Background: Phosphorylation and sumoylation are post-translational modifications of the Parkinson disease protein ␣-synuclein. Results: ␣-Synuclein inclusion clearance is impaired in yeast when sumoylation is inhibited; phosphorylation of ␣-synuclein can compensate SUMO impairment. Conclusion: Sumoylation stimulates autophagy clearance of ␣-synuclein inclusions, whereas phosphorylation promotes autophagy and proteasome degradation. Significance: A complex molecular post-translational cross-talk is required in yeast to clear toxic inclusions.

Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by αSyn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn that likely participates in PD pathology.

Abstract:The budding yeast Saccharomyces cerevisiae represents an established model system to study the molecular mechanisms associated to neurodegenerative disorders. A key-feature of Parkinson's disease is the formation of Lewy bodies, which are cytoplasmic protein inclusions. Misfolded Į-synuclein is one of their main constituents. Expression of Į-synuclein protein in yeast leads to protein aggregation and cellular toxicity, which is reminiscent to Lewy body containing human cells. The molecular mechanism involved in clearance of Į-synuclein aggregates is a central question for elucidating the Į-synuclein-related toxicity. Cellular clearance mechanisms include ubiquitin mediated 26S proteasome function as well as lysosome/vacuole associated degradative pathways as autophagy. Various modifications change Į-synuclein posttranslationally and alter its inclusion formation, cytotoxicity and the distribution to different clearance pathways. Several of these modification sites are conserved from yeast to human. In this review, we summarize recent findings on the effect of phosphorylation and sumoylation of Į-synuclein to the enhanced channeling to either the autophagy or the proteasome degradation pathway in yeast model of Parkinson's disease.