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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

Yhb1 protects mitochondria from A30P toxicity.(A) Live-cell fluorescence microscopy of YHB1 compared to Δyhb1 yeast cells expressing GFP (control), αSyn or A30P after 6 h induction in galactose-containing medium. MitoTracker Red was used to visualize mitochondria in the cells (MT panel). αSyn expressing cells with plasma membrane localization (PM) and with aggregates are visualized. Scale bar = 1 μM. (B) Quantification of yeast cells with tubular mitochondrial network. GFP: percentage of all cells with tubular mitochondria; αSyn* and A30P*: percentage of cells with aggregates, showing tubular mitochondria. At least 50 cells were counted per cell type and experiment. Significance of differences was calculated with t-test (*, p < 0.05, n = 4). (C) Quantification of yeast cells with tubular mitochondrial network for rescue of A30P phenotype. A30P with empty vector (EV) in YHB1 and Δyhb1 strain and A30P co-transformed with YHB1 on low-copy vector in Δyhb1 strain. A30P*: percentage of cells with aggregates, showing tubular mitochondria. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).
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pgen.1006098.g008: Yhb1 protects mitochondria from A30P toxicity.(A) Live-cell fluorescence microscopy of YHB1 compared to Δyhb1 yeast cells expressing GFP (control), αSyn or A30P after 6 h induction in galactose-containing medium. MitoTracker Red was used to visualize mitochondria in the cells (MT panel). αSyn expressing cells with plasma membrane localization (PM) and with aggregates are visualized. Scale bar = 1 μM. (B) Quantification of yeast cells with tubular mitochondrial network. GFP: percentage of all cells with tubular mitochondria; αSyn* and A30P*: percentage of cells with aggregates, showing tubular mitochondria. At least 50 cells were counted per cell type and experiment. Significance of differences was calculated with t-test (*, p < 0.05, n = 4). (C) Quantification of yeast cells with tubular mitochondrial network for rescue of A30P phenotype. A30P with empty vector (EV) in YHB1 and Δyhb1 strain and A30P co-transformed with YHB1 on low-copy vector in Δyhb1 strain. A30P*: percentage of cells with aggregates, showing tubular mitochondria. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).

Mentions: We examined whether deletion of YHB1 influences the mitochondrial morphology in αSyn and A30P αSyn expressing yeast cells. αSyn expression in the wild-type and Δyhb1 background was induced for 6 h in galactose medium and the mitochondria were visualized with a mitochondrial specific dye (MitoTracker Red). Cells expressing GFP were used as a control (Fig 8A). We could not detect co-localization of αSyn with mitochondria, which suggests that the described mitochondrial fraction of the protein might be small [78]. The mitochondrial morphology was classified as tubular, partially fragmented or fully fragmented. In the control cells, the mitochondria revealed a ribbon-like tubular architecture, typical for healthy mitochondria. Cells expressing αSyn showed a dramatic increase in the percentage of cells with fully fragmented mitochondria (Fig 8A and 8B). Cells with and without inclusions were considered separately for statistical evaluation. Cells with plasma-membrane localization of the GFP-signal, typical αSyn localization for early stages of expression or lower expression levels, revealed partially fragmented mitochondrial architecture. αSyn expressing cells with aggregates had fully fragmented mitochondria. In contrast to αSyn, A30P expressing cells with aggregates had mainly tubular mitochondria, similar to the control cells (Fig 8A). Deletion of YHB1 increased the percentage of cells with fully fragmented mitochondria almost two-fold (Fig 8B). Thus, the disrupted mitochondrial morphology in Δyhb1 correlates with the increased levels of RNS (Fig 7D–7F) and diminished growth behavior of A30P expressing cells (Fig 5B and 5D). Complementation of the Δyhb1 phenotype in A30P expressing cells rescued the defect (Fig 8C), as mitochondrial morphology was recovered by expression of YHB1 on a low-copy vector. The result suggests that Yhb1 protects against A30P-induced cytotoxicity by preventing the mitochondrial fragmentation.


