Linking Thrombin to α-Synuclein truncation reveals a molecular bridge between neuroinflammation and Parkinson disease

Linking Thrombin to α-Synuclein truncation reveals a molecular bridge between neuroinflammation and Parkinson disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Linking Thrombin to α-Synuclein truncation reveals a molecular bridge between neuroinflammation and Parkinson disease Patrizia Polverino de Laureto, Philipp Trolese, Benedetta Fongaro, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8050047/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Proteolytic processing of α-Synuclein (Syn) contributes to the molecular diversity and toxicity of its aggregates in Parkinson disease (PD), yet the proteases responsible for generating disease-relevant truncations remain incompletely defined. Here, we identify thrombin (Thb) — a serine protease best known for its role in haemostasis — as a highly selective Syn-processing enzyme with relevance to neuroinflammatory conditions. Thb predominantly cleaves Syn at Lys6–Gly7, producing a 7–140 fragment as the major proteolytic species. This reaction displays remarkable site specificity despite multiple lysine residues and is modulated by ionic strength and Syn conformational flexibility. The 7–140 fragment adopts a slightly more compact conformational ensemble, shows reduced membrane binding, and forms fibrils with slower aggregation kinetics and altered morphology compared to the full-length protein. These properties may extend the lifetime of soluble oligomeric intermediates, potentially contributing to chronic Syn-mediated toxicity. Our findings reveal a previously unrecognized link between inflammatory Thb activity and Syn proteostasis, suggesting that neuroinflammation-associated proteases may influence PD progression through selective truncation of Syn. Biological sciences/Biochemistry/Protein folding/Protein aggregation Biological sciences/Biochemistry/Proteases α-synuclein thrombin α-synuclein pathological mutants proteolysis truncated forms Parkinson disease neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Parkinson disease (PD) is a progressive neurodegenerative disorder affected by both genetic and environmental factors 1 . Its pathological hallmark is the presence of Lewy bodies (LB), intracellular fibrillar aggregates predominantly composed of α-synuclein (Syn) (Fig. 1 A). Familial mutations in Syn are associated with early-(A30P, E46K, A53T) and late-(H50Q) onset PD, highlighting the central role of the protein in disease pathogenesis 2 , 3 . Syn is a small (14.4 kDa) intrinsically disordered protein that exists in a dynamic equilibrium between cytosolic disordered conformations and a membrane-bound α-helical state 4 . Under pathological conditions, Syn aggregates into oligomers, protofibrils, and fibrils characterized by cross β-sheet structure (Fig. 1 B). Neurotoxicity is primarily attributed to soluble oligomers, which disrupt cellular homeostasis and exacerbate oxidative stress 5 . Structurally, Syn comprises three domains: a positively charged N-terminal region (residues 1–60) mediating membrane interactions 6 , 7 , a central hydrophobic Non-Amyloid-Component (NAC) domain (residues 61–96) essential for oligomerization and fibril formation 8 , and a negatively charged C-terminal domain (residues 97–140) that mediates interactions with ions and other proteins 9 . The consequences of the familial mutations on Syn structure, aggregation kinetics, and membrane affinity have been intensively studied. Variants such as A53T, E46K, and H50Q generally accelerate amyloid formation relative to the wild type, while the effect of A30P remains context dependent 3 . Notably, the E46K mutation produces a particularly severe clinical phenotype and increases the toxicity of aggregated Syn forms 10 , 11 . Syn aggregation is also influenced by environmental factors and post-translational modifications (PTMs). In PD, approximately 90% of Syn is phosphorylated 12 , and 10–30% of Syn in pathological inclusions is truncated 13 . C-terminal truncations are abundant and accelerate fibrillization 13 – 16 , whereas N-terminal truncations, though less studied, have been implicated in altered aggregate morphology, cytotoxicity, and aggregation kinetics 17 – 20 . Though, identifying the proteases responsible for Syn processing in vivo remains a major challenge. Although traditionally considered a cytoplasmic protein, both monomeric and oligomeric Syn have been found extracellularly in cerebrospinal fluid and blood plasma, especially in synaptic clefts and extracellular vesicles (EV) 21 , 22 . Unconventional pathways of Syn release include exosome release and micro-vesicle shedding. EV-associated Syn participates in intercellular communication and the propagation of neurodegenerative pathology. Moreover, extracellular Syn can trigger pro-inflammatory responses in astrocytes and microglia, linking protein aggregation to neuroinflammation 23 – 25 . Its extracellular presence increases the likelihood to act as a substrate for disease-associated proteases. Thrombin (Factor IIa, Thb) is a trypsin-like serine protease best known for its central role in haemostasis, where it exhibits both procoagulant and anticoagulant activities 26 , 27 . It is generated by proteolytic cleavage of its precursor, prothrombin (Factor II), which is primarily synthesised in the liver and circulates in plasma at concentrations of approximately 0.1 mg/mL. Beyond its well-established role in the coagulation cascade, accumulating evidence indicates that and its zymogen are also expressed within the central nervous system (CNS) 28 – 30 . Both neurons and glial cells have been reported to produce prothrombin mRNA and protein, suggesting the existence of a local, brain-derived Thb system that can be activated under physiological or pathological conditions 28 , 31 . Neuron-generated Thb has been implicated in modulating astrocyte activity and in driving neuroinflammatory responses following injury 31 , 32 . Thb is detectable in the brain, particularly under conditions involving during blood–brain barrier (BBB) disruption, injury, or neuroinflammatory conditions 29 , 33 . For instance, elevated Thb levels have been detected postmortem in senile plaques of Alzheimer disease (AD) patients 34 . Expression of protease nexin-1 (PN1), a Thb inhibitor is also reduced around cerebral blood vessels in AD 35 . Increased levels of both Thb and its receptor protease activated receptor-1 (PAR-1) have also been reported in PD patients 36 . This suggests a context in which Thb exerts increased activity in the CNS. Its extracellular presence allows it to modulate synaptic plasticity and inflammatory responses, thus raising the possibility of interactions with other proteins. Whether Thb directly processes Syn, thereby linking inflammatory signaling to protein misfolding, remains to be clarified. Here, we demonstrated that Thb predominantly cleaves Syn at Lys6–Gly7, generating a 7–140 fragment with altered conformational and aggregation properties. This mechanism defines a previously unrecognized pathway coupling inflammatory serine-protease activity with Syn proteostasis. Results Thrombin cleaves α-synuclein in a preferential site 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 conformations 37 . 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) environments 40 . 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 activity 42 . 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. Proteolysis of α-synuclein mutants by Thrombin occurs at different extent and is dictated by the conformational features of the protein substrates 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 reactions 43 , 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 states 46 . 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 data 17 . 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 vesicles 47 . 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. 6 A, B). Quantitative analysis (Fig. 6 C) 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. 