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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 forms dimers in vivo.(A) Spotting analysis of yeast cells expressing C-terminally HIS6-tagged αSyn and A30P αSyn on a high copy vector (2μ) driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Control cells expressed only the empty vector pME2795 (EV). (B) Western blotting of αSyn and A30P enriched from cell extracts by Ni2+ pull-down with anti-αSyn antibody. In vitro nitration was carried out with 15 μg of αSyn extracts using 1 μl peroxynitrite (PON) in the presence of 1 μl 0.3 M HCl. (C) Quantification of dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers in vivo and in PON-treated samples. The amount of dimers is presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with t-test (**, p < 0.01, n = 4).
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pgen.1006098.g001: αSyn forms dimers in vivo.(A) Spotting analysis of yeast cells expressing C-terminally HIS6-tagged αSyn and A30P αSyn on a high copy vector (2μ) driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Control cells expressed only the empty vector pME2795 (EV). (B) Western blotting of αSyn and A30P enriched from cell extracts by Ni2+ pull-down with anti-αSyn antibody. In vitro nitration was carried out with 15 μg of αSyn extracts using 1 μl peroxynitrite (PON) in the presence of 1 μl 0.3 M HCl. (C) Quantification of dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers in vivo and in PON-treated samples. The amount of dimers is presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with t-test (**, p < 0.01, n = 4).

Mentions: The first approach was to examine whether αSyn and A30P form dimers in vivo without additional exposure of the cells to nitrating or oxidative agents. αSyn and A30P expression was driven by the GAL1-promoter which was repressed in the presence of glucose and induced when shifted to 2% galactose-containing medium for 12 hours (h). High copy number expression of the HIS6-tagged αSyn resulted in growth inhibition whereas high expression of the A30P mutant resulted in a similar growth rate as the yeast control without any αSyn (Fig 1A). Similar results were previously reported with untagged or GFP-tagged αSyn and corroborate that the HIS6-tag does not interfere with the behavior of αSyn in yeast [60, 63]. αSyn proteins were enriched by Ni2+ pull-down under denaturing conditions in the presence of urea. Immunoblotting with anti-αSyn antibody revealed distinct bands, corresponding to monomeric (~17 kDa), dimeric (~35 kDa) and higher molecular weight αSyn species (oligomers), detected from in vivo samples (Fig 1B). This supports that αSyn and the A30P mutant form dimers and oligomers in vivo even without additional exposure of the cells to nitrating or oxidative agents.


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 forms dimers in vivo.(A) Spotting analysis of yeast cells expressing C-terminally HIS6-tagged αSyn and A30P αSyn on a high copy vector (2μ) driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Control cells expressed only the empty vector pME2795 (EV). (B) Western blotting of αSyn and A30P enriched from cell extracts by Ni2+ pull-down with anti-αSyn antibody. In vitro nitration was carried out with 15 μg of αSyn extracts using 1 μl peroxynitrite (PON) in the presence of 1 μl 0.3 M HCl. (C) Quantification of dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers in vivo and in PON-treated samples. The amount of dimers is presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with t-test (**, 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.g001: αSyn forms dimers in vivo.(A) Spotting analysis of yeast cells expressing C-terminally HIS6-tagged αSyn and A30P αSyn on a high copy vector (2μ) driven by the inducible GAL1-promoter on non-inducing (´OFF`: glucose) and inducing (´ON`: galactose) SC-Ura medium after 3 days. Control cells expressed only the empty vector pME2795 (EV). (B) Western blotting of αSyn and A30P enriched from cell extracts by Ni2+ pull-down with anti-αSyn antibody. In vitro nitration was carried out with 15 μg of αSyn extracts using 1 μl peroxynitrite (PON) in the presence of 1 μl 0.3 M HCl. (C) Quantification of dimers. Densitometric analysis of the immunodetection of αSyn and A30P αSyn dimers in vivo and in PON-treated samples. The amount of dimers is presented as percent of the total amount of αSyn detected per lane (monomer + dimer). Significance of differences was calculated with t-test (**, p < 0.01, n = 4).
Mentions: The first approach was to examine whether αSyn and A30P form dimers in vivo without additional exposure of the cells to nitrating or oxidative agents. αSyn and A30P expression was driven by the GAL1-promoter which was repressed in the presence of glucose and induced when shifted to 2% galactose-containing medium for 12 hours (h). High copy number expression of the HIS6-tagged αSyn resulted in growth inhibition whereas high expression of the A30P mutant resulted in a similar growth rate as the yeast control without any αSyn (Fig 1A). Similar results were previously reported with untagged or GFP-tagged αSyn and corroborate that the HIS6-tag does not interfere with the behavior of αSyn in yeast [60, 63]. αSyn proteins were enriched by Ni2+ pull-down under denaturing conditions in the presence of urea. Immunoblotting with anti-αSyn antibody revealed distinct bands, corresponding to monomeric (~17 kDa), dimeric (~35 kDa) and higher molecular weight αSyn species (oligomers), detected from in vivo samples (Fig 1B). This supports that αSyn and the A30P mutant form dimers and oligomers in vivo even without additional exposure of the cells to nitrating or oxidative agents.

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