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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

αSyn aggregate clearance after promoter shut-off.(A, C) Quantification of cells displaying aggregates of αSyn (A) and A30P (C) upon inhibition of autophagy by PMSF. Cells expressing αSyn (A) or A30P (C) and its 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to 2% glucose-containing media supplemented with 1 mM PMSF dissolved in EtOH and only EtOH as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut-off and presented as a ratio to the control (EtOH). Significance of differences was calculated with one-way ANOVA (*, p < 0.05; **, p < 0.01) or Dunnett’s multiple comparison test (#, p < 0.05; ##, p < 0.01 versus αSyn; n = 4). (B, D) Quantification of cells displaying aggregates of αSyn (B) and A30P (D) upon inhibition of the proteasome by MG132. Cells expressing αSyn (B) or A30P (D) and the indicated 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to glucose medium, supplemented with 75 μM MG132, dissolved in DMSO or only DMSO as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut off and presented as a ratio to the control (DMSO). 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 = 4).
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pgen.1006098.g013: αSyn aggregate clearance after promoter shut-off.(A, C) Quantification of cells displaying aggregates of αSyn (A) and A30P (C) upon inhibition of autophagy by PMSF. Cells expressing αSyn (A) or A30P (C) and its 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to 2% glucose-containing media supplemented with 1 mM PMSF dissolved in EtOH and only EtOH as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut-off and presented as a ratio to the control (EtOH). Significance of differences was calculated with one-way ANOVA (*, p < 0.05; **, p < 0.01) or Dunnett’s multiple comparison test (#, p < 0.05; ##, p < 0.01 versus αSyn; n = 4). (B, D) Quantification of cells displaying aggregates of αSyn (B) and A30P (D) upon inhibition of the proteasome by MG132. Cells expressing αSyn (B) or A30P (D) and the indicated 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to glucose medium, supplemented with 75 μM MG132, dissolved in DMSO or only DMSO as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut off and presented as a ratio to the control (DMSO). 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 = 4).

Mentions: GAL1 promoter shut-off experiments were performed to study the role of αSyn PTMs on autophagy/vacuole and proteasome-mediated aggregate clearance of αSyn. The impact of blocking these systems by drug treatments was examined. Expression of αSyn was induced for 4 h in galactose-containing medium and the cells were then shifted to glucose-containing medium in order to repress the promoter. Cells were imaged 4 h after promoter shut-off and the percentage of cells with inclusions was determined. Shut-off studies were performed with wild-type αSyn and the mutants 4(Y/F), S129A and Y133F. PMSF was used as an inhibitor of autophagy/vacuole to study the contribution of this pathway for aggregate clearance [63]. PMSF impairs the activity of many vacuolar serine proteases without affecting proteasome function [84, 85]. Inhibition of autophagy resulted in inefficient aggregate clearance of αSyn, as shown previously [41, 63]. Mutations of the codons for the four tyrosines as well as the S129 and Y133 single exchanges resulted in similar aggregate clearance by inhibition of the autophagic proteases as in the control cells without drug (ethanol) (Fig 13A). This suggests that autophagy is less involved in aggregate clearance of these mutants and shows that autophagy-mediated aggregate clearance requires modifications of the tyrosines and S129. The contribution of the proteasome on 4(Y/F), S129A and Y133F αSyn aggregate clearance was analyzed by applying the proteasome inhibitor MG132 [86]. In contrast to autophagy impairment, cells expressing 4(Y/F) and S129A αSyn cleared inclusions equally as the wild-type αSyn (Fig 13B) when the proteasome system was impaired. These results corroborate our previous findings showing a minor contribution of the proteasome to αSyn aggregate clearance [63]. However, cells expressing the Y133F mutant were unable to clear inclusions in a same manner as the wild-type, 4(Y/F) and S129A αSyn. This indicates that αSyn, which is not modified at Y133, is degraded by the proteasomal pathway. The results suggest that PTMs of tyrosine residues and S129 promote the autophagy-mediated aggregate clearance, whereas non-modified Y133 residue is a key determinant for the targeting of the protein to the proteasome.