6 D, 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. Removal of the first six residues affects both fibril kinetics and morphology Finally, we examined how the removal of the first six N-terminal residues affects aggregation, and how this truncated form may co-aggregate with the full-length protein. Under aggregation conditions, Syn, 7–140 Syn and the mix of the two proteins (3:1 ratio) exhibited distinct behaviors (Fig. 7A,B; Fig. S6A, Table S2). This ratio was selected to mimic the abundance of full-length Syn compared to truncated Syn species found in LB (~10–30 %) 13 . Syn displayed a lag phase of 43 ± 7 h and a half-time (t₁ / ₂) of 63 ± 16 h, with the highest overall fluorescence intensity. 7–140 Syn showed slower kinetics, with a lag phase of 50 ± 2 h, a t₁ / ₂ of 100 ± 1 h, and a fluorescence yield at plateau of approximately 60 % of that of Syn. The mixed sample exhibited intermediate behavior, with a lag phase of 47 ± 6 h, t 1/2 of 67 ± 2 h, and a fluorescence plateau ~80 % of that of Syn. After aggregation reached a plateau, TEM micrographs were acquired for each sample and the fibril width distribution measured (Fig. 7D, Fig. S6B). Interestingly, Syn fibrils appear with a smooth surface, whereas many of the 7–140 Syn and some of the mixed fibrils display a rugged surface. Additionally, Syn fibrils were the widest with an average width of 17.4 ± 4.0 nm, while 7–140 Syn were the thinnest (12.6 ± 3.2 nm). Fibrils from the mixed sample showed an intermediate width (15.0 ± 4.1 nm). Successively, samples were ultracentrifuged to separate insoluble protein fraction. The resulting pellets were resuspended in 7 M Gnd–HCl, and UV spectra were recorded to assess the amount of insoluble protein (Fig. S7A). The three conditions exhibited different extent of light scattering after resuspension: 7–140 Syn showed the lowest scattering contribution, Syn an intermediate level, and the mixed sample the highest. After baseline correction to remove scattering contribution (Fig. S7B), the insoluble fractions accounted for ~49 % of the total protein for both Syn and 7–140 Syn, and ~44 % for the mixed sample. To determine the composition of the insoluble fractions, the resuspended pellets were analyzed by RP–HPLC (Fig. 7C), and peak identities were confirmed by MS (not shown). In the Syn and 7–140 Syn samples, the main chromatographic peaks corresponded to the expected species. Minor additional peaks were attributed to secondary fragmentation probably generated during prolonged incubation, agitation and sample handling. Notably, in the mixed sample (blue line), the peak eluting at ~33 min was confirmed by MS to contain the 7–140 species. Despite the 3:1 ratio of the mixed sample, quantitative analysis of the chromatogram peaks revealed an approximately 1:1 ratio of Syn and 7–140 Syn. Discussion Neurodegenerative disorders such as PD are often correlated with neuroinflammatory states. Here, we propose a previously unrecognized interaction between Syn and Thb possibly linking inflammatory protease activity and protein misfolding in PD. Importantly, Thb has been previously implicated in PD pathology by contributing to neuronal cell death, activating microglia and contributing to oxidative stress 30 , 32 , 33 , 50 . Our data show that, when mixed in pathological mimicking conditions, Thb cleaves Syn and generates 7–140 Syn, as the predominant proteolytic species, selecting Lys6 despite the presence of 15 lysine residues in the protein sequence. The presence of a unique (main) site of proteolysis, the Lys6–Gly7 peptide bond, focussed our attention on both the specificity of Thb and the cleavage susceptibility of Syn. Thb has a preferential consensus cleavage site with Arg in position P1. After Arg the other residue recognized albeit with less preference is Lys 51 , 52 . Importantly, position P1’ is always a Gly and/or other hydrophilic not acidic residues. This aligns well with our observed binding site between Lys6 and Gly7. This also explains the further cleavages observed to a much lower extent (between Lys10–Ala11 and Lys32–Thr33) due to enzyme preference. Interestingly, Plasmin -another serine protease with similar specificity to Thb albeit less stringent – has been reported to generate the same fragments we observed (i.e. 11–140 and 33–140) without ever generating the 7–140 species 53 . Importantly, we also show that NaCl has a critical role on the reaction influencing the 7–140 Syn production yield. Na⁺ binding to Thb allosterically stabilizes its catalytically active conformation 27 , 41 . In parallel, it is known that NaCl accelerates Syn aggregation by stabilizing more extended conformations with an exposed NAC region 37 . Despite these notions, we observed slower Thb-mediated Syn proteolysis in the presence of NaCl. To further explore the interplay between structure and enzymatic processing, the conformational ensemble of Syn familiar mutants (A30P, E46K, H50Q, A53T) was examined. We found that mutants that preferentially choose a more extended Syn conformer like A53T give high proteolysis yields, while E46K which generally is more compact has lower yields. This is in line with what is typically expected for proteolysis, where a more flexible extended substrate is processed faster 43 . The effect of NaCl on Syn proteolysis by Thb is therefore multifaceted. While Na⁺ enhances Thb catalytic activity by stabilizing its “fast” conformational state and promoting more extended conformations of Syn, the concomitant presence of Cl⁻ ions likely shields the positively charged N-terminal region of Syn, resulting in an overall slower proteolysis reaction. Hence, Syn proteolysis is a complex reaction that depends on conformational flexibility and electrostatics. In our assay, the presence of 20 mM Na⁺ from phosphate buffer already saturates the Na⁺-binding site of Thb, so adding 150 mM NaCl primarily alters ionic strength rather than allosteric activation. This condition may shift the electrostatic landscape without significantly changing the intrinsic catalytic efficiency of the enzyme. Under neuroinflammatory conditions, Thb levels increase within CNS especially due to BBB breakdown 29 , 54 , even reaching up to 25 nM, sufficient to act on neuronal substrates 55 . While Syn is largely depicted as cytoplasmic protein an increasing number of studies report the presence of both monomeric and oligomeric forms in the extracellular space, including synaptic clefts and EVs are increasing 21 , 22 . The simultaneous extracellular presence of Thb and Syn raises the possibility of protease-mediated Syn processing, potentially affecting synaptic function and neurodegeneration. This interaction is likely compartment-specific (Scheme 1 ). Intracellularly, Syn is abundant whereas Thb is largely absent, limiting direct proteolysis; conversely, in the extracellular milieu, Thb concentrations are high while Syn levels remain relatively low. Such compartmental asymmetry suggests that Thb-mediated Syn processing may occur in restricted microdomains where both proteins transiently co-localize and accumulate, such as sites of neuron membrane leakage, vesicle release, synaptic clefts, or regions of BBB disruption. Notably, NaCl concentrations differ markedly between intracellular (~ 10 mM) and extracellular (~ 150 mM) environments 40 . Such ionic differences could attenuate Thb-mediated Syn proteolysis in the extracellular space, implying that sustained or repeated Thb exposure may be required for significant cleavage to occur in vivo. These localized interactions could influence the aggregation state, clearance, intercellular transmission and functions of Syn, thereby contributing to the propagation of synucleinopathies and associated neuroinflammatory processes. The removal of the first six residues of Syn would have a drastic impact on its characteristics and functions. On the one hand, 7–140 Syn shows a reduced helical stabilization upon membrane binding. Mechanistically, this may derive from a missing salt bridge between D2 and K6 identified by Fusco et al 56 to be important for helix nucleation. Cholak et al 57 even showed that the removal of residues 2–14 reduced protein membrane localization, confirming the N-terminal anchor has an impact in vivo. These differences would manifest in lacking membrane remodeling capabilities which facilitate vesicle fusion and neurotransmitter release 58 . On the other hand, 7–140 Syn lacks 2 Met residues