The role of methionine-sulfoxide reductases in protein aggregation and prion formation in S. cerevisiae

Oxidative stress is known to trigger protein misfolding and has been implicated in promoting spontaneous prion formation in both yeast and mammalian cells. Previous studies have suggested that oxidative stress causes protein misfolding and prion formation via oxidation of methionine residues. Methionine residues in proteins are particularly prone to oxidation, forming a racemic mixture of methionine- S-sulfoxide (Met-S-O) and methionine-R-sulfoxide (Met-R-O). In the current study, we directly tested the role of methionine oxidation in prion formation by mutating methionine residues in Sup35 and Rnq1, which are the soluble forms of the [PSI+] and [PIN+] prions, respectively. Substituting methionine residues for alanine residues in Sup35 or Rnq1 significantly reduced the frequency [PSI+] and [PIN+] prion formation consistent with the idea that methionine oxidation in a normally soluble protein underlies its transition to the amyloidogenic prion form. Previous studies have shown that methionine oxidation and prion formation is increased in mutants lacking antioxidant enzymes that act to detoxify reactive oxygen species (ROS) including peroxiredoxins, catalases and superoxide dismutases. Methionine-sulfoxide reductases (MXRs) are ubiquitous antioxidant enzymes that protect against methionine oxidation by directly reducing MetO. The current study looks at two yeast MXRs: Mxr1, which reduces Met-S-O, and Mxr2, which reduces Met-R-O. Mutants deleted for MXR1 or MXR2 are shown to have increased Sup35 methionine oxidation and an elevated frequency of [PSI+] prion formation consistent with the idea that methionine oxidation promotes prion formation. Surprisingly, [PSI+] prion formation is not so significantly increased in a double mutant lacking MXR1 and MXR2. [PSI+] prion formation can be induced by overexpression of Sup35 (NM-GFP) but this occurs at a much-reduced frequency in the double compared with the single mxr mutant strains. Visualisation of the Sup35 aggregates using fluorescence microscopy reveals that larger sized aggregates are formed in the double mxr mutant strain compared with the single and wild-type strains. The Hsp104 chaperone, that is required to generate [PSI+] propagons, is shown to localize to Sup35 aggregates and 15 a model is presented, where Hsp104 is less efficient at disaggregating the large Sup35 aggregates present in the mxr1 mxr2 mutant, which explains the decreased frequency of prion formation. Finally, we examined overall protein aggregation in mxr mutants by isolating aggregates and identifying the aggregated proteins using mass spectrometry. Similarly, high levels of protein aggregation are found in single and double mxr mutants compared with a wild-type strain. Analysis of the biophysical properties of the aggregates revealed that they are enriched for properties which have previously been described to promote aggregation including higher protein abundances, higher translation rates, and higher hydrophobicity. However, these biophysical properties are reduced in the mxr deletion strains compared to the wild-type strain indicating that the threshold for proteins to aggregate may be lower in the mutant strains. Analysis of the chaperones present in the aggregated fractions revealed that Hsp104 disaggregase is present in the aggregated fractions from the mxr2 and mxr1/2 deletion strain. Analysis of Hsp104 activity using a luciferase-GFP re-solubilisation assay shows that disaggregase activity is decreased in the mxr2 and mxr1/2 deletion strains suggesting one possible reason for the increased aggregation detected in these strains.