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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 deletion increases accumulation of reactive nitrogen species (RNS) in A30P expressing cells.(A) αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing medium for 6 h in YHB1 wild-type or Δyhb1 deletion yeast strains. Cells were incubated with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation for 1.5 h and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (B) Fluorescent intensity of cells from (A), assessed with flow cytometry analysis. Forward scatter (FSC) and DHR123 fluorescence of cells after 6 h induction of αSyn expression. (C) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying ROS stained by DHR123 using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. (D) Microscopy analysis of RNS stained cells. αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing SC-Ura medium for 6 h in YHB1 and Δyhb1 yeast strains. Cells were incubated with DAF-2 DA for 1 h at 30°C for visualization of RNS and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (E) Fluorescent intensity of cells from (D), assessed with flow cytometry analysis. Forward scatter (FSC) and DAF-2 DA fluorescence of cells after 6 h induction of αSyn expression. (F) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying RNS stained by DAF-2 DA using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).
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pgen.1006098.g007: YHB1 deletion increases accumulation of reactive nitrogen species (RNS) in A30P expressing cells.(A) αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing medium for 6 h in YHB1 wild-type or Δyhb1 deletion yeast strains. Cells were incubated with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation for 1.5 h and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (B) Fluorescent intensity of cells from (A), assessed with flow cytometry analysis. Forward scatter (FSC) and DHR123 fluorescence of cells after 6 h induction of αSyn expression. (C) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying ROS stained by DHR123 using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. (D) Microscopy analysis of RNS stained cells. αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing SC-Ura medium for 6 h in YHB1 and Δyhb1 yeast strains. Cells were incubated with DAF-2 DA for 1 h at 30°C for visualization of RNS and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (E) Fluorescent intensity of cells from (D), assessed with flow cytometry analysis. Forward scatter (FSC) and DAF-2 DA fluorescence of cells after 6 h induction of αSyn expression. (F) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying RNS stained by DAF-2 DA using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).

Mentions: Dihydrorhodamine 123 (DHR123) was used for ROS detection. The dye accumulates in cells, where it is oxidized by free radicals to the bright red fluorescent product rhodamine 123 (Fig 7A–7C). Expression of A30P and its derivative A30P/4(Y/F) did not significantly increase the number of cells accumulating ROS. In contrast, expression of wild-type αSyn as well as its 4(Y/F) derivative strongly increased the number of cells that accumulate red fluorescence indicative for ROS (Fig 7C). No difference in ROS accumulation was observed between the YHB1 wild-type and the Δyhb1 mutant strain.


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 deletion increases accumulation of reactive nitrogen species (RNS) in A30P expressing cells.(A) αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing medium for 6 h in YHB1 wild-type or Δyhb1 deletion yeast strains. Cells were incubated with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation for 1.5 h and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (B) Fluorescent intensity of cells from (A), assessed with flow cytometry analysis. Forward scatter (FSC) and DHR123 fluorescence of cells after 6 h induction of αSyn expression. (C) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying ROS stained by DHR123 using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. (D) Microscopy analysis of RNS stained cells. αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing SC-Ura medium for 6 h in YHB1 and Δyhb1 yeast strains. Cells were incubated with DAF-2 DA for 1 h at 30°C for visualization of RNS and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (E) Fluorescent intensity of cells from (D), assessed with flow cytometry analysis. Forward scatter (FSC) and DAF-2 DA fluorescence of cells after 6 h induction of αSyn expression. (F) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying RNS stained by DAF-2 DA using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. 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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getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g007: YHB1 deletion increases accumulation of reactive nitrogen species (RNS) in A30P expressing cells.(A) αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing medium for 6 h in YHB1 wild-type or Δyhb1 deletion yeast strains. Cells were incubated with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation for 1.5 h and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (B) Fluorescent intensity of cells from (A), assessed with flow cytometry analysis. Forward scatter (FSC) and DHR123 fluorescence of cells after 6 h induction of αSyn expression. (C) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying ROS stained by DHR123 using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. (D) Microscopy analysis of RNS stained cells. αSyn, A30P, 4(Y/F) and A30P/4(Y/F) were induced in galactose-containing SC-Ura medium for 6 h in YHB1 and Δyhb1 yeast strains. Cells were incubated with DAF-2 DA for 1 h at 30°C for visualization of RNS and analyzed by live-cell fluorescence microscopy. Scale bar = 5 μm. (E) Fluorescent intensity of cells from (D), assessed with flow cytometry analysis. Forward scatter (FSC) and DAF-2 DA fluorescence of cells after 6 h induction of αSyn expression. (F) Quantification of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) expressing cells displaying RNS stained by DAF-2 DA using flow cytometry. The percentage of the sub-population of yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with t-test (**, p < 0.01, n = 3).
Mentions: Dihydrorhodamine 123 (DHR123) was used for ROS detection. The dye accumulates in cells, where it is oxidized by free radicals to the bright red fluorescent product rhodamine 123 (Fig 7A–7C). Expression of A30P and its derivative A30P/4(Y/F) did not significantly increase the number of cells accumulating ROS. In contrast, expression of wild-type αSyn as well as its 4(Y/F) derivative strongly increased the number of cells that accumulate red fluorescence indicative for ROS (Fig 7C). No difference in ROS accumulation was observed between the YHB1 wild-type and the Δyhb1 mutant strain.

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