implicated in the scavenging of lipid hydroperoxides 59 . Missing reconversion of the lipids to their natural form would result in accumulation of oxidative damage in brains of PD patients. As a final remark, the removal of the first six N-terminal residues markedly slows aggregation kinetics consistent with a more compact starting conformation that is recruited by fibril ends with reduced efficiency. Consequently, the truncated species may persist longer in soluble oligomeric states and act as seeding competent intermediates. Hence, these species might contribute to a more chronic form of Syn–mediated toxicity. Fully understanding the structural and toxic properties of these intermediates will be essential to assess whether neuroinflammation enhances Syn toxicity through proteolytic processing. Importantly, even fibril morphology seems altered in fibrils formed by the fragment alone and by the co-aggregated species at physiological ratios. This contributes to existing literature describing different fibril morphology of Syn species lacking increasingly larger parts of the N-terminal fragment 17 , 60 . Of note, Dewison et al 61 studied a recombinant Syn construct missing residues 2–6, effectively differing only in the Met1 residue from the Thb-generated 7–140 species. In contrast to our work, they report no differences in membrane binding and helix formation, suggesting either the importance of the first Met residues and/or of the SUV lipid composition in this regard. In accordance with our data, they observe a slower fibril assembly, highlighting the critical sensitivity of fibril growth on the precise sequence of the N-terminal region. In conclusion, our work further strengthens the notion that the N-terminal residues have a stark impact on Syn biology in PD. Conclusion Together, these findings expand our understanding of how Thb interacts with Syn and establish a functional connection between Thb activity, proteolytic processing, ionic homeostasis, and Syn structural flexibility. Our data show that Thb-mediated proteolysis of Syn is a multifactorial process shaped by protein conformation, electrostatic interactions, and the surrounding molecular environment, each potentially contributing to PD pathology. Our findings provide mechanistic insight into the generation and significance of N-terminal truncations of Syn and may be extended to related serine proteases in vivo especially during states of chronic neuroinflammation. Defining the local, temporal and structural determinants of these truncations will be essential for understanding their contribution to disease progression and may direct the development of targeted therapeutic strategies. In particular, our results highlight the critical influence of ionic homeostasis on Thb–Syn interactions. Variations in Na⁺ and Cl⁻ levels, such as those occurring during neuroinflammation or impaired ion regulation in PD, could therefore differentially affect Thb activity and Syn susceptibility to proteolysis. This suggests that ionic imbalance within the CNS may not only alter Syn aggregation pathways but also tune its enzymatic processing, ultimately influencing the course and heterogeneity of synucleinopathies. Material and Methods Material. Thrombin, trypsin, PPACK, (D)-Phe-Pip-Arg-pNA (S-2238), and other reagents were provided by Merck (Darmstadt, Germany). L-alpha-Phosphatidylcholine (egg, PC) and L-alpha-Phosphatidylserine (bovine liver, PS) were purchased from Avanti Lipid (Alabaster, Alabama, USA). Expression and purification of recombinant α-synuclein and its mutants 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 described 45 . Protein identity and integrity were assessed by mass spectrometry (MS). Proteolysis of α-synuclein Proteolysis was carried out in 20 mM sodium phosphate buffer, pH 7.4, using Thb at an enzyme to substrate (E/S) ratio of 1:100 (mol/mol) at 22°C and 1:400 (mol/mol) at 37°C, in the presence and in the absence of 150 mM NaCl or the Thb inhibitor PPACK (E/I 1:1). Proteolysis by trypsin was conducted at an E/S of 1:1000 (by weight). All reactions were quenched at specific time by acidification with TFA in water (0.2%, by volume) and analysed by RP-HPLC. RP-HPLC analyses were carried out on a 1200 series Agilent Technologies chromatographer (Santa Clara, CA, USA), using a Jupiter C18 column (4.6 mm × 250 mm, 5.0 µm; Phenomenex, CA, USA). The elution was performed by a gradient of acetonitrile/0.085% TFA and water/0.1% TFA (5%–25% in 5 min, 25%–28% in 13 min, 28%-39% in 3 min, 39–45% in 21 min), recording the eluate absorbance at 226 nm. Thrombin activity The activity of Thb was tested by monitoring the release of p-nitroaniline (pNA) from (D)-Phe-Pip-Arg-pNA (S-2238) spectrophotometrically at 405 nm over time. The assay was conducted in 20 mM sodium phosphate, pH 7.4, in the presence and in the absence of 150 mm NaCl, at 37°C, using an E/S 1:200000. The extinction coefficient of pNA is 8270 M − 1 cm − 1 . Chemical-physical characterization 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 Hippel 62 . 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 cm 2 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 : $$\:{\theta\:}_{i}^{222}=\:{\theta\:}_{coil}^{222}+\:\frac{{\theta\:}_{helix}^{222}-{\theta\:}_{coil}^{222}}{2}\:\left[1+\:\frac{X}{n}+\frac{1}{n{k}_{B}{P}_{t}}-\sqrt{{\left(1+\frac{X}{n}+\frac{1}{n{k}_{B}{P}_{t}}\right)}^{2}-\frac{4X}{n}}\right]$$ where \(\:{\theta\:}_{i}^{222}\) is the ellipticity at each time point, \(\:{\theta\:}_{coil}^{222}\) is the ellipticity at the beginning of the titration, \(\:{\theta\:}_{helix}^{222}\) is the ellipticity at saturating conditions, X is the protein to phospholipids molar ratio, \(\:n\) is the number of lipids interacting, \(\:{k}_{B}\) is the binding constant and \(\:{P}_{t}\) is the initial protein concentration. Percentage of alpha-helix content was estimated with following Eq. 6 4 : $$\:Percentage\:helicity=100\:x\:\frac{\left({\theta\:}_{222}^{exp}-{\theta\:}_{222}^{u}\right)}{\left({\theta\:}_{222}^{h}-{\theta\:}_{222}^{u}\right)}$$ ; where \(\:{\theta\:}_{222}^{exp}\) is the ellipticity at 222 nm under saturating conditions, \(\:{\theta\:}_{222}^{h}\) and \(\:{\theta\:}_{222}^{u}\) correspond to the ellipticity at 222 nm of a protein with 100% and 0% of helical content (estimated to be -39500 deg cm 2 dmol − 1 and − 3000 deg cm 2 dmol − 1 , respectively). Mass spectrometry measurements 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% D 2 O) 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 from 49 . 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 33 amino acids were excluded, due to identification ambiguity and poor sequence localization. Aggregation study 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 Amylofit 66 , 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. Cell culture and viability test 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. Declarations Competing Interests. All authors declare no competing interests. Funding. This project was supported by a competitive grant from University of Padova (Progetti di Ateneo, 2022, #BIRD222840); by the Italian Ministry of Education, University and Research (MUR) Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2022, #2022FPWYA8); and by AFM (Association contre les Myopathies, research grant #28892). Author Contribution. Conceptualization: P.T., B.F., L.A., P.P.d.L. Methodology: P.T., B.F., V.S., A.S., M.S. Data analysis: P.T., B.F., V.S., A.S. Supervision: L.A., D.S., P.P.d.L. Writing: P.T., L.A., P.P.d.L. All authors reviewed, edited and approved. Acknowledgements. We are thankful to the Mass Spectrometry Facility of Department of Pharmaceutical and Pharmacological Science, and Imaging Facility of Department of Biology, at University of Padova, for technical support. References Kalia, L. V. & Lang, A. E. Parkinson’s disease. The Lancet 386 , 896–912 (2015). Conway, K. A. et al. Acceleration of oligomerization, not fibrillization, is a shared property of both α-synuclein mutations linked to early-onset Parkinson’s disease: Implications for pathogenesis and therapy. Proceedings of the National Academy of Sciences 97 , 571–576 (2000). Flagmeier, P. et al. Mutations associated with familial Parkinson’s disease alter the initiation and amplification steps of α-synuclein aggregation. Proceedings of the National Academy of Sciences 113 , 10328–10333 (2016). Meade, R. M., Fairlie, D. P. & Mason, J. M. Alpha-synuclein structure and Parkinson’s disease – lessons and emerging principles. Molecular Neurodegeneration 14 , 29 (2019). Winner, B. et al. In vivo demonstration that α-synuclein oligomers are toxic. Proceedings of the National Academy of Sciences 108 , 4194–4199 (2011). Eliezer, D., Kutluay, E., Bussell, R. & Browne, G. Conformational properties of α-synuclein in its free and lipid-associated states1. Journal of Molecular Biology 307 , 1061–1073 (2001). Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S. & Langen, R. Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement. Proceedings of the National Academy of Sciences 105 , 19666–19671 (2008). Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. & Lansbury, P. T. NACP, A Protein Implicated in Alzheimer’s Disease and Learning, Is Natively Unfolded. Biochemistry 35 , 13709–13715 (1996). Mahul-Mellier, A.-L. et al. Differential role of C-terminal truncations on alpha-synuclein pathology and Lewy body formation. npj Parkinsons Dis. 11 , 261 (2025). Zarranz, J. J. et al. The new mutation, E46K, of α-synuclein causes parkinson and Lewy body dementia. Annals of Neurology 55 , 164–173 (2004). Greenbaum, E. A. et al. The E46K Mutation in α-Synuclein Increases Amyloid Fibril Formation*. Journal of Biological Chemistry 280 , 7800–7807 (2005). Machiya, Y. et al. Phosphorylated α-Synuclein at Ser-129 Is Targeted to the Proteasome Pathway in a Ubiquitin-independent Manner*. Journal of Biological Chemistry 285 , 40732–40744 (2010). Ma, L. et al. C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Synuclein: A vicious cycle in Parkinson’s disease. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1864 , 3714–3725 (2018). Sorrentino, Z. A. et al. Physiological C-terminal truncation of α-synuclein potentiates the prion-like formation of pathological inclusions. Journal of Biological Chemistry 293 , 18914–18932 (2018). Murray, I. V. J. et al. Role of α-Synuclein Carboxy-Terminus on Fibril Formation in Vitro. Biochemistry 42 , 8530–8540 (2003). Liu, C.-W. et al. A Precipitating Role for Truncated α-Synuclein and the Proteasome in α-Synuclein Aggregation: IMPLICATIONS FOR PATHOGENESIS OF PARKINSON DISEASE*. Journal of Biological Chemistry 280 , 22670–22678 (2005). Thrush, R. J., Vadukul, D. M. & Aprile, F. A. A Facile Method to Produce N-Terminally Truncated α-Synuclein. Front. Neurosci. 16 , (2022). Thrush, R. J., Vadukul, D. M., Allerton, S. C., Storch, M. & Aprile, F. A. Surface Wetting Is a Key Determinant of α-Synuclein Condensate Maturation. 2024.06.06.597302 Preprint at https://doi.org/10.1101/2024.06.06.597302 (2025). Vamvaca, K., Volles, M. J. & Lansbury, P. T. The First N-terminal Amino Acids of α-Synuclein Are Essential for α-Helical Structure Formation In Vitro and Membrane Binding in Yeast. Journal of Molecular Biology 389 , 413–424 (2009). Santos, J. et al. A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion. J. Am. Chem. Soc. 146 , 12702–12711 (2024). Lööv, C., Scherzer, C. R., Hyman, B. T., Breakefield, X. O. & Ingelsson, M. α-Synuclein in Extracellular Vesicles: Functional Implications and Diagnostic Opportunities. Cell Mol Neurobiol 36 , 437–448 (2016). Lee, H.-J., Bae, E.-J. & Lee, S.-J. Extracellular α-synuclein—a novel and crucial factor in Lewy body diseases. Nat Rev Neurol 10 , 92–98 (2014). Klegeris, A. et al. Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells. The FASEB Journal 20 , 2000–2008 (2006). Lee, H.-J. et al. Direct Transfer of α-Synuclein from Neuron to Astroglia Causes Inflammatory Responses in Synucleinopathies*. Journal of Biological Chemistry 285 , 9262–9272 (2010). Roodveldt, C., Christodoulou, J. & Dobson, C. M. Immunological features of α-synuclein in Parkinson’s disease. Journal of Cellular and Molecular Medicine 12 , 1820–1829 (2008). Davie, E. W. & Kulman, J. D. An Overview of the Structure and Function of Thrombin. Semin Thromb Hemost 32 , 3–15 (2006). Di Cera, E. Thrombin. Molecular Aspects of Medicine 29 , 203–254 (2008). Arai, T., Miklossy, J., Klegeris, A., Guo, J.-P. & McGeer, P. L. Thrombin and Prothrombin Are Expressed by Neurons and Glial Cells and Accumulate in Neurofibrillary Tangles in Alzheimer Disease Brain. J Neuropathol Exp Neurol 65 , 19–25 (2006). Krenzlin, H., Lorenz, V., Danckwardt, S., Kempski, O. & Alessandri, B. The Importance of Thrombin in Cerebral Injury and Disease. International Journal of Molecular Sciences 17 , 84 (2016). Shlobin, N. A., Har-Even, M., Itsekson-Hayosh, Z., Harnof, S. & Pick, C. G. Role of Thrombin in Central Nervous System Injury and Disease. Biomolecules 11 , 562 (2021). Rajput, P. S. et al. Neuron-generated thrombin induces a protective astrocyte response via protease activated receptors. Glia 68 , 246–262 (2020). Chen, X. et al. Thrombin induces morphological and inflammatory astrocytic responses via activation of PAR1 receptor. Cell Death Discov. 8 , 189 (2022). Ye, F., Garton, H. J. L., Hua, Y., Keep, R. F. & Xi, G. The Role of Thrombin in Brain Injury After Hemorrhagic and Ischemic Stroke. Transl. Stroke Res. 12 , 496–511 (2021). Turgeon, V. L. & Houenou, L. J. The role of thrombin-like (serine) proteases in the development, plasticity and pathology of the nervous system. Brain Research Reviews 25 , 85–95 (1997). Vaughan, P. J., Su, J., Cotman, C. W. & Cunningham, D. D. Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer’s disease. Brain Research 668 , 160–170 (1994). Johnson, S. L., Iannucci, J., Seeram, N. P. & Grammas, P. Inhibiting thrombin improves motor function and decreases oxidative stress in the LRRK2 transgenic Drosophila melanogaster model of Parkinson’s disease. Biochemical and Biophysical Research Communications 527 , 532–538 (2020). Ramis, R., Ortega-Castro, J., Vilanova, B., Adrover, M. & Frau, J. Unraveling the NaCl Concentration Effect on the First Stages of α-Synuclein Aggregation. Biomacromolecules 21 , 5200–5212 (2020). Pelc, L. A., Koester, S. K., Kukla, C. R., Chen, Z. & Di Cera, E. The active site region plays a critical role in Na+ binding to thrombin. Journal of Biological Chemistry 298 , 101458 (2022). Ayala, Y. & Cera, E. D. Molecular Recognition by Thrombin. Role of the Slows→Fast Transition, Site-specific Ion Binding Energetics and Thermodynamic Mapping of Structural Components. Journal of Molecular Biology 235 , 733–746 (1994). Egidi, I., Guidi, M. & Giove, F. Compartmentalization of sodium in the human brain: a mini-review of 23Na-MRI methods. Front. Phys. 13 , (2025). Dang, O. D., Vindigni, A. & Di Cera, E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proceedings of the National Academy of Sciences 92 , 5977–5981 (1995). Stojanovski, B. M., Pelc, L. A. & Di Cera, E. Thrombin has dual trypsin-like and chymotrypsin-like specificity. Journal of Thrombosis and Haemostasis 22 , 1009–1015 (2024). Hubbard, S. J. The structural aspects of limited proteolysis of native proteins. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology 1382 , 191–206 (1998). Borthakur, K., Sisk, T. R., Panei, F. P., Bonomi, M. & Robustelli, P. Determining accurate conformational ensembles of intrinsically disordered proteins at atomic resolution. Nat Commun 16 , 9036 (2025). Fongaro, B., Cappelletto, E., Sosic, A., Spolaore, B. & Polverino de Laureto, P. 3,4-Dihydroxyphenylethanol and 3,4-dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α-synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect. Protein Science 31 , e4356 (2022). Heuvel, R. H. van den & Heck, A. J. Native protein mass spectrometry: from intact oligomers to functional machineries. Current Opinion in Chemical Biology 8 , 519–526 (2004). Davidson, W. S., Jonas, A., Clayton, D. F. & George, J. M. Stabilization of α-Synuclein Secondary Structure upon Binding to Synthetic Membranes*. Journal of Biological Chemistry 273 , 9443–9449 (1998). Fusco, G. et al. Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour. Nat Commun 5 , 3827 (2014). Rizzotto, E. et al. DOPAC as a modulator of α-Synuclein and E46K interactions with membrane: Insights into binding dynamics. International Journal of Biological Macromolecules 294 , 139427 (2025). Choi, S.