Phosphorylation of α-Synuclein at Y125 and S129 Alters Its Metal Binding Properties: Implications for Understanding the Role of α-Synuclein in the Pathogenesis of Parkinson’s Disease and Related Disorders

α-Synuclein (α-syn) is a 140-amino acid protein that plays a central role in the pathogenesis of Parkinson’s disease (PD) and other synucleinopathies. However, the molecular determinants that are responsible for triggering and/or propagating α-syn aggregation and toxicity remain poorly understood. Several studies have suggested that there are direct interactions between different metals and α-syn, but the role of metal ions and α-syn in the pathogenesis of PD is not firmly established. Interestingly, the majority of disease-associated post-translational modifications (PTMs) (e.g., truncation, phosphorylation, and nitration) of α-syn occur at residues within the C-terminal region (Y125, S129, Y133, and Y136) and in very close proximity to the putative metal binding sites. Therefore, we hypothesized that phosphorylation within this domain could influence the α-syn–metal interactions. In this paper, we sought to map the interactions between the di- and trivalent cations, Cu(II), Pb(II), Fe(II), and Fe(III), and the C-terminal region of α-syn encompassing residues 107–140 and to determine how phosphorylation at S129 or Y125 alters the specificity and binding affinity of metals using electrospray ionization-mass spectrometry (ESI-MS) and fluorescence spectroscopy. We demonstrate that D115-M116 and P128-S129 act as additional Cu(II) binding sites and show for the first time that the residues P128-S129 and D119 are also involved in Pb(II) and Fe(II) coordination, although D119 is not essential for binding to Fe(II) and Pb(II). Furthermore, we demonstrate that phosphorylation at either Y125 or S129 increases the binding affinity of Cu(II), Pb(II), and Fe(II), but not Fe(III). Additionally, we also show that phosphorylations at these residues lead to a shift in the binding sites of metal ions from the N-terminus to the C-teminus. Together, our findings provide critical insight into and expand our understanding of the molecular and structural bases underlying the interactions between α-syn and metal ions, including the identification of novel metal binding sites, and highlight the potential importance of cross-talk between post-translational modifications and metal ion binding in modulating α-syn functional and aggregation properties that are regulated by its C-terminal domain

Elucidating the Role of C-Terminal Post-Translational Modifications Using Protein Semisynthesis Strategies: α-Synuclein Phosphorylation at Tyrosine 125

Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of α-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine → glutamate or aspartate substitutions, there are no natural amino acids that can mimic phospho-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of α-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding, and subcellular localization of α-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in α-syn structure, aggregation, and functions in heath and disease

X-ray crystallography demonstrates that the three-dimensional structure of Leu46 mutants is very similar to the wt protein.

<p>(A) Overlay of crystal structures of L46F (blue) L46A (green) and L46G (red). (B, C, D) Secondary structure disruptions induced by the Leu46 mutants are shown by superimposition of the wt human and L46F (B), L46A (C) and L46G (D) MIF monomers. Wt and Leu46 mutant monomers are represented in pink and cyan respectively. Black arrows highlight the structural changes induced in the Leu46 variants.</p

Summarized enzymatic and biophysical data collection of wt and Leu46 huMIF mutants.

<p>Each data represented is the average of three independent measurements; apparent Tm values reported are measured at protein concentrations ranging from 5 to 30 µM; apparent Cm values reported by circular dichroism and fluorescence are measured at protein concentration of 10 and 3 µM respectively.</p

Mutation of Leu46 does not alter the quaternary structure of MIF.

<p>Sedimentation rate distributions as determined by Analytical Ultracentrifugation/Sedimentation Velocity experiments indicating similar sedimentation rates for the wt and Leu46 huMIF mutants (15 µM in PBS 1X, pH 7.4 buffer).</p

NMR chemical shift perturbations by Leu46 mutants reveal higher fluctuation of the Leu46 pocket.

<p>(A) Normalized changes in ¹H, ¹5N chemical shifts of L46A and L46F MIF compared to the wild-type protein from wild-type huMIF. Normalized shift changes are calculated according to √(ΔH2+(ΔN/5)2 ). (B) Two-dimensional representation of chemical shift deviations of L46F and L46G MIF from those of the L46A mutant. The gray square is drawn at +/−0.2 ppm in ¹⁵N, +/−0.02 ppm in the ¹H dimension and separates very small from larger chemical shift changes. Arrows indicate residues belonging to the hydrophobic pocket. (C) Residues with chemical shift deviations larger than +/−0.2 ppm in ¹⁵N, +/−0.02 ppm in the ¹H dimension in L46F and L46A MIF (relative to wt MIF) are mapped onto the MIF crystal structure. Leu46 is shown as black sphere, catalytic core residues (1, 32, 64) as blue sticks, residues with strong chemical shift changes are colored in red.</p

Disrupting the hydrophobic interactions via mutating Leu46 alters the structural stability of MIF.

