C-terminal tyrosine residue modifications modulate α-synuclein toxicity in yeast as unicellular model for Parkinson´s disease
von Alexandra Kleinknecht
Datum der mündl. Prüfung:2016-06-30
Betreuer:Prof. Dr. Gerhard Braus
Gutachter:Prof. Dr. Tiago Fleming Outeiro
Gutachter:Prof. Dr. Blanche Schwappach
EnglischThe presence of protein inclusions called Lewy bodies (LBs) that are mainly composed of misfolded and accumulated α-synuclein (αSyn) represents a hallmark of Parkinson´s disease (PD). Oligomeric αSyn species are thought to play a central role in the neurodegeneration of PD. Elevated levels of oxidative or nitrative stresses have been implicated in αSyn-related toxicity. Phosphorylation of αSyn on serine 129 (S129) is prominently found in Lewy bodies and modulates autophagic aggregates clearance. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Overexpression of αSyn in the unicellular eukaryotic model Saccharomyces cerevisiae results in growth impairment and cytoplasmic protein inclusions resembling the aggregates observed within LBs. In this study, yeast was used as reference cell to study the contribution of tyrosine modifications on αSyn-related toxicity. Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and forms stable dimers originating from covalent crosslinking of two tyrosine residues. LC-MS 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 than the A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn supporting dimer formation as a cellular detoxification pathway in yeast. In contrast to A30P, expression of αSyn significantly increased the accumulation of reactive oxygen species, which was independent from tyrosine modifications. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and enhanced aggregation and cytotoxicity of A30P. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation. Deletion of YHB1 elevated the level of reactive nitrogen species in A30P expressing cells, which can be diminished by mutating the nitration sites. Protein analysis showed that Yhb1 affects nitration but not dimerization levels of A30P indicating that nitrated tyrosine residues, but not di-tyrosine crosslinked dimers, contribute to αSyn cytotoxicity and aggregation. Under nitrative stress, deletion of YHB1 severely inhibited yeast growth in cells expressing wild-type and A30P αSyn. A30P was as toxic as wild-type αSyn indicating that increase in nitrative stress converts A30P to a toxic protein. Overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. This study suggests 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 be partially detoxified by αSyn di-tyrosine dimers. This novel complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of αSyn likely participates in PD pathology.
Keywords: α-synuclein, tyrosine, dimers, nitration, phosphorylation, yeast, mitochondria, cytotoxicity