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)

Yhb1 protects mitochondria from A30P toxicity.(A) Live-cell fluorescence microscopy of YHB1 compared to Δyhb1 yeast cells expressing GFP (control), αSyn or A30P after 6 h induction in galactose-containing medium. MitoTracker Red was used to visualize mitochondria in the cells (MT panel). αSyn expressing cells with plasma membrane localization (PM) and with aggregates are visualized. Scale bar = 1 μM. (B) Quantification of yeast cells with tubular mitochondrial network. GFP: percentage of all cells with tubular mitochondria; αSyn* and A30P*: percentage of cells with aggregates, showing tubular mitochondria. At least 50 cells were counted per cell type and experiment. Significance of differences was calculated with t-test (*, p < 0.05, n = 4). (C) Quantification of yeast cells with tubular mitochondrial network for rescue of A30P phenotype. A30P with empty vector (EV) in YHB1 and Δyhb1 strain and A30P co-transformed with YHB1 on low-copy vector in Δyhb1 strain. A30P*: percentage of cells with aggregates, showing tubular mitochondria. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).
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Related In: Results  -  Collection

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pgen.1006098.g008: Yhb1 protects mitochondria from A30P toxicity.(A) Live-cell fluorescence microscopy of YHB1 compared to Δyhb1 yeast cells expressing GFP (control), αSyn or A30P after 6 h induction in galactose-containing medium. MitoTracker Red was used to visualize mitochondria in the cells (MT panel). αSyn expressing cells with plasma membrane localization (PM) and with aggregates are visualized. Scale bar = 1 μM. (B) Quantification of yeast cells with tubular mitochondrial network. GFP: percentage of all cells with tubular mitochondria; αSyn* and A30P*: percentage of cells with aggregates, showing tubular mitochondria. At least 50 cells were counted per cell type and experiment. Significance of differences was calculated with t-test (*, p < 0.05, n = 4). (C) Quantification of yeast cells with tubular mitochondrial network for rescue of A30P phenotype. A30P with empty vector (EV) in YHB1 and Δyhb1 strain and A30P co-transformed with YHB1 on low-copy vector in Δyhb1 strain. A30P*: percentage of cells with aggregates, showing tubular mitochondria. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).
Mentions: We examined whether deletion of YHB1 influences the mitochondrial morphology in αSyn and A30P αSyn expressing yeast cells. αSyn expression in the wild-type and Δyhb1 background was induced for 6 h in galactose medium and the mitochondria were visualized with a mitochondrial specific dye (MitoTracker Red). Cells expressing GFP were used as a control (Fig 8A). We could not detect co-localization of αSyn with mitochondria, which suggests that the described mitochondrial fraction of the protein might be small [78]. The mitochondrial morphology was classified as tubular, partially fragmented or fully fragmented. In the control cells, the mitochondria revealed a ribbon-like tubular architecture, typical for healthy mitochondria. Cells expressing αSyn showed a dramatic increase in the percentage of cells with fully fragmented mitochondria (Fig 8A and 8B). Cells with and without inclusions were considered separately for statistical evaluation. Cells with plasma-membrane localization of the GFP-signal, typical αSyn localization for early stages of expression or lower expression levels, revealed partially fragmented mitochondrial architecture. αSyn expressing cells with aggregates had fully fragmented mitochondria. In contrast to αSyn, A30P expressing cells with aggregates had mainly tubular mitochondria, similar to the control cells (Fig 8A). Deletion of YHB1 increased the percentage of cells with fully fragmented mitochondria almost two-fold (Fig 8B). Thus, the disrupted mitochondrial morphology in Δyhb1 correlates with the increased levels of RNS (Fig 7D–7F) and diminished growth behavior of A30P expressing cells (Fig 5B and 5D). Complementation of the Δyhb1 phenotype in A30P expressing cells rescued the defect (Fig 8C), as mitochondrial morphology was recovered by expression of YHB1 on a low-copy vector. The result suggests that Yhb1 protects against A30P-induced cytotoxicity by preventing the mitochondrial fragmentation.

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