genética , Levaduras/metabolismo , Genética Microbiana/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Biología Molecular/tendenciasRESUMEN
The amyloid-based yeast prions are folded in-register parallel ß-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.
Asunto(s)
Inositol/metabolismo , Polifosfatos/metabolismo , Priones/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Glutatión Peroxidasa/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The [PSI+] prion is a folded in-register parallel ß-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.
Asunto(s)
Inositol/química , Polifosfatos/metabolismo , Priones/genética , Metabolismo Energético , Polifosfatos/química , Biosíntesis de Proteínas , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero , Transcripción GenéticaRESUMEN
Prions, infectious proteins, can transmit diseases or be the basis of heritable traits (or both), mostly based on amyloid forms of the prion protein. A single protein sequence can be the basis for many prion strains/variants, with different biological properties based on different amyloid conformations, each rather stably propagating. Prions are unique in that evolution and selection work at both the level of the chromosomal gene encoding the protein, and on the prion itself selecting prion variants. Here, we summarize what is known about the evolution of prion proteins, both the genes and the prions themselves. We contrast the one known functional prion, [Het-s] of Podospora anserina, with the known disease prions, the yeast prions [PSI+] and [URE3] and the transmissible spongiform encephalopathies of mammals.
Asunto(s)
Amiloide/genética , Evolución Molecular , Priones/genética , Amiloide/análisis , Amiloide/metabolismo , Animales , Proteínas Fúngicas/análisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Humanos , Factores de Terminación de Péptidos/análisis , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Podospora/química , Podospora/genética , Podospora/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Priones/análisis , Priones/metabolismo , Conformación Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
[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.
Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Glutatión Peroxidasa/genética , Chaperonas Moleculares/metabolismo , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Técnicas de Inactivación de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Plásmidos/genéticaRESUMEN
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-(13)C 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 â¼0.5-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 ß-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.
Asunto(s)
Amiloide/química , Factores de Terminación de Péptidos/química , Priones/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética con Carbono-13 , Datos de Secuencia Molecular , Proteínas Mutantes/química , Estructura Secundaria de Proteína , TransfecciónRESUMEN
[PSI(+)] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion "strains" or "variants" are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [PSI(+)] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI(+)] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI(+)], all indistinguishably "weak" [PSI(+)], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI(+)]. Notably, a "strong [PSI(+)]" can have any of several different transmission efficiency patterns, showing that "strong" versus "weak" is insufficient to indicate prion variant uniformity.
Asunto(s)
Proteínas Amiloidogénicas , Factores de Terminación de Péptidos , Priones , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Alelos , Secuencia de Aminoácidos , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Regulación Fúngica de la Expresión Génica , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , Fenotipo , Polimorfismo Genético , Priones/química , Priones/genética , Priones/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Structures of the infectious form of prion protein (e.g. PrP(Sc) or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP(Sc) retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP(C)), but evidence is accumulating that these helices are absent in PrP(Sc) amyloid. Moreover, recombinant PrP(C) can form amyloid fibrils in vitro that have parallel in-register intermolecular ß-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily ß-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90-231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP(Sc) fibrils, can have stable parallel in-register ß-sheet cores. These simulations revealed that the C-terminal residues â¼124-227 more readily adopt stable tightly packed structures than the N-terminal residues â¼90-123 in the absence of cofactors. Variations in the placement of turns and loops that link the ß-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures.
Asunto(s)
Amiloide/metabolismo , Priones/metabolismo , Espectroscopía de Resonancia Magnética con Carbono-13 , Disulfuros/química , Microscopía Electrónica de Transmisión de Rastreo , Modelos Moleculares , Polisacáridos/química , Priones/química , ProteolisisRESUMEN
Even deadly prions may be widespread in nature if they spread by infection faster than they kill off their hosts. The yeast prions [PSI+] and [URE3] (amyloids of Sup35p and Ure2p) were not found in 70 wild strains, while [PIN+] (amyloid of Rnq1p) was found in â¼16% of the same population. Yeast prion infection occurs only by mating, balancing the detrimental effects of carrying the prion. We estimated the frequency of outcross mating as about 1% of mitotic doublings from the known detriment of carrying the 2-µm DNA plasmid (â¼1%) and its frequency in wild populations (38/70). We also estimated the fraction of total matings that are outcross matings (â¼23-46%) from the fraction of heterozygosity at the highly polymorphic RNQ1 locus (â¼46%). These results show that the detriment of carrying even the mildest forms of [PSI+], [URE3], or [PIN+] is greater than 1%. We find that Rnq1p polymorphisms in wild strains include several premature stop codon alleles that cannot propagate [PIN+] from the reference allele and others with several small deletions and point mutations which show a small transmission barrier. Wild strains carrying [PIN+] are far more likely to be heterozygous at RNQ1 and other loci than are [pin-] strains, probably reflecting its being a sexually transmitted disease. Because sequence differences are known to block prion propagation or ameliorate its pathogenic effects, we hypothesize that polymorphism of RNQ1 was selected to protect cells from detrimental effects of the [PIN+] prion.