<p>(A) Leu46 hydrophobic pocket of wt huMIF. VMD representations of the hydrophobic pocket, where Leu46 is mutated to a phenylalanine (L46F) (B), alanine (L46A) (C), or glycine (L46G) (D). (<i>E-H):</i> The three Leu46 mutants are structurally less stable than the wild type protein, but retain the same overall secondary structure. (E) Far-UV CD spectra of wt and Leu46 mutants. (F) Thermal denaturation of wt and Leu46 mutants (at 20 µM) followed by far-UV CD at 218 nm. (G) GdnHCl denaturation studies monitored by far-UV CD at 218 nm and fluorescence spectroscopy (H), excitation wavelength: 295 nm, protein concentration: 10 µM. All spectroscopic experiments were performed in PBS 1X, pH 7.4 buffer. Black lines, wt MIF; blue lines, L46F MIF; red lines, L46A MIF; green lines: L46G MIF.</p

Backbone root mean square deviation of Leu46 mutants based on the structure of wt huMIF, and hydrogen bonds distances stabilizing the internal β-sheet involving β-strands β2 and β3 (Figure 1); percentages represent the increase/decrease of the hydrogen bond distances in the mutants compared to the wt protein.

<p>Backbone root mean square deviation of Leu46 mutants based on the structure of wt huMIF, and hydrogen bonds distances stabilizing the internal β-sheet involving β-strands β2 and β3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045024#pone-0045024-g001" target="_blank">Figure 1</a>); percentages represent the increase/decrease of the hydrogen bond distances in the mutants compared to the wt protein.</p

SIRT2 regulates aSyn aggregation and toxicity.

<p><b>(A)</b> H4 cells were infected with lentiviruses encoding shRNAs against SIRT2 (T2.KD) or scramble shRNA (Ctrl) and selected with puromycin. Cells were then cotransfected with SynT and synphilin-1 (Synph1). SIRT2, synphilin-1, aSyn, and GAPDH levels were assessed by immunoblot analyses. <b>(B)</b> Ctrl and T2.KD cells transiently expressing SynT and synphilin-1 for 48 h were processed for immunocytochemistry (ICC) (aSyn, green). Data show percentage of cells with aSyn inclusions (<i>n</i> = 3). Scale bar 15 μm. <b>(C)</b> Triton X-100 insoluble and total fractions of cells as in (B) probed for aSyn and GAPDH. <b>(D)</b> Native protein extracts from H4 cells as in (B) were separated on a sucrose gradient. Fractions were immunoblotted and probed for aSyn. <b>(E)</b> Anti-aSyn IP from cells as in (B). Fractions were immunoblotted and probed for acetyl-lysine and aSyn. <b>(F)</b> Toxicity of Ctrl and T2.KD measured by lactate dehydrogenase (LDH) release assay (<i>n</i> = 3). Data in all panels are average ± SD, ** <i>p</i> < 0.01, **** <i>p</i> < 0.0001. For (B) and (F), unpaired, two-tailed <i>t</i> test with equal SD. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p

aSyn acetylation-resistant mutant induces nigral dopaminergic neuronal loss in vivo.

<p><b>(A)</b> AAV6-mediated delivery of EGFP and mutant aSyn (KQ or KR) into the SN of the rat brain. TH and GFP or aSyn expression was examined in brain sections 3 wk after injection by immunohistochemistry (TH, red; GFP or aSyn, green; DAPI, blue). Representative sections are shown. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm. <b>(B)</b> Stereological counting of the number of TH-positive neurons in the SN. The contralateral SN of the different groups of animals was used as a control (intact). Data in panels are average ± SD. <b>(C)</b> Brain sections stained for aSyn (green), pS129 aSyn (red), and DAPI (Blue). Representative sections are shown. Dashed square boxes delineate the magnification presented on the right. Scale bar for isolated channels 1,000 μm and for merged channels 500 μm and 50 μm. *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001, one-way ANOVA with Bonferroni correction used for statistical calculations. In (B), GFP was used as a control; <i>n</i> = 6–7 animals per condition; five sections from a one-in-six series were analyzed per brain. Data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000374#pbio.2000374.s010" target="_blank">S1 Data</a>.</p