RESUMO
Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.
Assuntos
Amiloidose/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Peptídeos/metabolismo , Agregação Patológica de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Amiloidose/genética , Humanos , Proteína Huntingtina/análise , Proteína Huntingtina/genética , Doença de Huntington/genética , Mutação , Fatores de Terminação de Peptídeos/análise , Fatores de Terminação de Peptídeos/metabolismo , Peptídeos/análise , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mammalian prions are infectious agents of proteinaceous nature that cause several incurable neurodegenerative diseases. Interspecies transmission of prions is usually impeded or impossible. Barriers in prion transmission are caused by small interspecies differences in the primary structure of prion proteins. The barriers can also depend on the strain (variant) of a transmitted prion. Interspecies barriers were also shown for yeast prions, which define some heritable phenotypes. Yeast prions reproduce all the main traits of prion transmission barriers observed for mammals. This allowed to show that the barrier in prion transmission can be observed even upon copolymerization of two prionogenic proteins. Available data allow elucidation of the mechanisms that impede prion transmission or make it impossible.
Assuntos
Doenças Priônicas/transmissão , Príons/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Glutationa Peroxidase/genética , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos/genética , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Príons/química , Príons/patogenicidade , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , ZoonosesRESUMO
More than 20 human diseases are related to protein misfolding which causes formation of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are called amyloid diseases or amyloidoses. Of them only prion diseases are transmissible. Amyloids of the prion type are described in lower eukaryotes. However, in contrast to mammalian prions, which cause incurable neurodegenerative diseases, prions of lower eukaryotes are related to some non-chromosomally inherited phenotypic traits. Here we summarize the results of studies of prions of the yeast Saccharomyces cerevisiae and of the use of yeast model for investigation of some human amyloidoses, such as prion diseases, Alzheimer's, Parkinson's, and Huntington's diseases.
Assuntos
Amiloidose/metabolismo , Modelos Biológicos , Doenças Neurodegenerativas/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , HumanosRESUMO
Recent data on the use of yeast as a model for studying the molecular basis of prion infection are summarized. In contrast to mammalian prions, which are related to incurable neurodegenerative diseases, yeast prions determine the appearance of non-chromosomally inherited phenotypic traits. Prions of yeast are structurally similar to amyloids of mammals and their replication involves not only growth, but also fragmentation of prion amyloid-like fibrils. In mammals the fragmentation should lead to an increase in infectious titer. The use of yeast for study of the mechanisms of human amyloidoses, development of new anti-prion drugs and search for new proteins with prion properties is described.
Assuntos
Amiloide/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Amiloide/genética , Animais , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Doenças Priônicas/patologia , Príons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl - dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation.
RESUMO
Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA. Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the [PSI+] prion. A screen of a multicopy yeast genomic library for genes that inactivate the [PSI+]-related suppressor phenotype resulted in the isolation of the SSB1 gene. Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the [PSI+] prion.
Assuntos
Proteínas Fúngicas/genética , Chaperonas Moleculares , Chaperonas Moleculares/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animais , Clonagem Molecular , Códon sem Sentido , Genes Fúngicos/genética , Genótipo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Fenótipo , Plasmídeos/genética , Príons/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Supressão GenéticaRESUMO
The yeast cytoplasmically-inherited nonsense suppressor [PSI(+)] determinant is presumed to be a manifestation of the aggregated prion-like state of the Sup35 protein. Overexpression of the Sup35 protein induces generation of [PSI(+)] determinants with various suppressor efficiency and mitotic stabilities. Here, we demonstrate that the relative frequency of appearance of [PSI(+)] with different properties depends on the SUP35 allele used to induce their generation. The difference in properties of [PSI(+)] determinants was preserved after their transmission from one yeast strain to another. This difference correlated with variation in properties of the Sup35 protein. A novel type of prion instability was observed: some [PSI(+)] with weak suppressor efficiency could convert spontaneously into strong suppressor determinants.
Assuntos
Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Supressão Genética/genética , Alelos , Western Blotting , Agregação Celular/genética , Ciclo Celular , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Fatores de Terminação de Peptídeos , Fenótipo , Plasmídeos , Príons/genética , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieRESUMO
The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases , Proteínas Fúngicas/genética , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares , Fatores de Terminação de Peptídeos , Pichia/genética , Pichia/metabolismoRESUMO
The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates.
Assuntos
Proteínas Fúngicas/isolamento & purificação , Pichia/química , Saccharomyces cerevisiae/química , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/análise , Concentração de Íons de Hidrogênio , Micologia/métodos , Pichia/crescimento & desenvolvimento , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C-terminal domain responsible for translation termination and N-terminal extensions of different structure. The N-terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N-terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], 'hybrid' [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a 'species barrier' for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family.
Assuntos
Proteínas Fúngicas/química , Pichia/metabolismo , Príons/química , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Cruzamentos Genéticos , Endopeptidase K , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Microscopia de Fluorescência , Mitose , Dados de Sequência Molecular , Fatores de Terminação de Peptídeos , Pichia/genética , Ploidias , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Supressão GenéticaRESUMO
The data on prions--proteinaceous infectious agents--are briefly summarized. Prions cause several incurable neurodegenerative diseases in mammals, while in lower eukaryotes the prion properties of proteins may be responsible for the inheritance of some phenotypic traits. The novel experimental models for finding and studying proteins with prion properties based on the yeast Saccharomyces cerevisiae and the fungus Podospora anserina are described. The significance of the prion phenomenon for biology and medicine is discussed.
