Heterologous Gln/Asn-Rich Proteins Affect The Stability And Toxicity of Yeast Prions

The accumulation of amyloids, formed by β-sheet-rich proteins, into plaques in the brain, is associated with neurodegenerative disorders such as transmissible spongiform encephalopathies (TSEs), Alzheimer’s, Parkinson’s, and Huntington’s diseases. Only TSEs are caused by self-propagating amyloid aggregates called prions. Yeast prions are infectious amyloids that cause distinct heritable phenotypic traits and often depend upon a glutamine (Q)/asparagine (N)-rich domain to assemble into β-sheet-rich amyloid fibers. We showed that overexpression of many Q/N-rich proteins that can facilitate the de novo prion appearance also destabilizes pre-existing yeast prions. We demonstrated that this antagonistic effect of prion-like aggregates on heterologous prions is caused by the sequestration of cellular chaperones available to sever prions for proper propagation. Yeast prions are not inherently toxic, although their co-presence with excess prion protein often produces an excess of soluble oligomers or amyloid aggregates that sequester essential cellular factors causing toxicity. We explored the mechanism of amyloid toxicity elicited by the interplay of heterologous Q/N-rich proteins and prions. We showed that the prion can drive the enhanced aggregation and phosphorylation of a heterologous Q/N-rich protein associated with toxicity. This toxicity is couple with impaired ubiquitin-proteasome system function, presumably due to proteasome capacity overload by hyperphosphorylated amyloid aggregates

Quantified expression of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) mRNA level in liver (A) and placenta (B).

<p>*P<0.05 compared with control. †P<0.05 compared with ApoC3+L-NA. #P<0.05 compared with ApoC3+NS. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Mean arterial blood pressure throughout pregnancy in Pre (A) and Mid subgroups (B) and 24-hr urinary protein levels (C).

<p>The arrow indicates time of injection except for the β2GPI group. #P<0.05 compared with control. *p<0.05 compared with control and ApoC3+NS. ‡p<0.05 compared with control and other experiment groups. †P<0.05, compared with corresponding Mid-gestation group. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Representative immunohistochemical staining of LCHAD in mouse placenta at pre-implantation (A) and mid-gestational age (B).

<p>(Original magnification ×100, scale bars 100 µm) (C): IOD of placenta immunohistochemical images in all groups. *P<0.05 compared with control. †P<0.05 compared with ApoC3+L-NA. #P<0.05 compared with ApoC3+NS. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Lipid deposition in liver tissues with Oil-red O staining at pre-implantation (A) and mid-gestational age (B).

<p>(Original magnification ×200, scale bars 50 µm) (C): Percentage of area stained in all groups. *p<0.05 compared with control. #P<0.05 compared with ApoC3+NS. †p<0.05 compared with ApoC3+L-NA. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Representative immunohistochemical staining of LCHAD in mouse liver at pre-implantation (A) and mid-gestational age (B).

<p>(Original magnification ×100, scale bars 100 µm) (C): Integrated optical density (IOD) of liver immunohistochemical images in all groups. *P<0.05 compared with control. †P<0.05 compared with ApoC3+L-NA. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Western blot analysis and quantification of protein level of LCHAD in liver (A-C) and placenta (D-F).

<p>*P<0.05 compared with control. #P<0.05 compared with ApoC3+NS. †P<0.05 compared with ApoC3+L-NA. Data are mean±SD, n = 10. Pre, pre-implantation. Mid, Mid-gestation.</p

Feto-placental outcomes in all treatment groups.

<p>Data are mean±SD or number (%) and n = 10 per group.</p>a<p> P<0.05 compared with Control and ApoC3+NS.</p>b<p> P<0.05 compared with corresponding Pre group.</p><p>Feto-placental outcomes in all treatment groups.</p

Quantified expression of p47phox mRNA and protein expression in liver (A-C) and placenta (D-F) in Mid groups.

<p>*P<0.05 compared with control. Data are mean±SD, n = 10.</p

Overexpressed Sis1 reduces elimination of [<i>PSI</i>⁺] by excess Pin4C.

<p>(A) Increased levels of Sis1 inhibit [<i>PSI⁺</i>] curing by excess Pin4C. Strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells co-transformed with pHR81<i>GAL</i>-<i>PIN4C</i> and pYES3<i>GAL</i>-<i>SISI</i> (↑Pin4C, ↑Sis1); or with pHR81<i>GAL</i>-<i>PIN4C</i> and empty vector pYES3<i>GAL</i> (↑Pin4C); or with pYES3<i>GAL</i>-<i>SIS1</i> and empty vector pHR81<i>GAL</i> (↑Sis1); or with two empty vectors pHR81<i>GAL</i> and pYES3<i>GAL</i> (↑vectors), were examined as described above (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003236#pgen-1003236-g006" target="_blank">Figure 6C</a>). (B) Overexpressed Sis1 prevents overproduced Pin4C from forming large aggregates. Representative fluorescent images of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] (GF657) cells harboring pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> and pYES3<i>GAL</i>-<i>SISI</i> (↑Pin4C, ↑Sis1), or pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> and empty vector pYES3<i>GAL</i> (↑Pin4C) after overnight induction in liquid galactose are shown.</p