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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

Determination of crosslinked peptides from αSyn and A30P.(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated αSyn samples. (B) Distribution of all identified di-tyrosine peptides for αSyn. Identified combinations of crosslinked peptides are presented as percentage of n (n = total number of MS2 spectra verified as crosslinked peptides). (C) Distribution of all identified di-tyrosine peptides for A30P.
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pgen.1006098.g003: Determination of crosslinked peptides from αSyn and A30P.(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated αSyn samples. (B) Distribution of all identified di-tyrosine peptides for αSyn. Identified combinations of crosslinked peptides are presented as percentage of n (n = total number of MS2 spectra verified as crosslinked peptides). (C) Distribution of all identified di-tyrosine peptides for A30P.

Mentions: The LC-MS spectra of αSyn and A30P migrating in SDS-PAGE with the size of the dimer band were analyzed to assess whether di-tyrosines cause dimer formation of αSyn or A30P. The presence of di-tyrosine peptide crosslinks was validated using StavroX2.3.4.5 software [73]. This software compares the masses of all potential crosslinked peptides with the precursor ion masses, calculates b- and y-type ions for all possible crosslinks and compares them to MS2 data of the precursor ion. Different combinations of crosslinked peptides with an identical mass are possible when multiple tyrosine residues are located on one and the same peptide. The crosslinked tyrosine pairs were assigned according to the scores calculated by StavroX based on the fragment ion series of the MS2 spectra. The MS data analysis verified that αSyn dimers are crosslinked by tyrosine residues. The detected combinations of crosslinked tyrosines are depicted in Fig 3A. The results indicate a strong preference for crosslinking of defined combinations of tyrosines (Figs 3A, 3B, 3C and S1). The most frequent combinations for either wild-type αSyn or A30P are Y125-Y136 and Y133-Y136 dimers which are all located in the C-terminus. Only the C-terminal tyrosine residues can mutually interact. Only a small fraction of Y39-Y39 dimers were found and there are no tyrosine dimers between the N-terminal Y39 and the C-terminal tyrosines of αSyn or A30P.


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)

Determination of crosslinked peptides from αSyn and A30P.(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated αSyn samples. (B) Distribution of all identified di-tyrosine peptides for αSyn. Identified combinations of crosslinked peptides are presented as percentage of n (n = total number of MS2 spectra verified as crosslinked peptides). (C) Distribution of all identified di-tyrosine peptides for A30P.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g003: Determination of crosslinked peptides from αSyn and A30P.(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated αSyn samples. (B) Distribution of all identified di-tyrosine peptides for αSyn. Identified combinations of crosslinked peptides are presented as percentage of n (n = total number of MS2 spectra verified as crosslinked peptides). (C) Distribution of all identified di-tyrosine peptides for A30P.
Mentions: The LC-MS spectra of αSyn and A30P migrating in SDS-PAGE with the size of the dimer band were analyzed to assess whether di-tyrosines cause dimer formation of αSyn or A30P. The presence of di-tyrosine peptide crosslinks was validated using StavroX2.3.4.5 software [73]. This software compares the masses of all potential crosslinked peptides with the precursor ion masses, calculates b- and y-type ions for all possible crosslinks and compares them to MS2 data of the precursor ion. Different combinations of crosslinked peptides with an identical mass are possible when multiple tyrosine residues are located on one and the same peptide. The crosslinked tyrosine pairs were assigned according to the scores calculated by StavroX based on the fragment ion series of the MS2 spectra. The MS data analysis verified that αSyn dimers are crosslinked by tyrosine residues. The detected combinations of crosslinked tyrosines are depicted in Fig 3A. The results indicate a strong preference for crosslinking of defined combinations of tyrosines (Figs 3A, 3B, 3C and S1). The most frequent combinations for either wild-type αSyn or A30P are Y125-Y136 and Y133-Y136 dimers which are all located in the C-terminus. Only the C-terminal tyrosine residues can mutually interact. Only a small fraction of Y39-Y39 dimers were found and there are no tyrosine dimers between the N-terminal Y39 and the C-terminal tyrosines of αSyn or A30P.

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