Sup35 methionine oxidation is a trigger for de novo [PSI(+)] prion formation.
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ABSTRACT:
SUBMITTER: Grant CM
PROVIDER: S-EPMC4601295 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet how amyloids form in vivo is still unclear. Here, we take advantage of the yeast prion, [PSI +], to uncover the early steps of amyloid formation in vivo. [PSI +] is the prion form of the Sup35 protein. While [PSI +] formation is quite rare, the prion can be greatly induced by overexpression of the prion domain of the Sup35 protein. This de novo induction of [PSI +] shows the appearance of fluorescent cytoplasmic rings when the prion domain is fused with GFP. Our current work shows that de novo induction is more complex than previously thought. Using 4D live cell imaging, we observed that fluorescent structures are formed by four different pathways to yield [PSI +] cells. Biochemical analysis of de novo induced cultures indicates that newly formed SDS resistant oligomers change in size over time and lysates made from de novo induced cultures are able to convert [psi -] cells to [PSI +] cells. Taken together, our findings suggest that newly formed prion oligomers are infectious.
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Project description:Peroxiredoxins (Prxs) are ubiquitous antioxidants that protect cells against oxidative stress. We show that the yeast Tsa1/Tsa2 Prxs colocalize to ribosomes and function to protect the Sup35 translation termination factor against oxidative stress-induced formation of its heritable [PSI(+)] prion conformation. In a tsa1 tsa2 [psi(-)] [PIN(+)] strain, the frequency of [PSI(+)] de novo formation is significantly elevated. The Tsa1/Tsa2 Prxs, like other 2-Cys Prxs, have dual activities as peroxidases and chaperones, and we show that the peroxidase activity is required to suppress spontaneous de novo [PSI(+)] prion formation. Molecular oxygen is required for [PSI(+)] prion formation as growth under anaerobic conditions prevents prion formation in the tsa1 tsa2 mutant. Conversely, oxidative stress conditions induced by exposure to hydrogen peroxide elevates the rate of de novo [PSI(+)] prion formation leading to increased suppression of all three termination codons in the tsa1 tsa2 mutant. Altered translational fidelity in [PSI(+)] strains may provide a mechanism that promotes genetic variation and phenotypic diversity (True HL, Lindquist SL (2000) Nature 407:477-483). In agreement, we find that prion formation provides yeast cells with an adaptive advantage under oxidative stress conditions, as elimination of the [PSI(+)] prion from tsa1 tsa2 mutants renders the resulting [psi(-)] [pin(-)] cells hypersensitive to hydrogen peroxide. These data support a model in which Prxs function to protect the ribosomal machinery against oxidative damage, but when these systems become overwhelmed, [PSI(+)] prion formation provides a mechanism for uncovering genetic traits that aid survival during oxidative stress conditions.
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Project description:Prion diseases are characterized by the self-templated misfolding of the cellular prion protein (PrPC) into infectious aggregates (PrPSc). The detailed molecular basis of the misfolding and aggregation of PrPC remains incompletely understood. It is believed that the transient misfolding of PrPC into partially structured intermediates precedes the formation of insoluble protein aggregates and is a critical component of the prion misfolding pathway. A number of environmental factors have been shown to induce the destabilization of PrPC and promote its initial misfolding. Recently, oxidative stress and reactive oxygen species (ROS) have emerged as one possible mechanism by which the destabilization of PrPC can be induced under physiological conditions. Methionine residues are uniquely vulnerable to oxidation by ROS and the formation of methionine sulfoxides leads to the misfolding and subsequent aggregation of PrPC. Here, we provide a review of the evidence for the oxidation of methionine residues in PrPC and its potential role in the formation of pathogenic prion aggregates.
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Project description:Prions are self-propagating, misfolded forms of proteins associated with various neurodegenerative diseases in mammals and heritable traits in yeast. How prions form spontaneously into infectious amyloid-like structures without underlying genetic changes is poorly understood. Previous studies have suggested that methionine oxidation may underlie the switch from a soluble protein to the prion form. In this current study, we have examined the role of methionine sulfoxide reductases (MXRs) in protecting against de novo formation of the yeast [PSI+] prion, which is the amyloid form of the Sup35 translation termination factor. We show that [PSI+] formation is increased during normal and oxidative stress conditions in mutants lacking either one of the yeast MXRs (Mxr1, Mxr2), which protect against methionine oxidation by reducing the two epimers of methionine-S-sulfoxide. We have identified a methionine residue (Met124) in Sup35 that is important for prion formation, confirming that direct Sup35 oxidation causes [PSI+] prion formation. [PSI+] formation was less pronounced in mutants simultaneously lacking both MXR isoenzymes, and we show that the morphology and biophysical properties of protein aggregates are altered in this mutant. Taken together, our data indicate that methionine oxidation triggers spontaneous [PSI+] prion formation, which can be alleviated by methionine sulfoxide reductases.
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Project description: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.
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Project description:Prions are important disease agents and epigenetic regulatory elements. Prion formation involves the structural conversion of proteins from a soluble form into an insoluble amyloid form. In many cases, this structural conversion is driven by a glutamine/asparagine (Q/N)-rich prion-forming domain. However, our understanding of the sequence requirements for prion formation and propagation by Q/N-rich domains has been insufficient for accurate prion propensity prediction or prion domain design. By focusing exclusively on amino acid composition, we have developed a prion aggregation prediction algorithm (PAPA), specifically designed to predict prion propensity of Q/N-rich proteins. Here, we show not only that this algorithm is far more effective than traditional amyloid prediction algorithms at predicting prion propensity of Q/N-rich proteins, but remarkably, also that PAPA is capable of rationally designing protein domains that function as prions in vivo.
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Project description:A prion's pathogenic character is enciphered in its conformation, which also defines the chemical environments of its amino acids. Differences in chemical environments influence the reactivity of amino acid side chains, in a conformation-dependent manner. Chemical oxidation of susceptible methionines would identify those methionines on the surface of a prion, which would reveal conformation-dependent information. We identified a set of methionine-containing peptides derived from the tryptic, chymotryptic, or tryptic/chymotryptic digestion of recombinant prion protein and the Sc237 strain of hamster-adapted scrapie. We developed a multiple reaction monitoring-based method of quantifying the extent of the methionine oxidation in those peptides. This approach can be used to define a prion's conformation and to distinguish among prion strains, which is an important component of food safety.
| S-EPMC8793083 | biostudies-literature
Project description:Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.
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Project description:Differences in the clinical pathology of mammalian prion diseases reflect distinct heritable conformations of aggregated PrP proteins, called prion strains. Here, using the yeast [PSI(+) ] prion, we examine the de novo establishment of prion strains (called variants in yeast). The [PSI(+) ] prion protein, Sup35, is efficiently induced to take on numerous prion variant conformations following transient overexpression of Sup35 in the presence of another prion, e.g. [PIN(+) ]. One hypothesis is that the first [PSI(+) ] prion seed to arise in a cell causes propagation of only that seed's variant, but that different variants could be initiated in different cells. However, we now show that even within a single cell, Sup35 retains the potential to fold into more than one variant type. When individual cells segregating different [PSI(+) ] variants were followed in pedigrees, establishment of a single variant phenotype generally occurred in daughters, granddaughters or great-granddaughters - but in 5% of the pedigrees cells continued to segregate multiple variants indefinitely. The data are consistent with the idea that many newly formed prions go through a maturation phase before they reach a single specific variant conformation. These findings may be relevant to mammalian PrP prion strain establishment and adaptation.
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Project description:Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.
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