31 research outputs found

    C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of ╬▒-Synuclein in a Yeast Model of Parkinson's Disease

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    <div><p>Parkinson┬┤s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of ╬▒-synuclein (╬▒Syn). Elevated levels of oxidative or nitrative stresses have been implicated in ╬▒Syn related toxicity. Phosphorylation of ╬▒Syn 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. ╬▒Syn 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 ╬▒Syn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of ╬▒Syn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type ╬▒Syn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type ╬▒Syn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene <i>YHB1</i> resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P ╬▒Syn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of <i>YHB1</i>, protected against ╬▒Syn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in ╬▒Syn 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 ╬▒Syn di-tyrosine dimers. Our findings uncover a complex interplay between S129 phosphorylation and C-terminal tyrosine modifications of ╬▒Syn that likely participates in PD pathology.</p></div

    The human <i>NGB</i> gene for neuroglobin alters A30P and ╬▒Syn aggregation in yeast and mammalian cells.

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    <p>(A) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing αSyn and GFP (control) with either empty vector as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (B) Quantification of the percentage of cells displaying αSyn aggregates after 6 h induction in galactose-containing medium (n = 3). (C) Spotting analysis of <i>YHB1</i> and Δ<i>yhb1</i> yeast cells co-expressing A30P and GFP (control) with either empty vector (pME2788) as control or <i>YHB1</i> and <i>NGB</i>, respectively, on non-inducing and galactose-inducing SC-Ura medium after 3 days. (D) Quantification of the percentage of cells displaying A30P aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (**, <i>p</i> < 0.01, n = 3). (E) Fluorescence microscopy of H4 cells co-expressing SynT, synphilin-1 and pcDNA (control) or NGB-mCherry. Nuclei are stained with Hoechst dye (blue). Scale bar = 30 μm. (F) Quantification of the percentage of H4 cells displaying αSyn inclusions after 48 h after transfection. Cells were classified into three groups according to the number of αSyn-immunoreactive inclusions observed: cells with 10 inclusions, cells with less than 10 inclusions and cells without inclusions. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05, n = 3). (G) Lactate dehydrogenase (LDH) activity measurements support that <i>NGB</i> is non-toxic for H4 cells. H4 cells transfected with empty mammalian expression vector pcDNA3.1, with empty pcDNA3.1 or pcDNA3.1 encoding neuroglobin-mCherry (<i>NGB</i>) together with SynT and synphilin-1 (SynT+Synphilin-1) were analyzed. Media from indicated H4 cells were collected and the secretion of lactate LDH was determined as a measure of cytotoxicity. Significance of differences was calculated with t-test (not significant (n.s.); n = 3).</p

    Determination of crosslinked peptides from ╬▒Syn and A30P.

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    <p>(A) Analysis of di-tyrosine dimers. Exemplary heat map diagram of the number (N) of identified di-tyrosine crosslinked peptides of the non-treated ╬▒Syn samples. (B) Distribution of all identified di-tyrosine peptides for ╬▒Syn. 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.</p

    Tyrosine 133 mutation does not alter the accumulation of reactive oxygen and nitrogen species.

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    <p>(A, B) Quantification of cells expressing different ╬▒Syn variants displaying ROS and RNS assessed with flow cytometry analysis. ╬▒Syn expression was induced for 6 h and the cells were stained for 1.5 h with DHR123 to visualize ROS (A) or with DAF-2 DA to visualize RNS (B). Forward scatter (FSC) and DHR123 (A) or DAF-2 DA (B) fluorescence of the cells, showing one representative result from at least four independent experiments. The percentage of the sub-populations of yeast cells with higher fluorescent intensities (P1) than the background are presented in the lower panels. Significance of differences was calculated with one-way ANOVA (****, <i>p</i> < 0.0001).</p

    ╬▒Syn aggregate clearance after promoter shut-off.

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    <p>(A, C) Quantification of cells displaying aggregates of ╬▒Syn (A) and A30P (C) upon inhibition of autophagy by PMSF. Cells expressing ╬▒Syn (A) or A30P (C) and its 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to 2% glucose-containing media supplemented with 1 mM PMSF dissolved in EtOH and only EtOH as a control. Cells with aggregates were counted after 4 h <i>GAL1</i>-promoter shut-off and presented as a ratio to the control (EtOH). Significance of differences was calculated with one-way ANOVA (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01) or DunnettÔÇÖs multiple comparison test (#, <i>p</i> < 0.05; ##, <i>p</i> < 0.01 versus ╬▒Syn; n = 4). (B, D) Quantification of cells displaying aggregates of ╬▒Syn (B) and A30P (D) upon inhibition of the proteasome by MG132. Cells expressing ╬▒Syn (B) or A30P (D) and the indicated 4(Y/F), S129A and Y133F variants were incubated in 2% galactose-containing media for 4 h and shifted to glucose medium, supplemented with 75 ╬╝M MG132, dissolved in DMSO or only DMSO as a control. Cells with aggregates were counted after 4 h <i>GAL1</i>-promoter shut off and presented as a ratio to the control (DMSO). Significance of differences was calculated with one-way ANOVA (***, <i>p</i> < 0.001) or DunnettÔÇÖs multiple comparison test (#, <i>p</i> < 0.05; ##, <i>p</i> < 0.01 versus ╬▒Syn; n = 4).</p

    Yhb1 protects mitochondria from A30P toxicity.

