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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

Blocking of αSyn tyrosine nitration decreases aggregation and cytotoxicity.(A) Expression of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) αSyn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni2+ pull-down from yeast cell extracts. For in vitro nitration, 1 μl peroxynitrite (PON) was mixed with 15 μg of αSyn extracts in the presence of 1 μl 0.3 M HCl. Western blotting with di-tyrosine antibody reveals a major band at about 36 kDa, corresponding to dimers. Additional bands with lower molecular weights are observed, probably due to intramolecular di-tyrosine crosslinking. The same membrane was stripped and re-probed with αSyn antibody. (B) Western blotting using 3-nitro-tyrosine antibody (3-NT). Phenylalanine codon substitutions eliminate immunoreactivity. The same membrane was stripped and re-probed with αSyn antibody. (C) Spotting analysis of yeast cells expressing GAL1-driven αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (αSyn ‘OFF’) or galactose (αSyn ‘ON’). (D) Cell growth analysis of yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control) in galactose-containing SC-Ura medium for 40 h. Error bars represent standard deviations of three independent experiments. (E) Fluorescence microscopy of yeast cells, expressing indicated αSyn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 μm. (F) Quantification of the percentage of cells displaying aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (*, p < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged αSyn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.
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pgen.1006098.g004: Blocking of αSyn tyrosine nitration decreases aggregation and cytotoxicity.(A) Expression of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) αSyn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni2+ pull-down from yeast cell extracts. For in vitro nitration, 1 μl peroxynitrite (PON) was mixed with 15 μg of αSyn extracts in the presence of 1 μl 0.3 M HCl. Western blotting with di-tyrosine antibody reveals a major band at about 36 kDa, corresponding to dimers. Additional bands with lower molecular weights are observed, probably due to intramolecular di-tyrosine crosslinking. The same membrane was stripped and re-probed with αSyn antibody. (B) Western blotting using 3-nitro-tyrosine antibody (3-NT). Phenylalanine codon substitutions eliminate immunoreactivity. The same membrane was stripped and re-probed with αSyn antibody. (C) Spotting analysis of yeast cells expressing GAL1-driven αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (αSyn ‘OFF’) or galactose (αSyn ‘ON’). (D) Cell growth analysis of yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control) in galactose-containing SC-Ura medium for 40 h. Error bars represent standard deviations of three independent experiments. (E) Fluorescence microscopy of yeast cells, expressing indicated αSyn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 μm. (F) Quantification of the percentage of cells displaying aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (*, p < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged αSyn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.

Mentions: The codons for the four tyrosine sites of αSyn and A30P (Y39, Y125, Y133 and Y136) were replaced in the corresponding genes by phenylalanine codons (4(Y/F)) to analyze the role of the tyrosine residues on αSyn dimer formation, cytotoxicity or aggregation. Fusion genes with GFP-tags or HIS6-tags were constructed and expressed. We assessed whether the quadruple Y to F replacements influence the dimerization of αSyn and A30P. Expression of αSyn and A30P as well as their 4(Y/F) mutants was induced for 12 h. Tagged proteins were enriched by Ni2+ pull-down under denaturing conditions. Immunoblotting using αSyn antibodies as well as antibodies that specifically recognize di-tyrosines revealed that 4(Y/F) mutants of αSyn or A30P had lost the potential to form dimers in vivo (Fig 4A). Additional in vitro nitration with PON did also not result in any dimer or oligomer formation and served as control (Fig 4A). Immunoblotting analysis was carried out to determine in vivo nitrated αSyn using 3-nitro-tyrosine specific antibodies (Fig 4B). The results demonstrated that the 4(Y/F) variants of wild-type αSyn or A30P did not result in any nitration signal even after additional PON treatment. This is in contrast to wild-type αSyn with its four original tyrosine residues as control where nitration is present in vivo and can be further increased by additional PON treatment.


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)

Blocking of αSyn tyrosine nitration decreases aggregation and cytotoxicity.(A) Expression of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) αSyn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni2+ pull-down from yeast cell extracts. For in vitro nitration, 1 μl peroxynitrite (PON) was mixed with 15 μg of αSyn extracts in the presence of 1 μl 0.3 M HCl. Western blotting with di-tyrosine antibody reveals a major band at about 36 kDa, corresponding to dimers. Additional bands with lower molecular weights are observed, probably due to intramolecular di-tyrosine crosslinking. The same membrane was stripped and re-probed with αSyn antibody. (B) Western blotting using 3-nitro-tyrosine antibody (3-NT). Phenylalanine codon substitutions eliminate immunoreactivity. The same membrane was stripped and re-probed with αSyn antibody. (C) Spotting analysis of yeast cells expressing GAL1-driven αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (αSyn ‘OFF’) or galactose (αSyn ‘ON’). (D) Cell growth analysis of yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control) in galactose-containing SC-Ura medium for 40 h. Error bars represent standard deviations of three independent experiments. (E) Fluorescence microscopy of yeast cells, expressing indicated αSyn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 μm. (F) Quantification of the percentage of cells displaying aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (*, p < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged αSyn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4920419&req=5

pgen.1006098.g004: Blocking of αSyn tyrosine nitration decreases aggregation and cytotoxicity.(A) Expression of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) αSyn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni2+ pull-down from yeast cell extracts. For in vitro nitration, 1 μl peroxynitrite (PON) was mixed with 15 μg of αSyn extracts in the presence of 1 μl 0.3 M HCl. Western blotting with di-tyrosine antibody reveals a major band at about 36 kDa, corresponding to dimers. Additional bands with lower molecular weights are observed, probably due to intramolecular di-tyrosine crosslinking. The same membrane was stripped and re-probed with αSyn antibody. (B) Western blotting using 3-nitro-tyrosine antibody (3-NT). Phenylalanine codon substitutions eliminate immunoreactivity. The same membrane was stripped and re-probed with αSyn antibody. (C) Spotting analysis of yeast cells expressing GAL1-driven αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (αSyn ‘OFF’) or galactose (αSyn ‘ON’). (D) Cell growth analysis of yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control) in galactose-containing SC-Ura medium for 40 h. Error bars represent standard deviations of three independent experiments. (E) Fluorescence microscopy of yeast cells, expressing indicated αSyn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 μm. (F) Quantification of the percentage of cells displaying aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (*, p < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged αSyn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.
Mentions: The codons for the four tyrosine sites of αSyn and A30P (Y39, Y125, Y133 and Y136) were replaced in the corresponding genes by phenylalanine codons (4(Y/F)) to analyze the role of the tyrosine residues on αSyn dimer formation, cytotoxicity or aggregation. Fusion genes with GFP-tags or HIS6-tags were constructed and expressed. We assessed whether the quadruple Y to F replacements influence the dimerization of αSyn and A30P. Expression of αSyn and A30P as well as their 4(Y/F) mutants was induced for 12 h. Tagged proteins were enriched by Ni2+ pull-down under denaturing conditions. Immunoblotting using αSyn antibodies as well as antibodies that specifically recognize di-tyrosines revealed that 4(Y/F) mutants of αSyn or A30P had lost the potential to form dimers in vivo (Fig 4A). Additional in vitro nitration with PON did also not result in any dimer or oligomer formation and served as control (Fig 4A). Immunoblotting analysis was carried out to determine in vivo nitrated αSyn using 3-nitro-tyrosine specific antibodies (Fig 4B). The results demonstrated that the 4(Y/F) variants of wild-type αSyn or A30P did not result in any nitration signal even after additional PON treatment. This is in contrast to wild-type αSyn with its four original tyrosine residues as control where nitration is present in vivo and can be further increased by additional PON treatment.

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