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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 is required for phosphorylation of αSyn at serine 129.(A) Western blotting of αSyn and A30P expressed in YHB1 and Δyhb1 yeast enriched by Ni2+ pull-down, using Y133 phosphorylation-specific αSyn antibody (pY133) and S129 phosphorylation-specific αSyn antibody (pS129). The same membrane was stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P Y133- and S129-phosphorylation levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA test (**, p < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different αSyn variants after 6 h induction of protein expression using S129 phosphorylation-specific αSyn antibody (pS129) and αSyn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of αSyn and indicated mutant strains, driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, p < 0.001) or Dunnett’s multiple comparison test (#, p < 0.05, ##, p < 0.01 versus αSyn; n = 6). (F) Cell growth analysis of cells expressing different αSyn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001, n = 4). (G) Quantification of cells expressing different αSyn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of αSyn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001 versus αSyn; n = 4).
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pgen.1006098.g011: Tyrosine 133 is required for phosphorylation of αSyn at serine 129.(A) Western blotting of αSyn and A30P expressed in YHB1 and Δyhb1 yeast enriched by Ni2+ pull-down, using Y133 phosphorylation-specific αSyn antibody (pY133) and S129 phosphorylation-specific αSyn antibody (pS129). The same membrane was stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P Y133- and S129-phosphorylation levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA test (**, p < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different αSyn variants after 6 h induction of protein expression using S129 phosphorylation-specific αSyn antibody (pS129) and αSyn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of αSyn and indicated mutant strains, driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, p < 0.001) or Dunnett’s multiple comparison test (#, p < 0.05, ##, p < 0.01 versus αSyn; n = 6). (F) Cell growth analysis of cells expressing different αSyn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001, n = 4). (G) Quantification of cells expressing different αSyn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of αSyn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001 versus αSyn; n = 4).

Mentions: Immunoblotting with an antibody that specifically recognizes αSyn phosphorylated at Y133 showed that both αSyn and A30P are phosphorylated at these residues, in accordance with our results from MS analysis (Fig 11A). Quantification of Y133 phosphorylation revealed similar phosphorylation level of αSyn and A30P variant both in presence and absence of Yhb1 (Fig 11B). We analyzed whether there is a difference between S129 phosphorylation level of αSyn and A30P. S129 phosphorylation level of αSyn was much higher than that of A30P (Fig 11A and 11B). Tyrosine to phenylalanine (Y/F) substitutions were analyzed for their effects on the phosphorylation level at S129. Y/F mutation of the N-terminal tyrosine 39 as well as of the C-terminal Y125 and Y136 did not affect the phosphorylation status of S129. In contrast, mutation of Y133 had a drastic impact and resulted in complete loss of phosphorylation at S129 (Fig 11C). Yeast growth was compared in spotting assay as well as in liquid culture between yeast cells, expressing Y/F single mutants and S129A phosphorylation deficient mutant (Fig 11D and 11F). Yeast growth was measured after 20 h induction of protein expression. Expression of Y133F resulted in significant growth inhibition in comparison with αSyn and other tyrosine mutants. S129A showed slight growth inhibition (Fig 11D and 11F) and significant increase in the number of cells with aggregates (Fig 11E). In addition to growth analysis, membrane integrity of the Y/F single mutants and S129A phosphorylation deficient mutant was examined to assess the cell viability of the mutants (Fig 11G and S2 Fig). Propidium iodide (PI) staining was used after 20 h of protein induction as a sensitive method to determine the fraction of cells with compromised membrane integrity. Expression of Y133F and S129A significantly diminished membrane integrity, corroborating that expression of these mutants results in increased cytotoxicity.


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 is required for phosphorylation of αSyn at serine 129.(A) Western blotting of αSyn and A30P expressed in YHB1 and Δyhb1 yeast enriched by Ni2+ pull-down, using Y133 phosphorylation-specific αSyn antibody (pY133) and S129 phosphorylation-specific αSyn antibody (pS129). The same membrane was stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P Y133- and S129-phosphorylation levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA test (**, p < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different αSyn variants after 6 h induction of protein expression using S129 phosphorylation-specific αSyn antibody (pS129) and αSyn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of αSyn and indicated mutant strains, driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, p < 0.001) or Dunnett’s multiple comparison test (#, p < 0.05, ##, p < 0.01 versus αSyn; n = 6). (F) Cell growth analysis of cells expressing different αSyn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001, n = 4). (G) Quantification of cells expressing different αSyn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of αSyn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001 versus αSyn; n = 4).
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pgen.1006098.g011: Tyrosine 133 is required for phosphorylation of αSyn at serine 129.(A) Western blotting of αSyn and A30P expressed in YHB1 and Δyhb1 yeast enriched by Ni2+ pull-down, using Y133 phosphorylation-specific αSyn antibody (pY133) and S129 phosphorylation-specific αSyn antibody (pS129). The same membrane was stripped and re-probed with αSyn antibody. (B) Quantification of αSyn and A30P Y133- and S129-phosphorylation levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 αSyn and A30P relative to the intensity obtained for αSyn. Significance of differences was calculated with one-way ANOVA test (**, p < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different αSyn variants after 6 h induction of protein expression using S129 phosphorylation-specific αSyn antibody (pS129) and αSyn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of αSyn and indicated mutant strains, driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, p < 0.001) or Dunnett’s multiple comparison test (#, p < 0.05, ##, p < 0.01 versus αSyn; n = 6). (F) Cell growth analysis of cells expressing different αSyn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001, n = 4). (G) Quantification of cells expressing different αSyn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of αSyn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, p < 0.0001) or Dunnett’s multiple comparison test (#, p < 0.05; ###, p < 0.001 versus αSyn; n = 4).
Mentions: Immunoblotting with an antibody that specifically recognizes αSyn phosphorylated at Y133 showed that both αSyn and A30P are phosphorylated at these residues, in accordance with our results from MS analysis (Fig 11A). Quantification of Y133 phosphorylation revealed similar phosphorylation level of αSyn and A30P variant both in presence and absence of Yhb1 (Fig 11B). We analyzed whether there is a difference between S129 phosphorylation level of αSyn and A30P. S129 phosphorylation level of αSyn was much higher than that of A30P (Fig 11A and 11B). Tyrosine to phenylalanine (Y/F) substitutions were analyzed for their effects on the phosphorylation level at S129. Y/F mutation of the N-terminal tyrosine 39 as well as of the C-terminal Y125 and Y136 did not affect the phosphorylation status of S129. In contrast, mutation of Y133 had a drastic impact and resulted in complete loss of phosphorylation at S129 (Fig 11C). Yeast growth was compared in spotting assay as well as in liquid culture between yeast cells, expressing Y/F single mutants and S129A phosphorylation deficient mutant (Fig 11D and 11F). Yeast growth was measured after 20 h induction of protein expression. Expression of Y133F resulted in significant growth inhibition in comparison with αSyn and other tyrosine mutants. S129A showed slight growth inhibition (Fig 11D and 11F) and significant increase in the number of cells with aggregates (Fig 11E). In addition to growth analysis, membrane integrity of the Y/F single mutants and S129A phosphorylation deficient mutant was examined to assess the cell viability of the mutants (Fig 11G and S2 Fig). Propidium iodide (PI) staining was used after 20 h of protein induction as a sensitive method to determine the fraction of cells with compromised membrane integrity. Expression of Y133F and S129A significantly diminished membrane integrity, corroborating that expression of these mutants results in increased cytotoxicity.

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