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Copper induced radical dimerization of #-synuclein requires histidine Dinendra L Abeyawardhane, Ricardo D Fernández, Denver R Heitger, Madeleine K Crozier, Julia C Wolver, and Heather R. Lucas J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08947 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 13, 2018
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Journal of the American Chemical Society
Copper induced radical dimerization of α-synuclein requires histidine Dinendra L. Abeyawardhane, Ricardo D. Fernández, Denver R. Heitger, Madeleine K. Crozier, Julia C. Wolver†, Heather R. Lucas* Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284 ABSTRACT: Aggregation of the neuronal protein α-synuclein (αS) is a critical factor in the pathogenesis of Parkinson’s disease. Analytical methods to detect post-translational modifications of αS are under development, yet the mechanistic underpinnings of biomarkers like dityrosine formation within αS have yet to be established. In our work, we demonstrate that Cu Ibound N-terminally acetylated αS (NAcαS) activates O2 resulting in both intermolecular dityrosine crosslinking within the fibrillar core as well as intramolecular crosslinking within the C-terminal region. Substitution of the H50 residue with a disease relevant Q mutation abolishes intermolecular dityrosine crosslinking and limits the Cu I/O2 promoted crosslinking to the Cterminal region. Such a dramatic change in reaction behavior establishes a previously unidentified role for H50 in facilitating intermolecular crosslinking. Involvement of H50 in the reaction profile implies that long range histidine coordination with the upstream CuI coordination site is necessary to stabilize the transition of Cu I to CuII as is a required mechanistic outcome of CuI/O2 reactivity. The aggregation propensity of NAcH50Q-CuI is also enhanced in comparison to NAcαS-CuI, suggesting a potential functional role for both copper and intermolecular crosslinking in attenuating NAcαS fibrillization.
INTRODUCTION In Parkinson’s disease (PD) inflicted brain tissue from the substantia nigra, copper ion levels are diminished while the accumulation of misfolded N-terminally acetylated α-synuclein (NAcαS) into proteinaceous inclusions termed Lewy bodies is the cardinal feature.1, 2 NAcαS is a dynamic neuronal protein implicated in PD as well as a family of other neurodegenerative diseases known as synucleinopathies.3 Cerebral nerve terminals in healthy individuals are rich in cuprous ions for normal neurotransmission,4 with CuI present in concentrations approximately 6-fold higher than NAcαS.5 Changes in copper compartmentalization are also known to occur with aging, which has prompted various studies on the link between metal dyshomeostasis and neurodegeneration.6-10 Much of the research on α-synuclein has focused on the non-acetylated recombinant construct (αS), however it has now been well-documented that there are key conformational differences between this unmodified form and the native human form which is universally acetylated at the Nterminus.11 Both the helical content and the copper coordination sites of αS change upon N-terminal acetylation.6, 12 CuII and CuI were previously established as binding to the Nterminus of αS, while the principal CuII and CuI coordination sites in NAcαS are separated by 40+ amino acids. In particular, the high affinity CuII binding site shifts from the N-terminal amine in the most well explored non-acetylated recombinant αS form to a lower affinity H50 residue in the native human NAcαS form.13-17 Conversely, the principal CuI binding site of NAcαS remains at the N-terminus, anchored by the M1 and M5 residues (Chart 1).18, 19 An innate characteristic of metallobiochemistry is the redox state cycling (CuII/CuI) that occurs to facilitate oxidative mechanisms.20 Thus, the research described herein aims to take a fresh look at the oxidative chemistry associated with these NAcαS
sites, with a principal focus on CuI as it is the most physiologically relevant in a reducing intracellular environment. NAcαS containing the post-translational modification (PTM) dityrosine is becoming an increasingly recognized biomarker of PD.21-23 Detailed analyses of clinically derived samples of Lewy bodies have revealed that both dityrosine and NAcαS are colocalized, while in vitro studies using recombinant non-acetylated αS protein have demonstrated a role for copper in generating dityrosine crosslinked αS.14, 21 In converse to copper, iron levels are elevated in Lewy body plaques.24-26 Previously, we reported that FeII induces a disease relevant oligomeric conformation in the presence of O2 Chart 1. Structural representation pinpointing the CuI binding site of NAcαS based on modifications of PDB entry 2KKW27.
