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Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division.

ABSTRACT: Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers.

SUBMITTER: Wu YX

PROVIDER: S-EPMC1192827 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division.

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