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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 posttranslational modifications and nitrative stress in yeast.Enhanced intracellular nitrative stress increases the protein nitration level and influences yeast growth and aggregation. The nitration of tyrosine residues acts as trigger for αSyn and A30P toxicity. Wild-type αSyn, which is highly nitrated, inhibits growth and shows a high aggregation rate. A30P is weakly nitrated and therefore, does not inhibit yeast growth and has a low aggregation propensity. Yhb1 and its human homolog NGB protect the cells against accumulation of nitrative species and diminish the aggregate formation. Di-tyrosine crosslinked dimers are formed in reverse correlation to cytotoxicity and do not depend on Yhb1. A30P forms twice as many dimers as the toxic αSyn variant, suggesting that the di-tyrosine crosslinked dimers are not toxic species and are presumably part of a cellular detoxification pathway, sequestering the protein off-pathway of αSyn nucleation. The C-terminal tyrosine modifications have dual effect on the toxicity of the protein. Y133, which is nitrated and phosphorylated, is required for the protective phosphorylation at S129 and for the autophagy degradation of αSyn aggregates. Non-modified Y133 promotes the proteasomal degradation of αSyn aggregates. N: nitration; P: phosphorylation.
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pgen.1006098.g014: αSyn posttranslational modifications and nitrative stress in yeast.Enhanced intracellular nitrative stress increases the protein nitration level and influences yeast growth and aggregation. The nitration of tyrosine residues acts as trigger for αSyn and A30P toxicity. Wild-type αSyn, which is highly nitrated, inhibits growth and shows a high aggregation rate. A30P is weakly nitrated and therefore, does not inhibit yeast growth and has a low aggregation propensity. Yhb1 and its human homolog NGB protect the cells against accumulation of nitrative species and diminish the aggregate formation. Di-tyrosine crosslinked dimers are formed in reverse correlation to cytotoxicity and do not depend on Yhb1. A30P forms twice as many dimers as the toxic αSyn variant, suggesting that the di-tyrosine crosslinked dimers are not toxic species and are presumably part of a cellular detoxification pathway, sequestering the protein off-pathway of αSyn nucleation. The C-terminal tyrosine modifications have dual effect on the toxicity of the protein. Y133, which is nitrated and phosphorylated, is required for the protective phosphorylation at S129 and for the autophagy degradation of αSyn aggregates. Non-modified Y133 promotes the proteasomal degradation of αSyn aggregates. N: nitration; P: phosphorylation.

Mentions: Phosphorylation at serine residue S129 represents the major protective phosphorylation site of αSyn which is conserved from man to the baker’s yeast as a eukaryotic Morbus Parkinson cell model. The effect of nitrative modifications of αSyn and their contribution towards αSyn-induced cytotoxicity was investigated. A complex interplay was discovered between modifications of the C-terminal tyrosine residues and S129 phosphorylation (Fig 14). These tyrosine residues of αSyn can be phosphorylated or nitrated with drastic consequences for cellular growth. There is a strong preference of the C-terminus of αSyn for nitration or di-tyrosine formation. Nitration interferes with protective phosphorylation of S129, whereas di-tyrosine formation protects yeast cells. The yeast nitric oxidoreductase Yhb1 as well as its related human protein neuroglobin play protective roles against αSyn aggregation. Yhb1 decreases the nitration level of the A30P variant of αSyn by reducing the accumulation of reactive nitrogen species, resulting that yeast cells can tolerate increased levels of this αSyn variant without significant growth inhibition.


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 posttranslational modifications and nitrative stress in yeast.Enhanced intracellular nitrative stress increases the protein nitration level and influences yeast growth and aggregation. The nitration of tyrosine residues acts as trigger for αSyn and A30P toxicity. Wild-type αSyn, which is highly nitrated, inhibits growth and shows a high aggregation rate. A30P is weakly nitrated and therefore, does not inhibit yeast growth and has a low aggregation propensity. Yhb1 and its human homolog NGB protect the cells against accumulation of nitrative species and diminish the aggregate formation. Di-tyrosine crosslinked dimers are formed in reverse correlation to cytotoxicity and do not depend on Yhb1. A30P forms twice as many dimers as the toxic αSyn variant, suggesting that the di-tyrosine crosslinked dimers are not toxic species and are presumably part of a cellular detoxification pathway, sequestering the protein off-pathway of αSyn nucleation. The C-terminal tyrosine modifications have dual effect on the toxicity of the protein. Y133, which is nitrated and phosphorylated, is required for the protective phosphorylation at S129 and for the autophagy degradation of αSyn aggregates. Non-modified Y133 promotes the proteasomal degradation of αSyn aggregates. N: nitration; P: phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g014: αSyn posttranslational modifications and nitrative stress in yeast.Enhanced intracellular nitrative stress increases the protein nitration level and influences yeast growth and aggregation. The nitration of tyrosine residues acts as trigger for αSyn and A30P toxicity. Wild-type αSyn, which is highly nitrated, inhibits growth and shows a high aggregation rate. A30P is weakly nitrated and therefore, does not inhibit yeast growth and has a low aggregation propensity. Yhb1 and its human homolog NGB protect the cells against accumulation of nitrative species and diminish the aggregate formation. Di-tyrosine crosslinked dimers are formed in reverse correlation to cytotoxicity and do not depend on Yhb1. A30P forms twice as many dimers as the toxic αSyn variant, suggesting that the di-tyrosine crosslinked dimers are not toxic species and are presumably part of a cellular detoxification pathway, sequestering the protein off-pathway of αSyn nucleation. The C-terminal tyrosine modifications have dual effect on the toxicity of the protein. Y133, which is nitrated and phosphorylated, is required for the protective phosphorylation at S129 and for the autophagy degradation of αSyn aggregates. Non-modified Y133 promotes the proteasomal degradation of αSyn aggregates. N: nitration; P: phosphorylation.
Mentions: Phosphorylation at serine residue S129 represents the major protective phosphorylation site of αSyn which is conserved from man to the baker’s yeast as a eukaryotic Morbus Parkinson cell model. The effect of nitrative modifications of αSyn and their contribution towards αSyn-induced cytotoxicity was investigated. A complex interplay was discovered between modifications of the C-terminal tyrosine residues and S129 phosphorylation (Fig 14). These tyrosine residues of αSyn can be phosphorylated or nitrated with drastic consequences for cellular growth. There is a strong preference of the C-terminus of αSyn for nitration or di-tyrosine formation. Nitration interferes with protective phosphorylation of S129, whereas di-tyrosine formation protects yeast cells. The yeast nitric oxidoreductase Yhb1 as well as its related human protein neuroglobin play protective roles against αSyn aggregation. Yhb1 decreases the nitration level of the A30P variant of αSyn by reducing the accumulation of reactive nitrogen species, resulting that yeast cells can tolerate increased levels of this αSyn variant without significant growth inhibition.

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