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Figure 1. Mass spectrometric analysis following tryptic digestion of NAcαS displaying (A) the annotated CID mass spectrum for fragmentation of the N-terminal peptide that demonstrates successful acetylation; (B) fragmentation map of the N-terminal peptide; and (C) ion fragmentation tables of the observed and theoretical b- and y-ion series. due to the formation of an oligomer locked aggregate stabilized by intermolecular Schiff base crosslinks.24 Now, we report herein the first study on the CuI oxidative pathway of NAcαS. Through both immunoblotting and mass spectrometric analyses, we have identified that the downstream oxidative modifications that result following NAcαS-CuI activation of O2 are notably different than that previously reported for αSCuII.14, 21 Moreover, parallel experiments and further biophysical analyses performed on the N-terminally acetylated PD-relevant disease mutant NAcH50Q reveal a critical role for the native histidine in modulating the pattern of dityrosine PTM installation. Through our research findings reported herein, new details on the oxidative capacity of NAcαS-CuI invoke key insights into the structural and mechanistic requirements for dityrosine crosslinking. Dityrosine crosslinking represents a molecular level detail of PD pathophysiology that could provide insight into the mechanistic underpinnings of the role of abundant cuprous ions in healthy neurons. RESULTS AND DISCUSSION Confirmation of α-synuclein N-terminal acetylation. The human form of NAcαS was recombinantly prepared by co-transforming pET-28 containing a SNCA gene insert, which codes for non-acetylated αS, with a pACYCduet plasmid coding for the eukaryotic acetylase complex from fission yeast.24 In situ enzymatic N-terminal acetylation of αS is then achievable by the acetylase NatB, which targets proteins beginning with specific starter amino acid sets, including Met1-Asp2 as is found in αS.28 For the co-transformation, each plasmid was selected to have different antibiotic re-
sistance cassettes (pET-28, kanamycin; pNatB, chloramphenicol) to ensure plasmid maintenance and to aid in efficient acetylation. After expression of NAcαS, traditional purification methods using anion exchange chromatography resulted in purely monomeric NAcαS.24, 29 High definition mass spectrometry was then used to confirm complete N-terminal acetylation of NAcαS (Figure 1). Following trypsin digestion, the peptides were analyzed by UPLC-MS/MS and the fragmentation patterns were analyzed against Uniprot’s human database. MS analysis of the corresponding fragments confirmed the identity of NAcαS with 100% probability based on 92% coverage. Acetylation of the 1MDVFMK6 peptide was confirmed with high accuracy (2.5 ppm). As shown in Figure 1A, the collision-induced dissociation (CID) spectra of ions are consistent with the expected fragmentation pattern for an acetylated 1MDVFMK6 peptide (Figure 1B). Based on theoretical masses determined using the ProteinProspector data mining program,30 the observed b- and y-ion fragmentation pattern well matches the predicted values (Figure 1C). The unacetylated N-terminal tryptic peptide was not observed, indicating complete and efficient in situ acetylation of αS by NatB. Immunoblotting analysis of crosslinked NAcαS. In order to characterize the metal induced dityrosine PTM of the physiologically relevant N-terminally acetylated form of αS, the protein was treated with stoichiometric metal-triflate salts of the most prevalent redox active brain metal cations. Monomeric NAcαS was coordinated with equimolar transition metal cations, incubated at 37 ˚C, and agitated at 500 rpm for approximately one week in order to elucidate the changes induced by metals within this aggregation-prone protein. Following analysis by SDS-PAGE (Figure 2), only
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Journal of the American Chemical Society NAcαS-CuI
and NAcαS-CuII produce dimeric NAcαS crosslinks in comparison to NAcαS-FeII and NAcαS-FeIII. After transfer of the separated proteins to nitrocellulose membrane and treatment with the monoclonal dityrosine antibody (clone, 1C3),31 we discovered that the NAcαS-CuI aggregate resulted in intramolecular dityrosine crosslinks in addition to dimeric intermolecular dityrosine crosslinks (Figure 2). Conversely, NAcαS-CuII aggregation resulted in the generation of intermolecular dityrosine crosslinks, however, intramolecular dityrosine crosslinks were not observed. After multiple repetitions (n=20+), intramolecular crosslinks were observed consistently for NAcαS-CuI and on occasion (
