The yeast Sup35NM domain propagates as a prion in mammalian cells.
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ABSTRACT:
SUBMITTER: Krammer C
PROVIDER: S-EPMC2626725 | biostudies-literature | 2009 Jan
REPOSITORIES: biostudies-literature
Publications
The yeast Sup35NM domain propagates as a prion in mammalian cells.
Krammer Carmen C Kryndushkin Dmitry D Suhre Michael H MH Kremmer Elisabeth E Hofmann Andreas A Pfeifer Alexander A Scheibel Thomas T Wickner Reed B RB Schätzl Hermann M HM Vorberg Ina I
Proceedings of the National Academy of Sciences of the United States of America 20081229 2
Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained solu ...[more]
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Project description:Simple Summary DnaJB6 is a human cellular protein quality control factor that prevents diverse peptides from forming disease-associated amyloid, a highly ordered fibrous protein aggregate. DnaJB6 protects human cells, animals and yeast from toxicity caused by amyloids composed of Huntington’s-related polyglutamine, Parkinson’s-related α-synuclein, and ALS- associated TDP-43, and it cures yeast of endogenous prions (infectious amyloids). However, structurally different amyloids of one and the same prion protein, and prions composed of them, can be insensitive to anti-amyloid activity of DnaJB6. This limitation raises concerns about developing DnaJB6 as a therapeutic for amyloid diseases. Prions showing this insensitivity are highly toxic to yeast with reduced function of Sis1, a yeast protein related to DnaJB6. To begin assessing how DnaJB6 might act on amyloid in cells we tested if DnaJB6 could protect yeast from this toxicity. Although it does not eliminate the prion, it protects cells from prion toxicity, but differently than Sis1. Our work provides insight into how human DnaJB6 counteracts cellular toxicity caused by amyloid and establishes an in vivo genetic system useful for studying DnaJB6-amyloid interactions to decipher its mechanisms of action. Abstract Human J-domain protein (JDP) DnaJB6 has a broad and potent activity that prevents formation of amyloid by polypeptides such as polyglutamine, A-beta, and alpha-synuclein, related to Huntington’s, Alzheimer’s, and Parkinson’s diseases, respectively. In yeast, amyloid-based [PSI+] prions, which rely on the related JDP Sis1 for replication, have a latent toxicity that is exposed by reducing Sis1 function. Anti-amyloid activity of DnaJB6 is very effective against weak [PSI+] prions and the Sup35 amyloid that composes them, but ineffective against strong [PSI+] prions composed of structurally different amyloid of the same Sup35. This difference reveals limitations of DnaJB6 that have implications regarding its therapeutic use for amyloid disease. Here, we find that when Sis1 function is reduced, DnaJB6 represses toxicity of strong [PSI+] prions and inhibits their propagation. Both Sis1 and DnaJB6, which are regulators of protein chaperone Hsp70, counteract the toxicity by reducing excessive incorporation of the essential Sup35 into prion aggregates. However, while Sis1 apparently requires interaction with Hsp70 to detoxify [PSI+], DnaJB6 counteracts prion toxicity by a different, Hsp70-independent mechanism.
| S-EPMC9776390 | biostudies-literature
Project description:Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.
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Project description:Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.
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Project description:BackgroundPrions are transmissible, propagating alternative states of proteins, and are usually made from the fibrillar, beta-sheet-rich assemblies termed amyloid. Prions in the budding yeast Saccharomyces cerevisiae propagate heritable phenotypes, uncover hidden genetic variation, function in large-scale gene regulation, and can act like diseases. Almost all these amyloid prions have asparagine/glutamine-rich (N/Q-rich) domains. Other proteins, that we term here 'prionogenic amyloid formers' (PAFs), have been shown to form amyloid in vivo, and to have N/Q-rich domains that can propagate heritable states in yeast cells. Also, there are >200 other S.cerevisiae proteins with prion-like N/Q-rich sequence composition. Furthermore, human proteins with such N/Q-rich composition have been linked to the pathomechanisms of neurodegenerative amyloid diseases.ResultsHere, we exploit the increasing abundance of complete fungal genomes to examine the ancestry of prions/PAFs and other N/Q-rich proteins across the fungal kingdom. We find distinct evolutionary behavior for Q-rich and N-rich prions/PAFs; those of ancient ancestry (outside the budding yeasts, Saccharomycetes) are Q-rich, whereas N-rich cases arose early in Saccharomycetes evolution. This emergence of N-rich prion/PAFs is linked to a large-scale emergence of N-rich proteins during Saccharomycetes evolution, with Saccharomycetes showing a distinctive trend for population sizes of prion-like proteins that sets them apart from all the other fungi. Conversely, some clades, e.g. Eurotiales, have much fewer N/Q-rich proteins, and in some cases likely lose them en masse, perhaps due to greater amyloid intolerance, although they contain relatively more non-N/Q-rich predicted prions. We find that recent mutational tendencies arising during Saccharomycetes evolution (i.e., increased numbers of N residues and a tendency to form more poly-N tracts), contributed to the expansion/development of the prion phenomenon. Variation in these mutational tendencies in Saccharomycetes is correlated with the population sizes of prion-like proteins, thus implying that selection pressures on N/Q-rich protein sequences against amyloidogenesis are not generally maintained in budding yeasts.ConclusionsThese results help to delineate further the limits and origins of N/Q-rich prions, and provide insight as a case study of the evolution of compositionally-defined protein domains.
| S-EPMC4727409 | biostudies-literature
Project description:Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer's, Parkinson's, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer's amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.
| S-EPMC3473064 | biostudies-literature
Project description:RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways.IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.
| S-EPMC7157824 | biostudies-literature