Sumoylation Protects Against ß-Synuclein Toxicity in Yeast.

Aggregation of α-synuclein (αSyn) plays a central role in the pathogenesis of Parkinson's disease (PD). The budding yeast Saccharomyces cerevisiae serves as reference cell to study the interplay between αSyn misfolding, cytotoxicity and post-translational modifications (PTMs). The synuclein family includes α, ß and γ isoforms. ß-synuclein (ßSyn) and αSyn are found at presynaptic terminals and both proteins are presumably involved in disease pathogenesis. Similar to αSyn, expression of ßSyn leads to growth deficiency and formation of intracellular aggregates in yeast. Co-expression of αSyn and ßSyn exacerbates the cytotoxicity. This suggests an important role of ßSyn homeostasis in PD pathology. We show here that the small ubiquitin-like modifier SUMO is an important determinant of protein stability and ßSyn-induced toxicity in eukaryotic cells. Downregulation of sumoylation in a yeast strain, defective for the SUMO-encoding gene resulted in reduced yeast growth, whereas upregulation of sumoylation rescued growth of yeast cell expressing ßSyn. This corroborates a protective role of the cellular sumoylation machinery against ßSyn-induced toxicity. Upregulation of sumoylation significantly reduced ßSyn aggregate formation. This is an indirect molecular process, which is not directly linked to ßSyn sumoylation because amino acid substitutions in the lysine residues required for ßSyn sumoylation decreased aggregation without changing yeast cellular toxicity. αSyn aggregates are more predominantly degraded by the autophagy/vacuole than by the 26S ubiquitin proteasome system. We demonstrate a vice versa situation for ßSyn, which is mainly degraded in the 26S proteasome. Downregulation of sumoylation significantly compromised the clearance of ßSyn by the 26S proteasome and increased protein stability. This effect is specific, because depletion of functional SUMO did neither affect ßSyn aggregate formation nor its degradation by the autophagy/vacuolar pathway. Our data support that cellular ßSyn toxicity and aggregation do not correlate in their cellular impact as for αSyn but rather represent two distinct independent molecular functions and molecular mechanisms. These insights into the relationship between ßSyn-induced toxicity, aggregate formation and degradation demonstrate a significant distinction between the impact of αSyn compared to ßSyn on eukaryotic cells.

C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease.

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.

Posttranslational Modifications and Clearing of α-Synuclein Aggregates in Yeast.

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 α-synuclein is one of their main constituents. Expression of α-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 α-synuclein aggregates is a central question for elucidating the α-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 α-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 α-synuclein to the enhanced channeling to either the autophagy or the proteasome degradation pathway in yeast model of Parkinson's disease.

Interplay between sumoylation and phosphorylation for protection against α-synuclein inclusions.

Parkinson disease is associated with the progressive loss of dopaminergic neurons from the substantia nigra. The pathological hallmark of the disease is the accumulation of intracytoplasmic inclusions known as Lewy bodies that consist mainly of post-translationally modified forms of α-synuclein. Whereas phosphorylation is one of the major modifications of α-synuclein in Lewy bodies, sumoylation has recently been described. The interplay between α-synuclein phosphorylation and sumoylation is poorly understood. Here, we examined the interplay between these modifications as well as their impact on cell growth and inclusion formation in yeast. We found that α-synuclein is sumoylated in vivo at the same sites in yeast as in human cells. Impaired sumoylation resulted in reduced yeast growth combined with an increased number of cells with inclusions, suggesting that this modification plays a protective role. In addition, inhibition of sumoylation prevented autophagy-mediated aggregate clearance. A defect in α-synuclein sumoylation could be suppressed by serine 129 phosphorylation by the human G protein-coupled receptor kinase 5 (GRK5) in yeast. Phosphorylation reduced foci formation, alleviated yeast growth inhibition, and partially rescued autophagic α-synuclein degradation along with the promotion of proteasomal degradation, resulting in aggregate clearance in the absence of a small ubiquitin-like modifier. These findings suggest a complex interplay between sumoylation and phosphorylation in α-synuclein aggregate clearance, which may open new horizons for the development of therapeutic strategies for Parkinson disease.