-H., Lee, D. Y., Kim, S. U. & Jin, B. K. Thrombin-Induced Oxidative Stress Contributes to the Death of Hippocampal Neurons In Vivo: Role of Microglial NADPH Oxidase. J. Neurosci. 25 , 4082–4090 (2005). Gallwitz, M., Enoksson, M., Thorpe, M. & Hellman, L. The Extended Cleavage Specificity of Human Thrombin. PLOS ONE 7 , e31756 (2012). Uliana, F. et al. Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen. Nat Commun 12 , 1693 (2021). Kim, K. S. et al. Proteolytic Cleavage of Extracellular α-Synuclein by Plasmin: IMPLICATIONS FOR PARKINSON DISEASE*. Journal of Biological Chemistry 287 , 24862–24872 (2012). Leem, E., Jeong, K. H., Won, S.-Y., Shin, W.-H. & Kim, S. R. Prothrombin Kringle-2: A Potential Inflammatory Pathogen in the Parkinsonian Dopaminergic System. Experimental Neurobiology 25 , 147–155 (2016). Ben Shimon, M. et al. Thrombin regulation of synaptic transmission and plasticity: implications for health and disease. Front. Cell. Neurosci. 9 , (2015). Fusco, G. et al. Structural Ensembles of Membrane-bound α-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity. Sci Rep 6 , 27125 (2016). Cholak, E. et al. Avidity within the N-terminal anchor drives α-synuclein membrane interaction and insertion. The FASEB Journal 34 , 7462–7482 (2020). Khounlo, R. et al. Membrane Binding of α-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore. Front. Cell Dev. Biol. 9 , (2021). Maltsev, A. S., Chen, J., Levine, R. L. & Bax, A. Site-Specific Interaction between α-Synuclein and Membranes Probed by NMR-Observed Methionine Oxidation Rates. J. Am. Chem. Soc. 135 , 2943–2946 (2013). McGlinchey, R. P., Ni, X., Shadish, J. A., Jiang, J. & Lee, J. C. The N terminus of α-synuclein dictates fibril formation. Proceedings of the National Academy of Sciences 118 , e2023487118 (2021). Dewison, K. M. et al. Residues 2 to 7 of α-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways. Proceedings of the National Academy of Sciences 121 , e2315006121 (2024). Gill, S. C. & von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182 , 319–326 (1989). Rovere, M. Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of α-Synuclein with Membranes. Methods Mol Biol 1948 , 123–143 (2019). Wei, Y., Thyparambil, A. A. & Latour, R. A. Protein helical structure determination using CD spectroscopy for solutions with strong background absorbance from 190 to 230 nm. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1844 , 2331–2337 (2014). Peterle, D., Wales, T. E. & Engen, J. R. Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen–Deuterium Exchange Mass Spectrometry Experiments. Anal. Chem. 94 , 10142–10150 (2022). Meisl, G. et al. Molecular mechanisms of protein aggregation from global fitting of kinetic models. Nat Protoc 11 , 252–272 (2016). Tables Table 1. Chemical characterization of the protein material corresponding to the main peaks of the chromatograms shown in Fig. 2. The calculated masses were obtained from the amino acid sequence of Syn. RP-HPLC RT (min) Molecular Mass (Da) Species Found Calculated 35.5 14 460.51 ± 0.34 14 460.16 Syn 15.4 769.33 ± 0.01 769.34 1–6 32.5 13 708.38 ± 0.60 13 708.20 7–140 Table 2. Chemical characterization of the protein material corresponding to the peaks of the chromatograms of the proteolysis mixtures shown in Fig. 4. The calculated masses were obtained from the amino acid sequence of Syn variants. The yield of reaction (last column) was calculated as the ratio between the area under the peak of the 7–140 from each species and that of full-length protein. Species RP-HPLC RT (min) Molecular Mass (Da) Yield % Found Calculated 7 – 140 Syn 32.5 13 708.38 ± 0.60 13 708.20 78.74 7 – 140 A30P 31.3 13 733.90 ± 0.28 13 734.24 75.66 7 – 140 E46K 30.3 13 706.53 ± 0.28 13 707.26 18.11 7 – 140 H50Q 34.8 13 698.27 ± 0.12 13 699.19 61.42 7 – 140 A53T 31.3 13 737.88 ± 0.09 13 738.23 100.00 Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files nrreportingsummary3.pdf Article File - Reporting Summary Scheme1.docx supplementary.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8050047","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":545342908,"identity":"ef71b8af-f8ac-4564-b63c-f7b5efe53cbd","order_by":0,"name":"Patrizia Polverino de Laureto","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0367-6781","institution":"Università degli Studi di Padova","correspondingAuthor":true,"prefix":"","firstName":"Patrizia","middleName":"Polverino","lastName":"de Laureto","suffix":""},{"id":545342909,"identity":"1c827251-e53a-4a22-bc46-d8d09a6c67d0","order_by":1,"name":"Philipp Trolese","email":"","orcid":"https://orcid.org/0009-0004-7243-6396","institution":"Università degli Studi di Padova, Università degli Studi di Verona","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Trolese","suffix":""},{"id":545342910,"identity":"ee9d3da7-75a0-4eb6-9c93-d38fab682c50","order_by":2,"name":"Benedetta Fongaro","email":"","orcid":"https://orcid.org/0000-0003-0275-0190","institution":"Università degli Studi di Padova","correspondingAuthor":false,"prefix":"","firstName":"Benedetta","middleName":"","lastName":"Fongaro","suffix":""},{"id":545342911,"identity":"be2e7745-6c8e-435f-b392-e9993a225573","order_by":3,"name":"Vittoria Scapin","email":"","orcid":"https://orcid.org/0009-0005-9176-507X","institution":"Università degli Studi di Padova","correspondingAuthor":false,"prefix":"","firstName":"Vittoria","middleName":"","lastName":"Scapin","suffix":""},{"id":545342912,"identity":"a455002f-44ce-4c2e-a6ac-c234976f4cad","order_by":4,"name":"Alice Sosic","email":"","orcid":"","institution":"University of Padua","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Sosic","suffix":""},{"id":545342913,"identity":"8645a431-eff7-43ae-87e4-c13569b96788","order_by":5,"name":"Martina Scano","email":"","orcid":"","institution":"Università degli Studi di Padova","correspondingAuthor":false,"prefix":"","firstName":"Martina","middleName":"","lastName":"Scano","suffix":""},{"id":545342914,"identity":"e25a924d-d61f-4115-8f71-8288c90e1e35","order_by":6,"name":"Dorianna Sandona","email":"","orcid":"https://orcid.org/0000-0002-9908-2442","institution":"University of Padova","correspondingAuthor":false,"prefix":"","firstName":"Dorianna","middleName":"","lastName":"Sandona","suffix":""},{"id":545342915,"identity":"eeb2ab22-aba2-4ae6-8814-ae1b6dd32b6c","order_by":7,"name":"Laura Acquasaliente","email":"","orcid":"https://orcid.org/0000-0001-6495-0871","institution":"Università degli Studi di Padova","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Acquasaliente","suffix":""}],"badges":[],"createdAt":"2025-11-06 16:31:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8050047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8050047/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96169226,"identity":"5668c9cb-0d18-4743-8f98-c1a01a70d327","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4890517,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/4235fe12a1cb084e02598c6a.docx"},{"id":96250413,"identity":"46dc28da-3779-4d15-8483-5dcea3cf915b","added_by":"auto","created_at":"2025-11-19 07:38:17","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8848,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS2589673.json","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/7cd7ae9437d2600fdbff61e9.json"},{"id":96169232,"identity":"743e0c7f-527b-40b3-8f39-3d59821b6224","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166276,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25896730enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/34f3d4ee0864901656eecb81.xml"},{"id":96252086,"identity":"56dad212-ce82-497b-9db6-78a28cdb0fbf","added_by":"auto","created_at":"2025-11-19 07:40:26","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":660189,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/b527a66ea964167c1c2b2a0a.png"},{"id":96169235,"identity":"49e07d9a-b1fc-4d2c-9dd3-383c0e27b678","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161794,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/7c713caf8e74a41d44dc1fa9.jpeg"},{"id":96169237,"identity":"97264b9d-776d-47bf-9844-2b23d26635d3","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103262,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/f0f9c661b7859a4a73594a88.png"},{"id":96251427,"identity":"9001cf35-1ab3-43ba-8303-f62dbc652716","added_by":"auto","created_at":"2025-11-19 07:39:43","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":292010,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage12.