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    <p>(A) Live-cell fluorescence microscopy of <i>YHB1</i> compared to Δ<i>yhb1</i> 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 (*, <i>p</i> < 0.05, n = 4). (C) Quantification of yeast cells with tubular mitochondrial network for rescue of A30P phenotype. A30P with empty vector (EV) in <i>YHB1</i> and <i>Δyhb1</i> strain and A30P co-transformed with <i>YHB1</i> on low-copy vector in <i>Δyhb1</i> strain. A30P*: percentage of cells with aggregates, showing tubular mitochondria. Significance of differences was calculated with t-test (**, <i>p</i> < 0.01, n = 3).</p

    Tyrosine 133 is required for phosphorylation of ╬▒Syn at serine 129.

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    <p>(A) Western blotting of ╬▒Syn and A30P expressed in <i>YHB1</i> and ╬ö<i>yhb1</i> yeast enriched by Ni<sup>2+</sup> pull-down, using Y133 phosphorylation-specific ╬▒Syn antibody (pY133) and S129 phosphorylation-specific ╬▒Syn antibody (pS129). The same membrane was stripped and re-probed with ╬▒Syn antibody. (B) Quantification of ╬▒Syn and A30P Y133- and S129-phosphorylation levels in <i>YHB1</i> and ╬ö<i>yhb1</i> yeast cells. Densitometric analysis of the immunodetection of pY133, pS129 ╬▒Syn and A30P relative to the intensity obtained for ╬▒Syn. Significance of differences was calculated with one-way ANOVA test (**, <i>p</i> < 0.01; n = 4). (C) Western blotting of crude extracts from yeast cells, expressing different ╬▒Syn variants after 6 h induction of protein expression using S129 phosphorylation-specific ╬▒Syn antibody (pS129) and ╬▒Syn antibody. Cells expressing S129A mutant served as control. (D) Spotting analysis of ╬▒Syn and indicated mutant strains, driven by the inducible <i>GAL1-</i>promoter on non-inducing (┬┤OFF`: glucose) and inducing (┬┤ON`: galactose) SC-Ura medium after 3 days. Cells expressing GFP served as control. (E) Quantification of the percentage of cells displaying ╬▒Syn aggregates after 6 h induction in galactose-containing SC-Ura medium. Significance of differences was calculated with one-way ANOVA (***, <i>p</i> < 0.001) or DunnettÔÇÖs multiple comparison test (#, <i>p</i> < 0.05, ##, <i>p</i> < 0.01 versus ╬▒Syn; n = 6). (F) Cell growth analysis of cells expressing different ╬▒Syn variants and GFP (control) after 20 h induction of expression. Significance of differences was calculated with one-way ANOVA (****, <i>p</i> < 0.0001) or DunnettÔÇÖs multiple comparison test (#, <i>p</i> < 0.05; ###, <i>p</i> < 0.001, n = 4). (G) Quantification of cells expressing different ╬▒Syn variants and GFP (control) displaying Propidium Iodide (PI) fluorescence after 20 h induction of ╬▒Syn expression, assessed by flow cytometry. The percentage of PI-positive yeast cells with higher fluorescent intensities (P1) than the background is presented. Significance of differences was calculated with one-way ANOVA (****, <i>p</i> < 0.0001) or DunnettÔÇÖs multiple comparison test (#, <i>p</i> < 0.05; ###, <i>p</i> < 0.001 versus ╬▒Syn; n = 4).</p

    Blocking of ╬▒Syn tyrosine nitration decreases aggregation and cytotoxicity.

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    <p>(A) Expression of ╬▒Syn, A30P, 4(Y/F) and A30P/4(Y/F) ╬▒Syn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni<sup>2+</sup> pull-down from yeast cell extracts. For <i>in vitro</i> nitration, 1 ╬╝l peroxynitrite (PON) was mixed with 15 ╬╝g of ╬▒Syn extracts in the presence of 1 ╬╝l 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 ╬▒Syn 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 ╬▒Syn antibody. (C) Spotting analysis of yeast cells expressing <i>GAL1</i>-driven ╬▒Syn, A30P, 4(Y/F), A30P/4(Y/F) ╬▒Syn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (╬▒Syn ÔÇśOFFÔÇÖ) or galactose (╬▒Syn ÔÇśONÔÇÖ). (D) Cell growth analysis of yeast cells expressing ╬▒Syn, A30P, 4(Y/F), A30P/4(Y/F) ╬▒Syn 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 ╬▒Syn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 ╬╝m. (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 (*, <i>p</i> < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged ╬▒Syn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.</p

    Determination of nitrated peptides from ╬▒Syn and A30P.

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    <p>╬▒Syn and A30P were enriched by Ni<sup>2+</sup> pull-down from yeast crude extracts and separated by SDS-PAGE. Monomeric and dimeric ╬▒Syn stained with Coomassie were excised from the gel and digested with trypsin and AspN. Untreated <i>(in vivo)</i> and subsequent peroxynitrite (PON) treated <i>(in vitro)</i> ╬▒Syn and A30P protein samples were analyzed with LC-MS for tyrosine nitration. 3-NT (3-nitrotyrosine) indicates identified nitration sites, supported by at least two peptides and two independent experiments.</p
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