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)

αSyn aggregate clearance after promoter shut-off.(A, C) Quantification of cells displaying aggregates of αSyn (A) and A30P (C) upon inhibition of autophagy by PMSF. Cells expressing αSyn (A) or A30P (C) and its 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to 2% glucose-containing media supplemented with 1 mM PMSF dissolved in EtOH and only EtOH as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut-off and presented as a ratio to the control (EtOH). Significance of differences was calculated with one-way ANOVA (*, p < 0.05; **, p < 0.01) or Dunnett’s multiple comparison test (#, p < 0.05; ##, p < 0.01 versus αSyn; n = 4). (B, D) Quantification of cells displaying aggregates of αSyn (B) and A30P (D) upon inhibition of the proteasome by MG132. Cells expressing αSyn (B) or A30P (D) and the indicated 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to glucose medium, supplemented with 75 μM MG132, dissolved in DMSO or only DMSO as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut off and presented as a ratio to the control (DMSO). 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 = 4).
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Related In: Results  -  Collection

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pgen.1006098.g013: αSyn aggregate clearance after promoter shut-off.(A, C) Quantification of cells displaying aggregates of αSyn (A) and A30P (C) upon inhibition of autophagy by PMSF. Cells expressing αSyn (A) or A30P (C) and its 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to 2% glucose-containing media supplemented with 1 mM PMSF dissolved in EtOH and only EtOH as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut-off and presented as a ratio to the control (EtOH). Significance of differences was calculated with one-way ANOVA (*, p < 0.05; **, p < 0.01) or Dunnett’s multiple comparison test (#, p < 0.05; ##, p < 0.01 versus αSyn; n = 4). (B, D) Quantification of cells displaying aggregates of αSyn (B) and A30P (D) upon inhibition of the proteasome by MG132. Cells expressing αSyn (B) or A30P (D) and the indicated 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to glucose medium, supplemented with 75 μM MG132, dissolved in DMSO or only DMSO as a control. Cells with aggregates were counted after 4 h GAL1-promoter shut off and presented as a ratio to the control (DMSO). 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 = 4).
Mentions: GAL1 promoter shut-off experiments were performed to study the role of αSyn PTMs on autophagy/vacuole and proteasome-mediated aggregate clearance of αSyn. The impact of blocking these systems by drug treatments was examined. Expression of αSyn was induced for 4 h in galactose-containing medium and the cells were then shifted to glucose-containing medium in order to repress the promoter. Cells were imaged 4 h after promoter shut-off and the percentage of cells with inclusions was determined. Shut-off studies were performed with wild-type αSyn and the mutants 4(Y/F), S129A and Y133F. PMSF was used as an inhibitor of autophagy/vacuole to study the contribution of this pathway for aggregate clearance [63]. PMSF impairs the activity of many vacuolar serine proteases without affecting proteasome function [84, 85]. Inhibition of autophagy resulted in inefficient aggregate clearance of αSyn, as shown previously [41, 63]. Mutations of the codons for the four tyrosines as well as the S129 and Y133 single exchanges resulted in similar aggregate clearance by inhibition of the autophagic proteases as in the control cells without drug (ethanol) (Fig 13A). This suggests that autophagy is less involved in aggregate clearance of these mutants and shows that autophagy-mediated aggregate clearance requires modifications of the tyrosines and S129. The contribution of the proteasome on 4(Y/F), S129A and Y133F αSyn aggregate clearance was analyzed by applying the proteasome inhibitor MG132 [86]. In contrast to autophagy impairment, cells expressing 4(Y/F) and S129A αSyn cleared inclusions equally as the wild-type αSyn (Fig 13B) when the proteasome system was impaired. These results corroborate our previous findings showing a minor contribution of the proteasome to αSyn aggregate clearance [63]. However, cells expressing the Y133F mutant were unable to clear inclusions in a same manner as the wild-type, 4(Y/F) and S129A αSyn. This indicates that αSyn, which is not modified at Y133, is degraded by the proteasomal pathway. The results suggest that PTMs of tyrosine residues and S129 promote the autophagy-mediated aggregate clearance, whereas non-modified Y133 residue is a key determinant for the targeting of the protein to the proteasome.

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