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/106f7a36d5796d87b960f5e1.jpeg"},{"id":96169236,"identity":"725a98ff-7dd5-4400-92f5-c2f1a473cd7b","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":327474,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/8200a30185ed5aec38882e9f.png"},{"id":96251199,"identity":"ae3947fc-494a-4974-9597-4ef50887fbd8","added_by":"auto","created_at":"2025-11-19 07:39:29","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":145210,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/2228eb70a516317a17bd0045.png"},{"id":96169242,"identity":"da2b90a8-55fe-433d-9d65-b312adb99174","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":317602,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage15.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/c2961e47e3981ad827e5b6d9.jpeg"},{"id":96252632,"identity":"84157a87-5b7a-4817-96e0-8a266f4115d1","added_by":"auto","created_at":"2025-11-19 07:41:17","extension":"jpeg","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":415968,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/177b90606a710d4a76565f02.jpeg"},{"id":96249290,"identity":"362af4a9-69af-4cd0-a6ce-7fcac9b532a7","added_by":"auto","created_at":"2025-11-19 07:32:51","extension":"jpeg","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":606492,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/d17b35cc2cfa3d465da85ad3.jpeg"},{"id":96169246,"identity":"9e723647-efb9-4dd5-8b9a-5733244506e0","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":321574,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/68a977b13f7be19fc5354506.png"},{"id":96169260,"identity":"6ef00179-c62c-41d1-b32f-08ab876de52b","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":220187,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/743ca163472f7caeb9acf856.png"},{"id":96169257,"identity":"ff4213f5-5371-4213-94d2-575a93e2b679","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"jpeg","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":551255,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/619d2b8c848c917d2883f835.jpeg"},{"id":96169247,"identity":"eecb6cf2-b804-4274-9c53-02a1d3c081f2","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1200054,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/5a162d12221dedab905fbb5e.png"},{"id":96252601,"identity":"3a562c25-f023-4da0-9bc8-8ab2947b10e9","added_by":"auto","created_at":"2025-11-19 07:41:16","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":544401,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/3ef6594c46abe7562180c441.png"},{"id":96169245,"identity":"12f213c7-44a9-48b9-a1a5-54bc9f8d6f1e","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"jpeg","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252717,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/9af5035e13d9b723a0de4334.jpeg"},{"id":96169264,"identity":"e8171b2c-4a6b-473e-8830-445321903b3d","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78867,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/cbc0fb11997f90c049a15af0.png"},{"id":96169240,"identity":"cf55bd02-362e-4b2b-8453-4d948154fca5","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33055,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/52b967bb0fceee91e33ddccd.png"},{"id":96169251,"identity":"502226c7-c9d8-4eb6-bd31-5fef21e47ac9","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26857,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/77a193e5d6fcf587abef3192.png"},{"id":96169265,"identity":"dda06503-42ec-455b-9df7-695b62b158f7","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":57740,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/968944707097e9650eb84bca.png"},{"id":96169253,"identity":"b7f06e8a-d20d-482e-b628-4a6b20029b32","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67835,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/c936633d21e005e90f234979.png"},{"id":96169250,"identity":"d4b766f5-5f24-41dd-a79e-30ddcd86e980","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":38451,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/0cb451ee92a00dfb7440efa7.png"},{"id":96250890,"identity":"7d985f85-c3cc-4c1c-bcb9-cd5eda86ff83","added_by":"auto","created_at":"2025-11-19 07:39:05","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65700,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/1d7d1291f934b6a10d7af88e.png"},{"id":96251196,"identity":"80a4b008-72bd-4c59-8c35-b4145abbef53","added_by":"auto","created_at":"2025-11-19 07:39:29","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105634,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/5f8d789ca17e2f7cc212612a.png"},{"id":96169244,"identity":"23e42e3c-797d-4248-9872-194b3b9e69a2","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163262,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/e3e1491b07545a154657d122.png"},{"id":96250455,"identity":"c0c37a9d-a551-41e8-870b-a39e549be00d","added_by":"auto","created_at":"2025-11-19 07:38:24","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90915,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/28efe6c995882484d4258323.png"},{"id":96252615,"identity":"3adf50f5-c3ce-4faa-b24c-5375f67f25f0","added_by":"auto","created_at":"2025-11-19 07:41:16","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59476,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/ee826a6f0a335fdb9d2ee45a.png"},{"id":96252665,"identity":"544f2339-946f-400c-851f-4f11b65d2b8e","added_by":"auto","created_at":"2025-11-19 07:41:20","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":113831,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/10b6ab14adc8b6ef4956d7db.png"},{"id":96169261,"identity":"243a240f-cb11-431c-91e9-171779834aa3","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":212268,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/11f6950d1a11593ed0f2199c.png"},{"id":96169266,"identity":"f96545df-5351-41d5-8c78-dcdcc7bbb035","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":65790,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/850ef4e3b6497c6f71a96c47.png"},{"id":96169255,"identity":"987d1a23-7a60-486e-a5e8-2c06d9d285fb","added_by":"auto","created_at":"2025-11-18 10:21:29","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61035,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/42f397e356d3d8b3e58d579e.png"},{"id":96250309,"identity":"726f8d49-7a0f-4bf2-8394-25064bdd65da","added_by":"auto","created_at":"2025-11-19 07:38:03","extension":"xml","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164874,"visible":true,"origin":"","legend":"","description":"","filename":"NCOMMS25896730structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/fc38a56e2f577a55ca0b095b.xml"},{"id":96249409,"identity":"64d02206-7c1f-4e93-b429-2216161c415e","added_by":"auto","created_at":"2025-11-19 07:33:27","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174580,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/574b4ed72012a174317752e7.html"},{"id":96249710,"identity":"054e658b-63b0-4aff-8213-7747fc860923","added_by":"auto","created_at":"2025-11-19 07:36:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":265419,"visible":true,"origin":"","legend":"\u003cp\u003eStructure details of Syn. (A) Primary sequence scheme. The structural domains composing the protein and the main pathological mutations are highlighted. (B) Conformations adopted by Syn in vivo. In the centre the dynamic ensemble of monomeric Syn is displayed. In the physiological state (left), Syn exists in equilibrium between the disordered monomer and the membrane-bound form, where the lipid-binding domain (highlighted in blue) adopts an α-helical conformation. In the pathological state (right), Syn misfolds and enters the aggregation pathway, ultimately forming amyloid fibrils characterized by a cross-β-sheet conformation. The NAC domain, primarily responsible for amyloid assembly, is shown in red (created with Biorender).