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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

The nitric oxide oxidoreductase Yhb1 reduces A30P aggregation and toxicity.(A) Cell growth comparison of wild-type YHB1 and mutant Δyhb1 yeast cells in the presence of the NO stress-mediating drug DETA-NONOate (1 mM) in liquid galactose-containing SC-Ura medium. Error bars indicate standard deviations of three independent experiments. (B) Spotting analysis of YHB1 and Δyhb1 yeast cells expressing αSyn (upper boxes) or A30P (lower boxes) compared to GFP and empty vector (EV) as control on non-inducing and galactose-inducing SC-Ura medium after 3 days. (C) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, p < 0.01, n = 6). (D) Cell growth analysis of YHB1 and Δyhb1 yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) and GFP (control) after 40 h induction in galactose-containing SC-Ura medium. (upper panel,—DETA-NONOate; lower panel, + 600 μM DETA-NONOate). Error bars show standard deviations of three independent experiments. (E) Western blotting analysis of protein crude extracts of αSyn and A30P expressed in YHB1 and Δyhb1 yeast after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control. (F) Quantification of αSyn and A30P levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of αSyn and A30P relative to the intensity obtained for GAPDH (n = 3).
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pgen.1006098.g005: The nitric oxide oxidoreductase Yhb1 reduces A30P aggregation and toxicity.(A) Cell growth comparison of wild-type YHB1 and mutant Δyhb1 yeast cells in the presence of the NO stress-mediating drug DETA-NONOate (1 mM) in liquid galactose-containing SC-Ura medium. Error bars indicate standard deviations of three independent experiments. (B) Spotting analysis of YHB1 and Δyhb1 yeast cells expressing αSyn (upper boxes) or A30P (lower boxes) compared to GFP and empty vector (EV) as control on non-inducing and galactose-inducing SC-Ura medium after 3 days. (C) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, p < 0.01, n = 6). (D) Cell growth analysis of YHB1 and Δyhb1 yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) and GFP (control) after 40 h induction in galactose-containing SC-Ura medium. (upper panel,—DETA-NONOate; lower panel, + 600 μM DETA-NONOate). Error bars show standard deviations of three independent experiments. (E) Western blotting analysis of protein crude extracts of αSyn and A30P expressed in YHB1 and Δyhb1 yeast after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control. (F) Quantification of αSyn and A30P levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of αSyn and A30P relative to the intensity obtained for GAPDH (n = 3).

Mentions: The effect of nitrative stress on the toxicity and aggregation of wild-type and A30P mutant αSyn was examined. A yeast strain carrying a deletion in the yeast flavohemoglobin gene (YHB1), responsible for stress signaling, was used for enhancement of nitrative stress. Yhb1 is a nitric oxide oxidoreductase, which protects against nitration of cellular targets and against cell growth inhibition under aerobic or anaerobic conditions. Deletion of YHB1 abolishes the nitric oxide (NO) consuming activity of yeast cells [74]. The compound DETA-NONOate causes nitrative stress by acting as a NO donor. The absence of the flavohemoglobin results in a growth impairment of the hypersensitive yhb1 deletion strain in comparison to wild-type under NO nitrative stress conditions (Fig 5A).


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)

The nitric oxide oxidoreductase Yhb1 reduces A30P aggregation and toxicity.(A) Cell growth comparison of wild-type YHB1 and mutant Δyhb1 yeast cells in the presence of the NO stress-mediating drug DETA-NONOate (1 mM) in liquid galactose-containing SC-Ura medium. Error bars indicate standard deviations of three independent experiments. (B) Spotting analysis of YHB1 and Δyhb1 yeast cells expressing αSyn (upper boxes) or A30P (lower boxes) compared to GFP and empty vector (EV) as control on non-inducing and galactose-inducing SC-Ura medium after 3 days. (C) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, p < 0.01, n = 6). (D) Cell growth analysis of YHB1 and Δyhb1 yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) and GFP (control) after 40 h induction in galactose-containing SC-Ura medium. (upper panel,—DETA-NONOate; lower panel, + 600 μM DETA-NONOate). Error bars show standard deviations of three independent experiments. (E) Western blotting analysis of protein crude extracts of αSyn and A30P expressed in YHB1 and Δyhb1 yeast after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control. (F) Quantification of αSyn and A30P levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of αSyn and A30P relative to the intensity obtained for GAPDH (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g005: The nitric oxide oxidoreductase Yhb1 reduces A30P aggregation and toxicity.(A) Cell growth comparison of wild-type YHB1 and mutant Δyhb1 yeast cells in the presence of the NO stress-mediating drug DETA-NONOate (1 mM) in liquid galactose-containing SC-Ura medium. Error bars indicate standard deviations of three independent experiments. (B) Spotting analysis of YHB1 and Δyhb1 yeast cells expressing αSyn (upper boxes) or A30P (lower boxes) compared to GFP and empty vector (EV) as control on non-inducing and galactose-inducing SC-Ura medium after 3 days. (C) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, p < 0.01, n = 6). (D) Cell growth analysis of YHB1 and Δyhb1 yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) and GFP (control) after 40 h induction in galactose-containing SC-Ura medium. (upper panel,—DETA-NONOate; lower panel, + 600 μM DETA-NONOate). Error bars show standard deviations of three independent experiments. (E) Western blotting analysis of protein crude extracts of αSyn and A30P expressed in YHB1 and Δyhb1 yeast after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control. (F) Quantification of αSyn and A30P levels in YHB1 and Δyhb1 yeast cells. Densitometric analysis of the immunodetection of αSyn and A30P relative to the intensity obtained for GAPDH (n = 3).
Mentions: The effect of nitrative stress on the toxicity and aggregation of wild-type and A30P mutant αSyn was examined. A yeast strain carrying a deletion in the yeast flavohemoglobin gene (YHB1), responsible for stress signaling, was used for enhancement of nitrative stress. Yhb1 is a nitric oxide oxidoreductase, which protects against nitration of cellular targets and against cell growth inhibition under aerobic or anaerobic conditions. Deletion of YHB1 abolishes the nitric oxide (NO) consuming activity of yeast cells [74]. The compound DETA-NONOate causes nitrative stress by acting as a NO donor. The absence of the flavohemoglobin results in a growth impairment of the hypersensitive yhb1 deletion strain in comparison to wild-type under NO nitrative stress conditions (Fig 5A).

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