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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

Yhb1 affects nitration but not dimerization of αSyn and A30P.(A) Immunoblotting analysis of 3-nitrotyrosine using 3-nitrotyrosine antibody (left) and nitro-Y39 αSyn antibody (right). Protein expression was induced for 12 h in galactose-containing SC-Ura medium. Concentrated protein extracts of Ni2+ pull down-enriched αSyn and A30P αSyn from YHB1 and Δyhb1 yeast cells were applied. Cells expressing empty vector (EV) served as control. The same membranes were stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P nitration levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of nitrated αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA with Bonferroni’s multiple comparison test (*, p < 0.05; ****, p < 0.0001; n = 3). (C) Western blotting of αSyn enriched by Ni2+ pull-down with αSyn antibody. (D) Ratio of dimers relative to the sum of monomers and dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers, presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with one-way ANOVA (**, p < 0.01; n = 4).
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pgen.1006098.g010: Yhb1 affects nitration but not dimerization of αSyn and A30P.(A) Immunoblotting analysis of 3-nitrotyrosine using 3-nitrotyrosine antibody (left) and nitro-Y39 αSyn antibody (right). Protein expression was induced for 12 h in galactose-containing SC-Ura medium. Concentrated protein extracts of Ni2+ pull down-enriched αSyn and A30P αSyn from YHB1 and Δyhb1 yeast cells were applied. Cells expressing empty vector (EV) served as control. The same membranes were stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P nitration levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of nitrated αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA with Bonferroni’s multiple comparison test (*, p < 0.05; ****, p < 0.0001; n = 3). (C) Western blotting of αSyn enriched by Ni2+ pull-down with αSyn antibody. (D) Ratio of dimers relative to the sum of monomers and dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers, presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with one-way ANOVA (**, p < 0.01; n = 4).

Mentions: We assessed whether the different cytotoxicity of αSyn and A30P in yeast correlates with different nitration levels of the two variants in wild-type yeast background and under increased nitrative stress in Δyhb1 strain. Immunoblotting analysis performed with two specific antibodies against nitro-tyrosine (3-nitrotyrosine and nitro-Y39 αSyn) showed that αSyn and A30P are nitrated in the YHB1 as well as in Δyhb1 yeast (Fig 10A). Quantification of band intensities of both nitrated αSyn variants revealed significantly higher nitration level of αSyn in comparison to A30P. Deletion of YHB1 resulted in increase of A30P nitration level, whereas αSyn nitration level was not affected (Fig 10B). The increased nitration level does not correlate with an increased dimerization level of both αSyn variants. The dimerization was not influenced by nitrative stress enhancement (Fig 10C and 10D). The results suggest that nitration of αSyn contributes to the cytotoxicity of the protein, whereas dimer formation is in reverse correlation to toxicity.


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)

Yhb1 affects nitration but not dimerization of αSyn and A30P.(A) Immunoblotting analysis of 3-nitrotyrosine using 3-nitrotyrosine antibody (left) and nitro-Y39 αSyn antibody (right). Protein expression was induced for 12 h in galactose-containing SC-Ura medium. Concentrated protein extracts of Ni2+ pull down-enriched αSyn and A30P αSyn from YHB1 and Δyhb1 yeast cells were applied. Cells expressing empty vector (EV) served as control. The same membranes were stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P nitration levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of nitrated αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA with Bonferroni’s multiple comparison test (*, p < 0.05; ****, p < 0.0001; n = 3). (C) Western blotting of αSyn enriched by Ni2+ pull-down with αSyn antibody. (D) Ratio of dimers relative to the sum of monomers and dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers, presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with one-way ANOVA (**, p < 0.01; n = 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g010: Yhb1 affects nitration but not dimerization of αSyn and A30P.(A) Immunoblotting analysis of 3-nitrotyrosine using 3-nitrotyrosine antibody (left) and nitro-Y39 αSyn antibody (right). Protein expression was induced for 12 h in galactose-containing SC-Ura medium. Concentrated protein extracts of Ni2+ pull down-enriched αSyn and A30P αSyn from YHB1 and Δyhb1 yeast cells were applied. Cells expressing empty vector (EV) served as control. The same membranes were stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P nitration levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of nitrated αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA with Bonferroni’s multiple comparison test (*, p < 0.05; ****, p < 0.0001; n = 3). (C) Western blotting of αSyn enriched by Ni2+ pull-down with αSyn antibody. (D) Ratio of dimers relative to the sum of monomers and dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers, presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with one-way ANOVA (**, p < 0.01; n = 4).
Mentions: We assessed whether the different cytotoxicity of αSyn and A30P in yeast correlates with different nitration levels of the two variants in wild-type yeast background and under increased nitrative stress in Δyhb1 strain. Immunoblotting analysis performed with two specific antibodies against nitro-tyrosine (3-nitrotyrosine and nitro-Y39 αSyn) showed that αSyn and A30P are nitrated in the YHB1 as well as in Δyhb1 yeast (Fig 10A). Quantification of band intensities of both nitrated αSyn variants revealed significantly higher nitration level of αSyn in comparison to A30P. Deletion of YHB1 resulted in increase of A30P nitration level, whereas αSyn nitration level was not affected (Fig 10B). The increased nitration level does not correlate with an increased dimerization level of both αSyn variants. The dimerization was not influenced by nitrative stress enhancement (Fig 10C and 10D). The results suggest that nitration of αSyn contributes to the cytotoxicity of the protein, whereas dimer formation is in reverse correlation to toxicity.

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