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/7721a8388a1f0031e8dc7ecd.png"},{"id":96250346,"identity":"7025b95a-d4eb-4ab8-9858-4fa54ecfc0c8","added_by":"auto","created_at":"2025-11-19 07:38:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":151007,"visible":true,"origin":"","legend":"\u003cp\u003eProteolysis of Syn by Thb. The reaction was performed at pH 7.4 at an E/S ratio of 1:100. (A) RP-HPLC chromatograms of the protein before (top) and after (bottom) incubation with the protease. (B) Profiles of the same reaction in the absence (top) and in the presence (bottom) of a Thb inhibitor. (C) The main site of proteolysis by Thb evidenced by an arrow.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/849f50da655099a9a65b613a.png"},{"id":96251417,"identity":"dbfef246-f4bd-48f4-8b7b-bf773af655c4","added_by":"auto","created_at":"2025-11-19 07:39:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":200795,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between the time course of the proteolysis of Syn by Thb in the absence (A) and in the presence (B) of 150 mM NaCl. (C) Proteolysis yield in the case of Syn (square) and 7–140 Syn (circle) in the absence (continuous line) and in the presence (dashed line) of NaCl as a function of time. (D) Proteolysis of Syn with Trypsin (E/S 1:1000). Reaction conditions were described in Method section. (E) Cartoon of the proteolysis event mediated by Thb on Syn as a substrate in the presence and in the absence of NaCl.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/50ed9102d7f8aabf685b7759.png"},{"id":96169227,"identity":"64d4cee3-2c12-43bd-be7a-d2c3fe20e73e","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":179608,"visible":true,"origin":"","legend":"\u003cp\u003eProteolysis by Thb (top) and Native MS (bottom) of Syn variants (A-D). The reactions were performed at pH 7.4 at an E/S ratio of 1:100. The RP-HPLC chromatograms of the proteins before (red) and after (black) incubation with the protease were shown. ESI-MS spectra of samples containing 10 μM of sample. The numbers 1, 2 and 3 close to the m/z indicate three different conformer populations. Histograms comparing the percentages of each population observed for the analysed species (E).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/b708fa8fc4482afde05fa948.png"},{"id":96249325,"identity":"2488240d-834e-47fc-8e98-b4b015197f13","added_by":"auto","created_at":"2025-11-19 07:32:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":147442,"visible":true,"origin":"","legend":"\u003cp\u003eConformational features and cytotoxicity of 7–140 Syn. (A) Size exclusion chromatography (SEC) of 7–140 (black line) and Syn (red line). Elution was conducted in 20 mM sodium phosphate buffer, 150 mM NaCl, pH 7.0. (B) Far-UV CD spectra of 7–140 in the absence (continuous line) and in the presence (dashed line) of 150 mM NaCl (B). Inset: Far-UV CD spectra of Syn under the same conditions. (C) Native ESI-MS. Histograms comparing the percentages of each population observed for Syn (dark grey) and 7–140 (light grey) determined by native MS. (D) Cell viability test on neuroblastoma SH-SY5Y cells. Cells were exposed to 7.0 μM Syn or 7–140 Syn and viability assessed after 24 (black) or 48 (white) hours, compared to not treated controls (NT). Statistical analysis was performed by one-way Anova followed by the Tukey test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/ea5ca117b9c9ff6dd54293ef.png"},{"id":96251175,"identity":"eeaf1b89-e2e1-471c-925d-6056745fa40c","added_by":"auto","created_at":"2025-11-19 07:39:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":215308,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction of Syn and 7–140 Syn with SUV membrane probed by CD (A-C) and HDX-MS (D-F). Far-UV CD spectra of Syn (A) and 7–140 Syn (B) at increasing SUV concentrations. Protein concentration was 3.5 µM. The isodichroic point was highlighted. The normalized ellipticity of Syn (red) and 7–140 Syn (black) measured at 222 nm from three independent measurements was plotted against the SUV concentration (C). Deuterium uptake percent of fragments of Syn (red) and 7–140 Syn (black) measured along the incubation time (D-F).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/2efb569c343253f86c4077d6.png"},{"id":96169234,"identity":"007073cd-b4fa-48f7-a1bc-7c3a7eba97b8","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":651185,"visible":true,"origin":"","legend":"\u003cp\u003e(A) ThT aggregation kinetics of Syn (red), 7–140 Syn (black) and the 3:1 mixed sample (blue). Symbols (squares, circles and triangles, respectively) represent the average experimental ThT fluorescence values, and solid lines show the corresponding fits obtained by AmyloFit using the fragmentation-dominated aggregation model. (B) Bar plot comparing the lag phase, half–time (t₁/₂) and time to plateau for Syn (red), the mixed sample (blue) and 7–140 Syn (black).\u003cbr\u003e\n(C) RP-HPLC chromatograms of the solubilized pellets obtained after ultracentrifugation of the aggregation mixtures, showing peaks corresponding to Syn (red), 7–140 Syn (black) and the mixed sample (blue). (D) Representative TEM micrographs of Syn, 7–140 Syn and the 3:1 mixed sample fibrils. The scale is reported on the left.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/e3f4074bfb0365b0e15c7267.png"},{"id":96256943,"identity":"e08b3526-e457-4531-b95e-55211eea6f59","added_by":"auto","created_at":"2025-11-19 07:51:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2705808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/e30da254-ded1-4a87-8ebd-ab7e2cd3247a.pdf"},{"id":96169223,"identity":"52617a7a-eb88-4b5c-a71c-29542732fbcd","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1665116,"visible":true,"origin":"","legend":"Article File - Reporting Summary","description":"","filename":"nrreportingsummary3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/110d150dafe3ef6566673edb.pdf"},{"id":96249568,"identity":"1d3b6d2c-ad98-4450-8d08-6cf98c5e4752","added_by":"auto","created_at":"2025-11-19 07:34:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":447238,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/ff797c7eb3c0c441a0cb5cfb.docx"},{"id":96169230,"identity":"88ab8296-57b8-4297-af66-1048a4d853e0","added_by":"auto","created_at":"2025-11-18 10:21:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1068204,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8050047/v1/5d94862943882de43c1691be.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Linking Thrombin to α-Synuclein truncation reveals a molecular bridge between neuroinflammation and Parkinson disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson disease (PD) is a progressive neurodegenerative disorder affected by both genetic and environmental factors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Its pathological hallmark is the presence of Lewy bodies (LB), intracellular fibrillar aggregates predominantly composed of α-synuclein (Syn) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Familial mutations in Syn are associated with early-(A30P, E46K, A53T) and late-(H50Q) onset PD, highlighting the central role of the protein in disease pathogenesis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSyn is a small (14.4 kDa) intrinsically disordered protein that exists in a dynamic equilibrium between cytosolic disordered conformations and a membrane-bound α-helical state\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Under pathological conditions, Syn aggregates into oligomers, protofibrils, and fibrils characterized by cross β-sheet structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Neurotoxicity is primarily attributed to soluble oligomers, which disrupt cellular homeostasis and exacerbate oxidative stress\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Structurally, Syn comprises three domains: a positively charged N-terminal region (residues 1\u0026ndash;60) mediating membrane interactions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, a central hydrophobic Non-Amyloid-Component (NAC) domain (residues 61\u0026ndash;96) essential for oligomerization and fibril formation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and a negatively charged C-terminal domain (residues 97\u0026ndash;140) that mediates interactions with ions and other