Assuntos
Príons/genética , Príons/patogenicidade , Proteínas de Saccharomyces cerevisiae , Amiloide/química , Animais , Ascomicetos/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Glutationa Peroxidase , Humanos , Mutação , Fatores de Terminação de Peptídeos , Doenças Priônicas/etiologia , Príons/química , Saccharomyces cerevisiae/genéticaRESUMO
The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.
Assuntos
Proteínas Fúngicas/genética , Mutação , Príons/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Cromossomos Fúngicos , Dosagem de Genes , Fatores de Terminação de Peptídeos/genéticaRESUMO
The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.
Assuntos
Proteínas Fúngicas/química , Príons/química , Conformação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Endopeptidases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Modelos Químicos , Fatores de Terminação de Peptídeos , Fenótipo , Proteínas PrPC/química , Proteínas PrPSc/química , Biossíntese de Proteínas , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Solubilidade , Transformação GenéticaRESUMO
The SUP45 and SUP35 genes of Saccharomyces cerevisiae encode polypeptide chain release factors eRF1 and eRF3, respectively. It has been suggested that the Sup35 protein (Sup35p) is subject to a heritable conformational switch, similar to mammalian prions, thus giving rise to the non-Mendelian [PSI+] nonsense suppressor determinant. In a [PSI+] state, Sup35p forms high-molecular-weight aggregates which may inhibit Sup35p activity, leading to the [PSI+] phenotype. Sup35p is composed of the N-terminal domain (N) required for [PSI+] maintenance, the presumably nonfunctional middle region (M), and the C-terminal domain (C) essential for translation termination. In this study, we observed that the N domain, alone or as a part of larger fragments, can form aggregates in [PSI+] cells. Two sites for Sup45p binding were found within Sup35p: one is formed by the N and M domains, and the other is located within the C domain. Similarly to Sup35p, in [PSI+] cells Sup45p was found in aggregates. The aggregation of Sup45p is caused by its binding to Sup35p and was not observed when the aggregated Sup35p fragments did not contain sites for Sup45p binding. The incorporation of Sup45p into the aggregates should inhibit its activity. The N domain of Sup35p, responsible for its aggregation in [PSI+] cells, may thus act as a repressor of another polypeptide chain release factor, Sup45p. This phenomenon represents a novel mechanism of regulation of gene expression at the posttranslational level.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Terminação de Peptídeos , Príons/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Proteínas de Choque Térmico/metabolismo , Peso Molecular , Fenótipo , Saccharomyces cerevisiaeRESUMO
The yeast Saccharomyces cerevisiae SUP35 gene that encodes the Sup35p protein homologous to the translation termination eRF3 factor of higher eukaryotes is essential to replication of the nonchromosomally inherited [psi+] determinant. The nonsense suppressor phenotype of this determinant was assumed to be dependent on a specific conformational state of the Sup35p protein; the transition to this state leads to partial inactivation of this protein. In terms of this hypothesis, the Sup35p protein can, like mammalian prions, induce its own specific conformation via protein-protein interactions in the newly synthesized Sup35p molecules; in this way, inheritance of the [psi+] phenotype is ensured in a series of cell generations. In recent years, this hypothesis has been experimentally verified. Allele substitution of the wild-type SUP35 gene by its chimeric GST-SUP35 version, which encodes the glutathione S-transferase sequence fused with the N end of Sup35p, was shown to cause elimination of the [psi+] determinant. The ability to eliminate [psi+] is a recessive trait, because fusions heterozygous for the GST-SUP35 allele did not lose this trait. Elimination of [psi+] seems to be caused by inability of the chimeric protein to bring about oligomerization. The obtained data indicate that the chimeric protein manifests attenuated terminating activity but can interact with the eRF1 translation termination factor encoded by the SUP45 gene.
Assuntos
Proteínas Fúngicas/metabolismo , Glutationa Transferase/metabolismo , Príons/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Replicação do DNA , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Recessivos , Glutationa Transferase/genética , Heterozigoto , Fatores de Terminação de Peptídeos , Deleção de Sequência , Regiões Terminadoras GenéticasRESUMO
Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRF1), which has a stop-codon-dependent peptidyl-release activity. Using a conditional-lethal mutant allele of SUP45 (sup45-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37 degrees C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRF1 plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.
Assuntos
Proteínas Fúngicas/genética , Genes Fúngicos , Fatores de Terminação de Peptídeos , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , MutaçãoRESUMO
We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to induce the appearance of the non-Mendelian factor [PSI]. This result was later interpreted by others as a crucial piece of evidence for a model postulating that [PSI] is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduction of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSI]. We also show that the "prion-inducing domain" of Sup35p is in the N-terminal region, which, like the "prion-inducing domain" of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.
Assuntos
DNA Fúngico/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Príons/genética , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Terminação de PeptídeosRESUMO
The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes. It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the [psi+] nonsense suppressor determinant, inherited in a non-Mendelian fashion. Here we present data confirming the prion-like nature of [psi+]. We show that Sup35p molecules interact with each other through their N-terminal domains in [psi+], but not [psi-] cells. This interaction is critical for [psi+] propagation, since its disruption leads to a loss of [psi+]. Similarly to mammalian prions, in [psi+] cells Sup35p forms high molecular weight aggregates, accumulating most of this protein. The aggregation inhibits Sup35p activity leading to a [psi+] nonsense-suppressor phenotype. N-terminally altered Sup35p molecules are unable to interact with the [psi+] Sup35p isoform, remain soluble and improve the translation termination in [psi+] strains, thus causing an antisuppressor phenotype. The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the [psi+] strain, also causing an antisuppressor phenotype. We propose that Hsp104p plays a role in establishing stable [psi+] inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.