Abstract
Human chaperone DnaJB6, an Hsp70 co-chaperone whose defects cause myopathies, protects cells from polyglutamine toxicity and prevents purified polyglutamine and Aβ peptides from forming amyloid. Yeast prions [URE3] and [PSI(+)] propagate as amyloid forms of Ure2 and Sup35 proteins, respectively. Here we find DnaJB6-protected yeast cells from polyglutamine toxicity and cured yeast of both [URE3] prions and weak variants of [PSI(+)] prions but not strong [PSI(+)] prions. Weak and strong variants of [PSI(+)] differ only in the structural conformation of their amyloid cores. In line with its anti-prion effects, DnaJB6 prevented purified Sup35NM from forming amyloids at 37 °C, which produce predominantly weak [PSI(+)] variants when used to infect yeast, but not at 4 °C, which produces mostly strong [PSI(+)] variants. Thus, structurally distinct amyloids composed of the same protein were differentially sensitive to the anti-amyloid activity of DnaJB6 both in vitro and in vivo. These findings have important implications for strategies using DnaJB6 as a target for therapy in amyloid disorders.
Highlights
In many mammalian diseases, including Alzheimer, Parkinson, and Huntington diseases, type 2 diabetes, and prion diseases, tissue pathology is associated with accumulation of amyloid, an insoluble, highly ordered fibrous protein aggregate
DnaJB6b Prevents PolyQ103GFP Toxicity in Yeast—Fig. 1A shows the domain structure of human DnaJB molecular chaperones used here compared with yeast Ydj1 and Sis1
We show that overexpression of the human J-protein DnaJB6b cured yeast of [URE3] prions very effectively
Summary
Yeast Strains and Growth Conditions—Yeast strains used are isogenic to 1075 (MATa, kar, PDAL5::ADE2, his3⌬200, leu2⌬1, trp1⌬63, ura, SUQ5, [URE3]) [22]. 779-6A is the same but has ade in place of PDAL5::ADE2, is [ure-o], and has endogenous strong [PSIϩ] and [PINϩ], both of uncertain origin. [PSIϩ] prion variants were cytoduced into a [rhoo] version of 779-6A that had been cured of prions by passage on medium containing 3 mM guanidine-HCl to generate strains 1566, 1567, 1586, and 1587. 779-6A is the same but has ade in place of PDAL5::ADE2, is [ure-o], and has endogenous strong [PSIϩ] and [PINϩ], both of uncertain origin. Strains 1566 and 1567 were made using strains JW129 [PSIϩ]Sc4 (strong) and JW127 [PSIϩ]Sc37 (weak) as prion donors [23]. Strains 1586 and 1587 were made using L2892 [PSIϩ]W (weak, here designated SL[PSIϩ]W) and L2885 [PSIϩ]S (strong, here designated SL[PSIϩ]S) as prion donors [24]. YPAD contains 1% yeast extract, 2% peptone, 400 mg/liter (excess) adenine and 2% dextrose. To monitor prion phenotypes cells were grown on 1/2YPD (0.5% yeast extract, 2% peptone, 2% dextrose), which contains a limiting but undefined amount of adenine. Cells were grown on minimal synthetic dextrose media, which contain 2% dextrose and 0.7% yeast nitrogen base supplemented with appropriate nutrients. Limiting adenine in selective media was 10 mg/liter.
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