Protein semi-synthesis is a powerful approach to site-specifically incorporate modifications of interest into a protein sequence(Thompson and Muir, 2020) and it has been a method of choice for many αS PTM studies(Moon et al., 2021), including Lys acetylation(de Oliveira et al., 2017). To test this approach to synthesizing acetylated αS, we chose ^AcK₈₀ as an example, and combined solid-phase peptide synthesis (SPPS),(Merrifield, 1963) by which χ-acetyllysine is incorporated, with the expression of protein fragments and a three-part NCL sequence using acyl hydrazides(Zheng et al., 2013) (Figure 2A, additional details in SI Scheme S1).

N-terminal thioester fragment αS_1-76-MES (1a) and C-terminal fragment αS_85-140-C₈₅ (4) were each recombinantly expressed as a fusion with Mxe GyrA intein. The N-terminal thioester was generated by adding excess sodium 2-mercaptoethane sulfonate (MESNa) to cleave the intein by N,S-acyl shift.(Muir et al., 1998) (reported yield 24.1 mg/L(Pan et al., 2020a)). Endogenous methionyl aminopeptidase in E. coli processes the N-terminus of the 85-140 peptide to expose the N-terminal cysteine,(Xiao et al., 2010) which further reacts with aldehydes or ketones in vivo to form thiazolidine derivatives.(Liu et al., 2016)

The thiazolidine derivatives were deprotected with methoxyamine to give a free N-terminal cysteine (4.40 mg/L, SI Figure S1B). The middle acyl hydrazide peptide αS_77-84-Pen₇₇^AcK₈₀-NHNH₂ (2, Pen: penicillamine(Haase et al., 2008)) was synthesized through SPPS (Yield: 12.4 mg, 12 μmol, 48%, SI Figure S1A).

αS_1-76-MES (1a) and αS_77-84-Pen₇₇^AcK₈₀-NHNH₂ (2) were ligated overnight under routine NCL conditions (NCL1) in the presence of 4-mercaptophenylacetic acid (MPAA). (Yield: 1.46 mg, 172 nmol, 57%, Figure 2B). The purified product (3a) was activated by oxidation to form a MES thioester (3b) (Yield: 1.29 mg, 126 nmol, 73%, SI Figure S1C). The second ligation (NCL2) between αS_1-84-Pen₇₇^AcK₈₀-MES (3b) and αS_85-140-C₈₅ (4) to form αS-Pen₇₇C₈₅^AcK₈₀ (5a) as performed in the presence of methyl thioglycolate to allow for desulfurization without intermediate purification (Figure 2C).(Huang et al., 2016) The product, αS-^AcK₈₀ (5b), was obtained in 43% yield (0.90 mg, 62 nmol, Figure 2D). Although we successfully completed this synthesis, it is notable that we encountered solubility issues of the intermediate fragments (3b, 5a) and the product (5b), after lyophilization.

Experiencing difficulties in sample handling and considering the inefficiency of applying NCL to scan 12 lysine acetylation sites distributed throughout the protein, we then sought to access site-specifically acetylated αS through ncAA mutagenesis (Figure 3A). We recombinantly expressed αS with lysine acetylation at site 80 in E. coli through amber codon suppression. We used a previously reported pair of aminoacyl tRNA synthetase (chAcK3RS with IPYE mutations) and cognate tRNA to incorporate ε-acetyllysine at a position dictated by an amber stop (TAG) codon(Bryson et al., 2017). In addition to 10 mM ε-acetyllysine, 50 mM nicotinamide, an inhibitor to endogenous deacetylases, was added to the media before inducing αS expression. The protein was expressed as an intein fusion as reported before for easy removal of truncated protein through affinity purification.(Batjargal et al., 2015) After traceless intein cleavage with 2-mercaptoethanol, the ^AcK-containing protein was purified by reverse phase high performance liquid chromatography (RP-HPLC) and exchanged into appropriate buffers for biophysical assays (Figure 3B).

We obtained 0.65 mg of pure αS-^AcK₈₀ per L of bacterial culture (Figure 3C,3D), a yield comparable to that obtained by NCL, but because no lyophilization or handling of ligation intermediates is required, we did not encounter the solubility problems observed in the NCL process. Therefore, we deemed the ncAA mutagenesis approach at least comparable to NCL for producing a specific construct. Since we wished to study 12 sites distributed throughout the αS sequence, ncAA mutagenesis was also advantageous because we avoided having to generate constructs for several different ligation sites and could simply perform site-directed mutagenesis to insert TAG codons for each new αS-^AcK_n variant.

Bolstered by our success with ^AcK₈₀, we generated TAG mutants at sites 12, 21, 23, 32, 34, 43, 45, 58, 60, 96, or 102. We expressed and purified these proteins, observing successful ncAA mutagenesis at each site (Figs. S2-S12), however, the yield varied significantly between different sites (0.11-1.5 mg). This is an interesting result in light of the large number of sites that were tested in the same protein and the fact that αS is an intrinsically disordered protein, so protein folding should not affect incorporation. Examination of the local RNA sequence context of the amber (TAG) codon did not explain the varied suppression efficiency, based either on previously identified sequence impacts (Pott et al., 2014) or by comparing the sites within αS (Figure S13). Given the pseudo-repeat nature of the αS sequence, many sites feature similar sequences, and a comparison of 21 and 58 is particularly striking with a 10-fold difference in expression levels despite near identity in the flanking sequences. While these observations are notable for users of ncAA technology, in the context of this study, our approach allowed us to acquire sufficient amounts of the 12 different authentically modified αS constructs for biophysical experiments.

Thus, in spite of low expression yields for some sites, ncAA mutagenesis was a preferred method for this work, due to the better efficiency in scanning 12 different modification sites and the ease of handling aggregation-prone protein fragments. The expression-based strategy also allows for low-cost access to isotopically labeled, PTM-modified αS constructs, as we have demonstrated previously.(Pan et al.)

αS is known to bind to lipid surfaces and form helical structures, part of its physiological role in modulating neurotransmitter vesicle trafficking(Ramirez et al., 2023). More specifically, on micelles, an NMR structure showed that micelle-bound αS forms a broken helix, where two helical strands are connected with a loop region (Figure 1B, PDB: 1xq8)(Ulmer et al., 2005). A helical wheel model, created based on this structure, shows that lysine residues are aligned on the membrane surface, and that they are likely involved in enhancing binding by interactions with negatively charged lipid head groups(Meade et al., 2019).

With each acetylated αS variant, we first examined the effects of Lys acetylation on the secondary structure of αS in the presence of micelles by wavelength scan circular dichroism (CD) spectroscopy. Each acetylated αS was compared to unmodified wild type (WT) αS in phosphate-based buffer, pH 7.4, with a large excess of sodium dodecyl sulfate (SDS). We normalized the molar ellipticity at 222 nm of each acetylated construct to that of WT to compare the effect on helicity at each site. We found that significant reduction of helicity was caused only by acetylation at site 43 and that acetylation at other sites had only minor effects on helicity (Figure 4, Figure S14). This study implies that only this site could potentially perturb αS function in neurotransmitter trafficking.

We then investigated whether lysine acetylation at different sites has impacts on αS in pathological contexts. To do this, we first performed in vitro aggregation experiments and assessed site-specific effects. A plate-based approach was taken to efficiently perform the assay, and each aggregation reaction was seeded by mixing with αS WT pre-formed fibrils (PFFs) that constituted 10% of the total monomer concentration. The monomer samples were prepared by mixing αS WT with acetylated αS at either 10% or 25% of the total monomer concentration. These concentrations were chosen because our quantitative studies of other PTMs in patient samples indicated that most were present in this range, rather than stoichiometrically(Zhang et al., 2023; Zhao et al., 2022) (see additional discussion in Conclusions). Aggregation was carried out at 37 °C with shaking and kinetics and thermodynamics (final fibril amounts) were monitored.

To examine the effects on aggregation kinetics, we took advantage of the change in fluorescence of the amyloid binding dye, thioflavin T (ThT), during aggregation to monitor the process in situ. We found that the effects differ between different modification sites (Figure 5, Figure S15-16). For Lys acetylation at 12, 23, 43, 80 and 102 we observed differential slowing effects – the effects were particularly significant at sites 12, 43 and 80, and the effects at 12, 23 and 43 were dose dependent. While we observed acceleration of aggregation for site 32 both at 10% and 25%, the effects were similar between the different dosages.

To confirm that these effects were not the result of reduced monomer incorporation, we isolated fibrils at the endpoint of 10% or 25% aggregations and SDS-PAGE gels were run and stained with the Coomassie Brilliant Blue dye to quantify total monomer incorporation into the fibrils. We found that there were no consistent reductions in monomer incorporation, and in fact there were some moderate apparent enhancements of incorporation. However, these were generally not consistent between the 10% and 25% aggregation experiments, except in the case of ^AcK₃₄ (Figure S17-18). Taking all of the aggregation kinetics and monomer incorporation data into account, we chose to investigate the kinetically perturbed sites 12, 43, and 80 further, since the cellular process will be unlikely to reach equilibrium and our previous study had shown that the Gln mimic mutation at position 34 did not alter aggregation in cells.(Zhang et al., 2023)

To investigate the impact of Lys acetylation on aggregation in more physiologically relevant contexts – in cultured neurons – we followed the approach that we have done previously with arginylated αS.(Pan et al., 2022; Zhao et al., 2022) We prepared PFFs with the following compositions: αS WT or αS WT mixed with 25% acetylated αS, ^AcK₁₂, ^AcK₄₃ or ^AcK₈₀. Mouse primary hippocampal neurons were grown for 8 days on a coated plate, to which 50 ng/µL PFFs were added, following established protocols.(Haney et al., 2016; Luk et al., 2009; Marotta et al., 2021) After 2 weeks, intracellular αS aggregates were quantified by staining with an antibody that recognizes phosphoserine 129 (pS₁₂₉), a commonly used pathological marker (Figure 6, Figure S19). Compared to the WT PFFs, all the acetylated PFFs tested resulted in significantly reduced aggregation seeding: the pS₁₂₉ signal (AU ± SEM (arbitrary units, standard error of the mean)) of PFF-seeded αS aggregates was 2347 ± 107.1 (WT), 1730 ± 83.67 (^AcK₁₂), 1854 ± 70.79 (^AcK₄₃), 1698 ± 54.41 (^AcK₈₀) with respect to DAPI. Notably, acetylation at these sites also slowed seeded aggregation in the in vitro fibrilization experiment, but did not reduce aggregates quantified at the endpoint. This supports the idea that aggregation in a cellular context is unlikely to reach saturation.

Having demonstrated that acetylation at K₁₂, K₄₃ or K₈₀ significantly reduced αS aggregation in vitro and in cells, we wished to gain information on the structural impact of acetylation at these sites. First, to give insights into the effects on monomer conformation, we acquired proton-nitrogen correlation spectra (¹H,¹⁵N – HSQC) for ^AcK₁₂, ^AcK_43, or ^AcK₈₀, an experiment that is facile with ncAA mutagenesis, but challenging to perform via NCL due to the high cost of isotopically-labeled amino acids for SPPS. To access ¹⁵N-labeled αS, we expressed the acetylated αS and αS WT in M9 minimal media containing ¹⁵N-labeled ammonium chloride (SI Figures S20-22). This afforded comparable protein yields to expressions in LB media. It is notable, however, that sub-stoichiometric isotopic labeling was observed at some Lys sites, depending on batches of expression, which could be due to deacetylation in E. coli cells followed by incorporation at Lys codons. Overlaying the HSQC spectra for αS-WT and αS-^AcK₁₂, ^AcK₄₃ or ^AcK₈₀, peak shifts were observed only in signals from surrounding residues, suggesting that the structural change was local and there is no major impact of lysine acetylation on monomer structure (SI Figures S23-S24).

We next wished to learn the effects of Lys acetylation at K₁₂, K₄₃ or K₈₀ on the native function of αS by investigating its lipid binding mode. To quantify conformational changes of αS upon vesicle binding, we acquired ¹H,¹⁵N – HSQC spectra for WT, ^AcK₁₂, ^AcK₄₃ or ^AcK₈₀ in the presence of small, unilamellar vesicles (SUVs) that are composed of 60:25:15 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine/1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPC/DOPE/DOPS). The NMR peak chemical shifts were similar for all constructs and consistent with spectra previously reported for WT αS(Eliezer et al., 2001). There was no notable chemical shift perturbation at the surrounding residues of each acetylation site (Figure S25).

In the presence of vesicles, a reduction of intensity for residues 1-100 was observed for all the constructs, which is caused by binding of this portion of αS to the slowly tumbling lipid vesicles and is again consistent with previous observations(Bodner et al., 2009; Bussell and Eliezer, 2004). αS-^AcK₈₀ had a similar intensity change to WT (∼40%, Figure 7A, Figure S26), whereas αS-^AcK₄₃ had a smaller intensity change, suggesting weaker vesicle binding (∼20%, Figure 7A, Figure S26). This is consistent with the acetylation effects observed with SDS micelles (Figure 4). αS-^AcK₁₂ had an intermediate intensity reduction (∼30%, Figure 7A, Figure S26), a more significant effect of K₁₂ acetylation on vesicle binding than what was observed with SDS micelles (Figure 4). It is possible that this is due to the differences in curvature and headgroup between the micelles and the vesicles, which is known to result in different αS binding modes(Cholak et al., 2020; Georgieva et al., 2010; Middleton and Rhoades, 2010; Rhoades et al., 2006; Trexler and Rhoades, 2009). It is also possible that this is due to the increased sensitivity of NMR to subtle differences in binding.

While NMR is a very valuable technique for characterizing vesicle binding with a non-perturbing label, to measure affinity, we turned to FCS, a well-established method for rigorously determining vesicle apparent dissociation constants (K_d,app) (Rhoades et al., 2006). To enable this experiment, we expressed the acetylated αS constructs at site 12, 43, or 80 or a non-acetylated construct (“WT”), bearing a Cys mutation at site 114 (8, αS–^AcK₁₂C₁₁₄, αS–^AcK₄₃C₁₁₄, αS–^AcK₈₀C_114, and αS–C₁₁₄) to allow for fluorescent labeling (Figure 7B, SI Figures S27-30). The fluorophore Atto488-maleimide was reacted with purified Cys mutants overnight at 4 °C or for a few hours at room temperature to yield labeled constructs (9, αS– ^AcK₁₂C^Atto488₁₁₄, αS–^AcK₄₃C^Atto488₁₁₄, αS–^AcK₈₀C ^Atto488_114, and αS–C ^Atto488₁₁₄), and the conversion was almost quantitative (SI Figures S27-30). We prepared synthetic lipid vesicles containing 50:50 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPS/POPC). The diffusion times of free αS and of the vesicles were obtained first, and in assessing the αS-vesicle binding, we added the same quantity of αS to varied concentrations of vesicles and then determined the protein fractions bound by fitting a two-component autocorrelation function. The fraction bound values at each vesicle concentration were used to fit a binding curve for each αS construct. We found that acetylation at site 43 leads to two-fold weaker binding and acetylation at site 12 or 80 did not significantly affect binding (Figure 7C, SI Figures S31-32; K_d,app^WT = 3.2 ± 0.5 μM, K_d,app^AcK12 = 3.9 ± 0.3 μM, K_d,app^AcK43 = 6.3 ± 1.3 μM, K_d,app^AcK80 = 4.0 ± 1.0 μM).

Slightly reduced binding due to acetylation at site 43 correlates with the reduced helicity we observed in the CD wavelength scan and the differences in NMR peak intensities in the presence of vesicles. The NMR experiments showed that ^AcK₄₃ reduced vesicle binding more significantly than ^AcK₁₂ (moderate) or ^AcK₈₀ (little to none). The FCS experiments supported this, showing that ^AcK₄₃ led to weaker binding than ^AcK₁₂ or ^AcK₈₀, which were similar to WT. Previous NMR experiments suggested that the N-terminal helix of αS (residues 6-25) drives association with lipid membranes and the 26-97 region modulates the affinity, depending on lipid composition(Fusco et al., 2014). The different effect between ^AcK₄₃ and ^AcK₁₂ or ^AcK₈₀ suggests that K₄₃ is more important in modulating the binding affinity.

Taken together, our results show that among all the disease-relevant acetylation sites, ^AcK₁₂, ^AcK₄₃, and ^AcK₈₀ each inhibit aggregation, but that ^AcK₄₃ also inhibits membrane binding (as does ^AcK₁₂, to a lesser degree). Thus, in the case of ^AcK₄₃, the potential benefits of reduced amyloidogenicity may be offset by compromising function in neurotransmitter release.

To get preliminary insights into fibril structure effects, we performed TEM imaging on fibrils formed from acetylated αS (αS-^AcK₁₂, ^AcK₄₃ or ^AcK₈₀), mixed with αS WT, at 25% of the total monomer concentration (SI Figure S33). Interestingly, we observed mixed morphology for PFFs prepared with αS-^AcK₁₂, with some very narrow fibrils. Both 25% ^AcK₁₂ and 25% ^AcK₄₃ PFFs have minimal helical twist, making them difficult to characterize by cryo-EM. On the other hand, for PFFs prepared with 25% αS-^AcK₈₀, we observed a slightly more twisted fibril morphology, so we attempted to solve a structure by single particle cryo-EM methods. We were able to solve a structure to 2.88 Å resolution, and comparison to previously published αS WT fibril structures (see SI for experimental details, density images, and atomic models, SI Figure S34) shows that the backbone fold is similar to the two-stranded polymorph typified by PDB ID 6a6b (Figure 8, inset, 6a6b),(Li et al., 2018b) similar to PDB IDs 6cu7(Li et al., 2018a) and 6h6b(Guerrero-Ferreira et al., 2018), which all have the “Greek key” protein fold first reported in solid state NMR studies of single stranded fibrils under PDB ID 2n0a.(Tuttle et al., 2016) However, the density clearly shows that K₈₀ is not acetylated in this structure, implying that the WT fibrils are forming this 6a6b-type fibril, while the ^AcK₈₀ fibrils are forming a separate, minority population that we are not able to resolve. In order to more clearly observe the structural impact of K₈₀ acetylation, we prepared fibrils with 100% ^AcK₈₀ αS in Tris-buffered saline (TBS) for cryo-EM studies. For these fibrils, we were able to solve structures of two different polymorphs, both composed of two strands (Figure 8, ^AcK₈₀-A and ^AcK₈₀-B, see SI for experimental details and density images, and atomic models, SI Figures S35, S37, S38). The protein fold is essentially the same in both polymorphs, but they differ in strand-strand packing. Since it is well-documented that differences in buffer composition and aggregation methods can lead to differences in fibril morphology, we also prepared ^AcK₈₀ αS fibrils in phosphate-buffered saline (PBS), the same conditions used in our aggregation kinetics studies. Gratifyingly, the ^AcK₈₀ αS fibrils prepared in PBS exhibited the same two polymorphs seen for TBS ^AcK₈₀ αS fibrils, with identical protein folds and strand-strand packings (Figure 8, and SI Figure S39). We were also able to solve cryo-EM structures of WT αS fibrils generated under the same conditions in TBS. We observed two WT polymorphs (Figure 8, WT-A and WT-B, see SI for experimental details and density images, and atomic models, SI Figures S35, S36, S38) which exhibited similar folds and strand-strand packings to the ^AcK₈₀ polymorphs, but with a notable change in morphology around K₈₀. Acetylation of K₈₀ disrupts a salt-bridge interaction with E₈₃ that can be clearly seen in the WT-B polymorph (Figure 8, WT Fold) and neutralizes the sidechain charge, allowing it to pack in a hydrophobic pocket formed by Ala₆₉ and Val₇₁ (Figure 8, ^AcK₈₀ Fold). This leads to a twist of the backbone in the T₇₅-A₉₀ segment, generating a modest change in the protein fold. Given that the ^AcK₈₀ protein fold is fairly similar to the WT protein fold, it is not surprising that they exhibit similar strand-strand packings and that K₈₀ acetylation has a moderate impact on aggregation rates. We can use these structures to consider ^AcK₈₀ effects in the context of other structural studies of αS fibrils.

Our WT-A and WT-B structures resemble those first reported under PDB IDs 6rtb and 6rto (Figure 8, inset, 6rtb; see SI for additional overlays and structural comparisons, Figure S40).(Guerrero-Ferreira et al., 2019) These polymorphs have been observed by several investigators for WT αS fibrils formed at near-neutral pH, along with the commonly observed 6a6b/2n0a form (Figure 8, inset, 6a6b).(Li et al., 2018b) Our ^AcK₈₀ αS fibril structures, ^AcK₈₀-A and ^AcK₈₀-B, resemble those recently reported for WT αS fibrils formed at pH ≤6.5 under PDB IDs 8pix and 8pic (Figure 8, inset, 8pix).(Frey et al., 2024) In the two fibril polymorphs commonly populated at pH 7, K₈₀ makes key stabilizing salt bridge interactions. For the 6a6b polymorph, K₈₀ makes a salt bridge with E₄₆; for the 6rtb polymorph, it makes a salt bridge with E₈₃. Disruption of these salt bridges by acetylation would destabilize either fold, favoring the ^AcK₈₀ fold that we observe. The fact that a similar fold (8pix) has been seen at lower pHs can be rationalized by assuming that acidification leads to protonation of the E₄₆ or E₈₃ sidechains, weakening their interactions with K₈₀ just as acetylation does to drive a change fibril polymorph. While pH 6.5 is significantly above the pK_a of a typical glutamate sidechain, it is possible that the pK_as are perturbed in the local environment of the fibril, and full deprotonation would not be required to destabilize interactions with K₈₀. Thus, one can rationalize our observation of a polymorph like 8pix for our ^AcK₈₀ at pH 7, when it had only been previously observed at lower pHs. It is further notable that the ^AcK₈₀ fold is robust to changes in buffer (PBS vs. TBS).

Finally, it is worth comparing the effects that we observe for ^AcK₈₀ acetylation to the structural and biophysical effects observed with other αS PTMs which have been structurally characterized, such as Y₃₉ phosphorylation (pY₃₉) or S₈₇ phosphorylation (pS₈₇) and N-Acetyl glucosamine glycosylation (gS₈₇). For pY₃₉ αS, a 4-fold increase in aggregation rate has been observed for αS 100% phosphorylation, with nuanced effects at lower phosphorylation percentages. We have shown that pY₃₉ leads to only modest changes in monomer conformation, based on NMR and single molecule FRET studies.(Pan et al.; Pan et al., 2020b) In contrast, Zhao et al’s cryo-EM structure shows a dramatic rearrangement of the fibril polymorph.(Zhao et al., 2020) This indicates that the effect of Y₃₉ phosphorylation is primarily at the fibril level, similar to our findings here for ^AcK₈₀ acetylation. Both pS₈₇ and gS₈₇ modifications inhibit aggregation much more drastically than pY₃₉. Two different structures of gS₈₇ αS fibrils have been reported, both showing a significant deviation from reported WT αS polymorphs.(Balana et al., 2024; Hu et al., 2024) Despite their differences, the structures both provide a clear rationale for the effects of S₈₇ glycosylation. Intriguingly, Hu et al. highlight the way in which gS₈₇ changes the structure of the 80-89 region of αS to disrupt the E₄₆-K₈₀, destabilizing the 6a6b fold.(Hu et al., 2024) The pS₈₇ structure is different from either gS₈₇ structure, and although the residue cannot be observed in the structure, it again demonstrates that a PTM can dramatically alter fibril morphology. Thus, like ^AcK₈₀ studied here, these PTMs seem to primarily exert their influence on αS aggregation through changes in fibril structure, which is sensible given the disordered nature of the αS monomer.

Since acetylation of K_12, K₄₃ and K₈₀ can reduce αS aggregation, we considered the potential of increasing acetylation at these sites by inhibiting a KDAC. Doing so would require that the KDAC had some specificity for these residues. Using previously published methods,(Decroos et al., 2014; Dowling et al., 2008; Osko et al., 2021) we expressed and purified recombinant human HDAC8, a Zn-dependent HDAC known to act on non-histone proteins, including cytosolic targets like tubulin in HeLa cells(Vanaja et al., 2018). We treated samples of each of the acetylated αS variants with HDAC8 and monitored deacetylation through a matric assisted laser desorption ionization (MALDI) MS assay using ¹⁵N-labeled αS as a standard. After 24 h, all of the constructs showed significant levels of deacetylation, but ^AcK₃₄, ^AcK₄₃, ^AcK₄₅, and ^AcK₈₀ retained an average of 44% acetylation, 3-fold greater levels than all other sites (Figure 9). These preliminary results indicate that inhibition of HDAC8 could increase acetylation levels of αS at specific lysine residues shown to retard aggregation in vitro and in PFF-seeded neurons.