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

Kleinknecht A, Popova B, Lázaro DF, Pinho R, Valerius O, Outeiro TF, Braus GH - PLoS Genet. (2016)

Bottom Line: Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies.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.The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Institute of Microbiology and Genetics, Georg-August-Universität, Göttingen, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Tyrosine 133 mutation does not alter the accumulation of reactive oxygen and nitrogen species.(A, B) Quantification of cells expressing different αSyn variants displaying ROS and RNS assessed with flow cytometry analysis. αSyn expression was induced for 6 h and the cells were stained for 1.5 h with DHR123 to visualize ROS (A) or with DAF-2 DA to visualize RNS (B). Forward scatter (FSC) and DHR123 (A) or DAF-2 DA (B) fluorescence of the cells, showing one representative result from at least four independent experiments. The percentage of the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background are presented in the lower panels. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001).
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pgen.1006098.g012: Tyrosine 133 mutation does not alter the accumulation of reactive oxygen and nitrogen species.(A, B) Quantification of cells expressing different αSyn variants displaying ROS and RNS assessed with flow cytometry analysis. αSyn expression was induced for 6 h and the cells were stained for 1.5 h with DHR123 to visualize ROS (A) or with DAF-2 DA to visualize RNS (B). Forward scatter (FSC) and DHR123 (A) or DAF-2 DA (B) fluorescence of the cells, showing one representative result from at least four independent experiments. The percentage of the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background are presented in the lower panels. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001).

Mentions: Flow cytometry measurements were performed to determine the accumulation of ROS and RNS in yeast cells, expressing the single mutants. DHR123 was used for detection of ROS (Fig 12A) and DAF-2 DA was used for detection of RNS (Fig 12B). Expression of all mutants revealed a significant increase in the levels of ROS and RNS in comparison with the control; however, no significant differences were observed between the single mutants revealing that the enhanced toxicity of Y133F and S129A mutant is not due to higher accumulation of ROS or RNS.


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

Kleinknecht A, Popova B, Lázaro DF, Pinho R, Valerius O, Outeiro TF, Braus GH - PLoS Genet. (2016)

Tyrosine 133 mutation does not alter the accumulation of reactive oxygen and nitrogen species.(A, B) Quantification of cells expressing different αSyn variants displaying ROS and RNS assessed with flow cytometry analysis. αSyn expression was induced for 6 h and the cells were stained for 1.5 h with DHR123 to visualize ROS (A) or with DAF-2 DA to visualize RNS (B). Forward scatter (FSC) and DHR123 (A) or DAF-2 DA (B) fluorescence of the cells, showing one representative result from at least four independent experiments. The percentage of the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background are presented in the lower panels. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g012: Tyrosine 133 mutation does not alter the accumulation of reactive oxygen and nitrogen species.(A, B) Quantification of cells expressing different αSyn variants displaying ROS and RNS assessed with flow cytometry analysis. αSyn expression was induced for 6 h and the cells were stained for 1.5 h with DHR123 to visualize ROS (A) or with DAF-2 DA to visualize RNS (B). Forward scatter (FSC) and DHR123 (A) or DAF-2 DA (B) fluorescence of the cells, showing one representative result from at least four independent experiments. The percentage of the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background are presented in the lower panels. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001).
Mentions: Flow cytometry measurements were performed to determine the accumulation of ROS and RNS in yeast cells, expressing the single mutants. DHR123 was used for detection of ROS (Fig 12A) and DAF-2 DA was used for detection of RNS (Fig 12B). Expression of all mutants revealed a significant increase in the levels of ROS and RNS in comparison with the control; however, no significant differences were observed between the single mutants revealing that the enhanced toxicity of Y133F and S129A mutant is not due to higher accumulation of ROS or RNS.

Bottom Line: Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies.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.The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics and Göttingen Center for Molecular Biosciences (GZMB), Institute of Microbiology and Genetics, Georg-August-Universität, Göttingen, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus