To explore a potential correlation between Thb and Syn, we examined the Thb-mediated proteolysis of Syn. Figure 2A reports the chromatograms corresponding to the proteolysis mixtures at the initial time point (top) and after an overnight incubation (bottom). The Syn chromatographic profile at 0 h of incubation showed a main peak at approximately at 35.5 min-retention time (RT). Extending Syn exposure to Thb to 3 h, novel peaks emerged at RT 15.1 and 32.5 min, indicating the formation of new species, with a more hydrophilic character than Syn. These species were characterized by ESI-Q-TOF-MS (Table 1). A mass matching that of a truncated form of Syn, lacking the first six residues, was found for the species eluting at 32.5 min, while the first peak corresponded to the complementary peptide 1–6. The truncated form was named 7–140 Syn, and the identity was then confirmed by MS analysis (not shown). Interestingly, by MS estimation this peak contained also trace amounts (~ 5%) of another co-eluting species (Fig. S1 A and B). It was identified as the peptide 11–140, where the residue 10 is a lysine. When prolonging the proteolysis to 24 h an additional shoulder containing the peptide 33–140 appeared right before the main peak at 32.5 min. The latter appeared only slightly enriched in 11–140 even after 24 h (see Fig. S1, Table S1). Figure 2B shows the profile of the reaction mixture of Syn and Thb, in the presence of Thb specific inhibitor PPACK. No cleavage event occurred under this condition, confirming the involvement of Thb in Syn truncation.

For a better comprehension of the mechanism of the substrate recognition, based on conformational or residue specificity, additional analyses were performed. The proteolysis was repeated in the presence of 150 mM NaCl (Fig. 3). Ionic strength is known to shift the compactness of Syn by reducing the electrostatic interaction between charged residues, resulting in altered conformations³⁷. In parallel, Na⁺ ions exert a direct allosteric effect on Thb, stabilizing its catalytically active conformation and modulating substrate recognition and enzymatic efficiency. Thus, the presence of NaCl may simultaneously affect both the structural dynamics of Syn and the catalytic state of Thb^38,39, influencing the overall proteolytic outcome. Importantly, NaCl concentrations differ markedly between intracellular (~ 10 mM) and extracellular (~ 150 mM) environments⁴⁰.

Proteolysis of the chromogenic substrate (D)-Phe-Pip-Arg-pNA (S-2238) was performed in the presence and absence of 150 mM NaCl, using 20 mM sodium phosphate buffer. No significant difference in Thb activity was observed under these conditions (Fig. S2). Given that the Na⁺-binding site of Thb exhibits a low-millimolar affinity for sodium ions (Kd ≈ 2–10 mM)^27,41, the 20 mM sodium already present in the buffer was likely sufficient to saturate this site. Consequently, the enzyme remained predominantly in its Na⁺-bound active conformation even under nominal “0 mM NaCl” conditions, explaining the absence of a measurable Na⁺-dependent modulation of catalytic activity.

However, the presence of NaCl seems to delay the proteolysis of Syn with Thb. In the absence of salt, after 3 h of incubation with the protease, Syn peak intensity decreased by ~ 50% (Fig. 3A), while in the presence of NaCl, only by ~ 15% (Fig. 3B). Prolonging the reaction for 24 h in the absence of NaCl, only small amounts of intact Syn were detectable in the proteolytic mixture, while not reacted Syn was still abundant in the presence of salt. Moreover, in the presence of salt the 33–140 fragment did not form. The amount of reacted species calculated from the area of peaks was reported as a function of time of incubation (Fig. 3C). Interestingly, 7–140 Syn exhibited a very scarce susceptibility to proteolytic cleavage by Thb in the absence and in the presence of NaCl compared to full length Syn. Further proteolysis of 7–140 generates the species 11–140 and 33–140 in similar amount (Fig. S1C).

Thb specifically catalyses the cleavage of peptide bond containing an arginine residue (Arg-X, where X is a generic residue), which is absent in Syn sequence. Arg is a basic residue, like Lys, but Thb exhibits a lower affinity for Lys residues compared to trypsin, despite its trypsin-like activity⁴². Therefore, a new reaction has been conducted by using trypsin (E/S 1:1000, w/w) and monitored over different times of incubation. Analysis of the chromatographic profile corresponding to 5-min of incubation revealed a distinct pattern compared to the proteolysis produced with Thb (Fig. 3D). Specifically, trypsin-generated proteolysis produced a variety of Syn-derived peptides with increasing signal intensity over time, while Syn peak (RT 35.5 min) decreased. Notably, a high peak at approximately RT 32.5 min corresponding to the RT of truncated 7–140 Syn was present, but MS analysis (Table S2) detected the presence of other species besides 7–140, especially when the reaction was prolonged up to 30 min. Therefore, trypsin recognized not only Lys6, but all Lys residues present along the sequence of the protein, producing fragments that span the entire protein.

The genetic variants of Syn are involved in familiar forms of PD and differ from the wild type protein by one single mutation^2,3. These mutations also alter Syn structural and biochemical characteristics. Their susceptibility to Thb activity was investigated and compared to that of Syn. Without incubation with the protease, all mutant proteins exhibited a different chromatographic profile consistent with the amino acid substitutions affecting the protein hydrophobicity (Fig. 4A-D, top, red line). After incubation with Thb, the chromatographic profiles of all mutant proteins showed the emergence of a new, more hydrophilic peak (Fig. 4A-D, top, black line), to the detriment of the peak corresponding to the intact protein. The RP-HPLC fraction corresponding to the novel species was collected and analysed by ESI-Q-TOF-MS (Table 2), showing that it contains the 7–140 fragment for each mutant. Comparing the area under the peaks, the reaction yield for the truncated form production was determined (Table 2). The calculated yield was 75.66, 18.11, 61.42, 100.00 and 78.74% for A30P, E46K, H50Q, A53T and Syn, respectively.

Since the flexibility of a protein substrate influences proteolysis reactions⁴³, we compared the varying compactness of Syn mutants. Although these proteins are intrinsically disordered, they can adopt multiple conformational ensembles^44,45. Therefore, samples of Syn mutants and of Syn were analysed by native-MS to monitor protein ion charge-state distribution that is correlated to the solvent-exposed area (Fig. 4A-D, bottom). The figure shows representative native ESI-MS spectra of Syn variants. Three different populations corresponding to multiple conformers appear for all proteins. They were indicated as 1, 2 and 3, where 1 corresponds to the most relaxed conformation and 3 to the most compact one, according to their charge states⁴⁶. The population named 2 has intermediate properties and represents the prevalent conformation for all the proteins with different percentage (Fig. 4E). E46K populates mostly the compact conformation, while A53T the more relaxed ones. Syn, A30P and H50Q showed an intermediate behaviour, where A30P is slightly more relaxed than Syn and H50Q slightly more compact. The presence of a mutation in Syn sequence resulted in alteration of the overall distribution of the protein conformers in solution, in comparison to Syn, affecting both structural flexibility and compactness and in turn, the extent of proteolysis. Proteolysis occurs on the fractions of more extended conformations in equilibrium with the compact ones.

The species 7–140 exhibits only slightly different conformational features in comparison to the full-length protein, but the 1–6 residues strongly affect the interaction with membrane

After purification, fragment 7–140 was characterized by SEC, CD and native MS (Fig. 5, Fig. S3). The measured hydrodynamic volume (Fig. 5A) of 7–140 in solution showed a behaviour similar to that of Syn with the monomeric species eluting with an apparent molecular weight corresponding to 53 kDa. Far-UV CD was used to determine the conformation of 7–140 species and its spectrum appeared superimposable with that of the full-length protein (Fig. 5B). At pH 7.5, 7–140 displays random secondary structure, according to previous data¹⁷. Interestingly, native MS (Fig. 5C) revealed differences in the conformational ensemble of Syn and 7–140. The truncated variant principally populated the most compact conformer (59% for 7–140 Syn vs 15% for Syn), while the full-length protein appears preferentially in the intermediate conformer (34% vs 65%). Additionally, Syn also populates the most extended conformer more (7% for 7–140 Syn vs 20% for Syn). These results indicate that the removal of the first six residues has a big impact on the conformational ensemble of 7–140 Syn, effectively biasing occupancy towards the more compact states. Finally, cells exposed to the monomeric form of 7–140 Syn showed no detectable toxic effects neither after 24 h nor 48 h of treatment, in analogy to the full-length protein (Fig. 5D).

Syn is known to undergo a disorder-to-helix transition upon binding to highly curved, negatively charged lipid vesicles⁴⁷. To investigate the role of the first six N-terminal residues in this process, we compared full-length Syn with the truncated variant 7–140. CD-monitored titrations with PC/PS SUVs of ~ 50 nm diameter revealed that both proteins undergo cooperative helix formation, as evidenced by the presence of minima at 222 and 208 nm and an isodichroic point at 205 nm (Fig. 6A, B). Quantitative analysis (Fig. 6C) showed that the binding affinities were comparable (Syn: Kd = 18 ± 20 µM; 7–140: Kd = 42 ± 8 µM), indicating that N-terminal truncation does not substantially impair membrane association. However, the overall helicity was reduced in 7–140 (25%) compared to full-length Syn (40%), suggesting that while the 6 N-terminal residues are not essential for lipid binding, they play a key role in efficient helix formation. Furthermore, we have observed that only Syn induced SUV clustering and aggregation as revealed by DLS (Fig. S4A), whereas 7–140 did not in the examined timeframe (4 hours). SUV fusion is accompanied by a loss in helical structure (Fig. S4B).

HDX–MS measurements provided higher-resolution insights into this difference (Fig. 6D, E, F; Fig. S5). The membrane bound form of Syn can be subdivided into three segments: a strongly bound N-terminal helix, a loosely bound central region, and a non-membrane associated C-terminal part^48,49. The mean deuterium uptake for these regions showed that the N-terminal region (residues 1–39) of the full-length Syn formed a stable helix that exchanged slowly (full deuteration after 10 min). In contrast, the same region in 7–140 (7–39) was destabilized, reaching full deuteration within 1 min. Protection in the central region (residues 40–98) was also reduced in 7–140, indicating that stable helix formation at the extreme N-terminus contributes to cooperative stabilization along the sequence. The C-terminal region (residues 99–140) was unaffected, remaining unstructured in both proteins.

In summary, our data reveal that the first six residues of Syn, although not essential for membrane binding, are critical for helix stabilization. This N-terminal segment thus promotes both structural ordering and functional membrane remodelling.

The expression of human α-Synuclein (Syn) and its mutants (A30P, E46K, H50Q, A53T) was conducted in E. coli BL21 and BL21-Gold cells, respectively. Overexpression of the proteins was achieved by growing cells in LB medium at 37°C to an A_600nm of 0.6, followed by induction with 0.5 mM isopropyl β-thiogalactopyranoside (IPTG). The purification of the proteins was conducted following a procedure previously described⁴⁵. Protein identity and integrity were assessed by mass spectrometry (MS).

Protein concentrations were determined by absorption measurements at 280 nm using a double-beam Lambda-20 spectrophotometer (Perkin Elmer Life Sciences). The molar absorptivity at 280 nm for Syn and mutants was 5960 cm^− 1 M^− 1, as evaluated from their amino acid composition by the method of Gill and von Hippel⁶².

Size exclusion chromatography (SEC) was performed by a Superdex TM 200 Increase (10/300 GL) column (Amersham Biosciences, Uppsala, Sweden), using an ÄKTA FPLC system (Amersham Biosciences, Uppsala, Sweden). Sample aliquots (200 µg) were centrifuged, loaded onto the column and eluted at 0.75 mL/min in 20 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl. The effluent was monitored by recording the absorbance at 214 nm. Column calibration was obtained by eluting a mixture of blue dextran (void volume), albumin (67 kDa), ovalbumin (45 kDa), α-lactalbumin (14.4 kDa) and aprotinin (6.5 kDa).

The secondary structure content of the proteins was assessed by far-UV circular dichroism (CD). The measurements were performed on a J-800 Series spectropolarimeter (JASCO, Japan) in a 1-mm quartz cuvette at room temperature. Data were recorded in the wavelength range of 250 − 190 nm, collecting data with high-tension voltage < 600 V, and avoiding noisy signals. All protein samples were measured at the same settings averaged in five and the buffer data subtracted. The mean residue ellipticity [θ] (degree cm² dmol^− 1) was calculated from the formula [θ] (θ_obs/10) (MRW/lc), where θ_obs is the observed ellipticity in degrees; MRW is the mean residue molecular weight of the protein; l is the optical path length in cm; and c is the protein concentration in g/mL. Vesicles (SUV) were obtained by mixing POPC (10 µmol) and POPG (10 µmol) suspended in 1 ml and extruded with 50 nm filter. Dynamic light scattering (DLS) was used to test the integrity and the diameter of SUV. For the titration experiments, a sample of the proteins at 3.5 µM was prepared and the protein to phospholipid ratio increased by 50 with each addition (i.e. 1:50, 1:100, 1:150). The measurement was carried out until the SUV addition did not result in any significative change of helicity. The raw spectrums were blank corrected and normalized keeping the dilution of the initial Syn concentration after each SUV addition in mind. The ellipticity value at 222 nm was plotted against the protein to phospholipid ratio, and the resulting plot fitted with the following equation described by Rovere et al.⁶³:

where is the ellipticity at each time point, is the ellipticity at the beginning of the titration, is the ellipticity at saturating conditions, X is the protein to phospholipids molar ratio, is the number of lipids interacting, is the binding constant and is the initial protein concentration. Percentage of alpha-helix content was estimated with following Eq. 6⁴: ;

where is the ellipticity at 222 nm under saturating conditions, and correspond to the ellipticity at 222 nm of a protein with 100% and 0% of helical content (estimated to be -39500 deg cm² dmol^− 1 and − 3000 deg cm² dmol^− 1, respectively).

Mass spectrometry analyses were performed on a Xevo® G2-XS ESI-Q-TOF mass spectrometer (Waters Corporation, Milford, Massachusetts, USA). Measurements were carried out in positive mode at 1.2–1.6 kV capillary voltage and 30–40 V cone voltage. The source temperature was set at 30°C. For native-MS, Syn and mutant samples were buffer exchanged by filtration using Amicon Ultra-0.5 centrifugal filters (Merck Millipore Ctd, Ireland). Samples (10 µM) in 200 mM ammonium acetate (pH 7.0) were analyzed by direct infusion electrospray ionization (ESI). The analyses were performed in nanoflow mode by using quartz emitter produced in-house by using a Sutter Instruments Co. (Novato, CA, USA) P2000 laser pipette puller. Up to 5.0 µL samples were typically loaded onto each emitter by using a gel-loader pipette tip. Data were processed by using Mass Lynx (v 4.2, SCN781, Waters, Manchester, UK) software. To evaluate conformer occurrence, abundances of the different populations in each experiment were calculated aided by in-house developed MATLAB (R2016b) script, expressed as percentage, and compared.

For Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), samples were injected by an Acquity M-class UPLC (Waters), an Automation 2.0 sample workstation (Waters), and an HDX PAL autosampler (Leap technologies, Carrboro, NC, USA). Leu-enkephalin (Waters) was continuously infused as the reference lock mass. The protein/liposome complexes (starting concentration 4 µM for the protein and 8 mM for the lipids) were left to incubate for 30 min at room temperature before starting with the exchange reactions. Immediately after the incubation, samples were thermostated at 4°C. For the H/D exchange reaction, an aliquot (5 µl) of each sample was diluted 10-fold in deuterated buffer (20 mM sodium phosphate, pD 7.4, in 99.9% D₂O) and was then allowed to exchange for either 10, 30, 60 sec or 10 min, at 4°C. H/D exchange was quenched at 0°C by adding an equal volume of quenching buffer, i.e. 0.8% formic acid, adjusted to pH 2.35. A total of 35 pmol were injected for each measurement. Parameters for the measurements were adapted from⁴⁹. Deuterium incorporation was determined according to the following equation: %D = (mt – m0)/ (m100 − m0), where mt is the mass of the fragments after labeling time t, m0, and m100 are the masses of the non-deuterated fragment and the maximally deuterated sample (maxD), respectively. The maximally deuterated samples used for back-exchange corrections were prepared as described by Peterle et al.⁶⁵. Fragments generated from online pepsin digestion were identified using the Protein Lynx Global Server 3.0 and then analyzed with DynamX 3.0 software (Waters). Only fragments matching the following criteria were considered for the analysis: i) 5%-retention time window in the chromatographic separation; ii) maximum MH⁺ error of 6 ppm; iii) at least 2 ion products identified for each peptic fragment; iv) a minimum of 0.2 ion products generated per amino acid in the fragment; v) fragments containing < 4 or > 33 amino acids were excluded, due to identification ambiguity and poor sequence localization.

Freshly resuspended proteins were centrifuged at 12.000 rpm for 15 min and filtered through 22 µm PVDF filter to obtain monomeric Syn and 7–140 Syn for the aggregation assays. Total protein concentration in each aggregation reaction was 70 µM. Three conditions were tested: i) Syn, ii) 7–140 Syn and iii) a mixture of Syn and 7–140 Syn at a 3:1 molar ratio. Aggregation was initiated by incubating the samples in low binding 1.5 ml microcentrifuge tube (Sarstedt) at 37° C with continuous shaking at 1000 rpm in a thermomixer. At regular intervals, aliquots were taken and diluted to a final Syn concentration of approximately 4 µM in 20 mM sodium phosphate buffer (pH 6.0) containing 25 µM ThT. ThT fluorescence was recorded on a Jasco FP 8250 (Tokyo, Japan) using an excitation wavelength of 440 nm, and emission spectra were collected from 450 to 600 nm. Fluorescence intensity values were plotted as a function of time and fitted using Amylofit⁶⁶, selecting in each case the model yielding the lowest residual error. All ThT values were normalized to the maximum observed fluorescence. Aggregation experiments were performed in biological triplicates. Upon reaching the ThT fluorescence plateau, samples were ultracentrifuged to separate soluble and insoluble fractions. The centrifugation was carried out at 100.000 rpm for 30 min (Optima MAX-XP Beckman, USA). The resulting pellets were washed once with Milli-Q water and then resuspended in 7 M guanidinium hydrochloride (Gnd-HCl). UV absorption spectra of the solubilized pellets were recorded from 230 to 350 nm and corrected for the contribution of Gnd-HCl. To evaluate the composition of the insoluble fractions, the Gnd-HCl–solubilized pellets were analyzed by RP-HPLC with the same gradient described above. The identity of the peaks was confirmed by ESI-MS.

Transmission electron microscopy (TEM) was performed by negative staining method, in which 5.0 µL of sample (0.5 mg/ml) was dried on the carbon grid and subsequently stained with 10 µL of 1.0% (w/v) uranyl acetate solution. A Tecnai G2 12 Twin TEM microscope (FEI Company, Hillsboro, OR) was used for sample imaging. Fibril measurements were performed with Fiji ImageJ software. A total of > 100 singular measurements were made for each sample. Only well-defined fibrils with high contrast were considered.

The cytotoxic effects of Syn and 7–140 Syn were evaluated in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM/F-12 medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, Waltham, MA, USA), 3.0 mM glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO₂ atmosphere. Cells were seeded in 96-well plates at a density of 10 000 cells/well. After 24 hours, cells were treated with 7.0 µM Syn or 7–140Syn for 24 or 48 hours. Following treatment, 20 µL of MTS reagent (CellTiter 96®AQueous One Solution Cell Proliferation Assay, Promega, Madison, Wisconsin, USA) were added to each well, and the plates were incubated for 3 hours at 37°C in 5% CO₂ atmosphere, according to manufacturer instructions. Cell viability was assessed by measuring the absorbance at 492 nm, which is directly proportional to the amount of formazan product generated by metabolically active cells. Data were expressed as mean ± standard deviation from 3 independent experiments, each one performed in sextuplicate.