proteins\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The consequences of the familial mutations on Syn structure, aggregation kinetics, and membrane affinity have been intensively studied. Variants such as A53T, E46K, and H50Q generally accelerate amyloid formation relative to the wild type, while the effect of A30P remains context dependent\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Notably, the E46K mutation produces a particularly severe clinical phenotype and increases the toxicity of aggregated Syn forms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSyn aggregation is also influenced by environmental factors and post-translational modifications (PTMs). In PD, approximately 90% of Syn is phosphorylated\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and 10\u0026ndash;30% of Syn in pathological inclusions is truncated\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. C-terminal truncations are abundant and accelerate fibrillization\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, whereas N-terminal truncations, though less studied, have been implicated in altered aggregate morphology, cytotoxicity, and aggregation kinetics\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Though, identifying the proteases responsible for Syn processing in vivo remains a major challenge.\u003c/p\u003e\u003cp\u003eAlthough traditionally considered a cytoplasmic protein, both monomeric and oligomeric Syn have been found extracellularly in cerebrospinal fluid and blood plasma, especially in synaptic clefts and extracellular vesicles (EV)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Unconventional pathways of Syn release include exosome release and micro-vesicle shedding. EV-associated Syn participates in intercellular communication and the propagation of neurodegenerative pathology. Moreover, extracellular Syn can trigger pro-inflammatory responses in astrocytes and microglia, linking protein aggregation to neuroinflammation\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Its extracellular presence increases the likelihood to act as a substrate for disease-associated proteases.\u003c/p\u003e\u003cp\u003eThrombin (Factor IIa, Thb) is a trypsin-like serine protease best known for its central role in haemostasis, where it exhibits both procoagulant and anticoagulant activities\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. It is generated by proteolytic cleavage of its precursor, prothrombin (Factor II), which is primarily synthesised in the liver and circulates in plasma at concentrations of approximately 0.1 mg/mL. Beyond its well-established role in the coagulation cascade, accumulating evidence indicates that and its zymogen are also expressed within the central nervous system (CNS)\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Both neurons and glial cells have been reported to produce prothrombin mRNA and protein, suggesting the existence of a local, brain-derived Thb system that can be activated under physiological or pathological conditions\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Neuron-generated Thb has been implicated in modulating astrocyte activity and in driving neuroinflammatory responses following injury\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thb is detectable in the brain, particularly under conditions involving during blood\u0026ndash;brain barrier (BBB) disruption, injury, or neuroinflammatory conditions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. For instance, elevated Thb levels have been detected postmortem in senile plaques of Alzheimer disease (AD) patients\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Expression of protease nexin-1 (PN1), a Thb inhibitor is also reduced around cerebral blood vessels in AD\u003csup\u003e35\u003c/sup\u003e. Increased levels of both Thb and its receptor protease activated receptor-1 (PAR-1) have also been reported in PD patients\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This suggests a context in which Thb exerts increased activity in the CNS. Its extracellular presence allows it to modulate synaptic plasticity and inflammatory responses, thus raising the possibility of interactions with other proteins.\u003c/p\u003e\u003cp\u003eWhether Thb directly processes Syn, thereby linking inflammatory signaling to protein misfolding, remains to be clarified. Here, we demonstrated that Thb predominantly cleaves Syn at Lys6\u0026ndash;Gly7, generating a 7\u0026ndash;140 fragment with altered conformational and aggregation properties. This mechanism defines a previously unrecognized pathway coupling inflammatory serine-protease activity with Syn proteostasis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eThrombin cleaves \u0026alpha;-synuclein in a preferential site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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\u0026ndash;6. The truncated form was named 7\u0026ndash;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\u0026ndash;140, where the residue 10 is a lysine. When prolonging the proteolysis to 24 h an additional shoulder containing the peptide 33\u0026ndash;140 appeared right before the main peak at 32.5 min. The latter appeared only slightly enriched in 11\u0026ndash;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor 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 conformations\u003csup\u003e37\u003c/sup\u003e. In parallel, Na⁺\u0026nbsp;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\u003csup\u003e38,39\u003c/sup\u003e, influencing the overall proteolytic outcome. Importantly, NaCl concentrations differ markedly between intracellular (~10 mM) and extracellular (~150 mM) environments\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eProteolysis of the chromogenic substrate (D)-Phe-Pip-Arg-pNA (S-2238) was performed in\u0026nbsp;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 \u0026asymp; 2\u0026ndash;10 mM)\u003csup\u003e27,41\u003c/sup\u003e, 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 \u0026ldquo;0 mM NaCl\u0026rdquo; conditions, explaining the absence of a measurable Na⁺-dependent modulation of catalytic activity.\u003c/p\u003e\n\u003cp\u003eHowever, 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\u0026ndash;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\u0026ndash;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\u0026ndash;140 generates the species 11\u0026ndash;140 and 33\u0026ndash;140 in similar amount (Fig. S1C).\u003c/p\u003e\n\u003cp\u003eThb 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 activity\u003csup\u003e42\u003c/sup\u003e. 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\u0026ndash;140 Syn was present, but MS analysis (Table S2) detected the presence of other species besides 7\u0026ndash;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteolysis of \u0026alpha;-synuclein mutants by Thrombin occurs at different extent and is dictated by the conformational features of the protein substrates\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genetic variants of Syn are involved in familiar forms of PD and differ from the wild type protein by one single mutation\u003csup\u003e2,3\u003c/sup\u003e. 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\u0026ndash;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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince the flexibility of a protein substrate influences proteolysis reactions\u003csup\u003e43\u003c/sup\u003e, we compared the varying compactness of Syn mutants. Although these proteins are intrinsically disordered, they can adopt multiple conformational ensembles\u003csup\u003e44,45\u003c/sup\u003e. 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 states\u003csup\u003e46\u003c/sup\u003e. 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe species 7\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e140 exhibits only slightly different conformational features in comparison to the full-length protein, but the 1\u0026ndash;6 residues strongly affect the interaction with membrane\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter purification, fragment 7\u0026ndash;140 was characterized by SEC, CD and native MS (Fig. 5, Fig. S3). The measured hydrodynamic volume (Fig. 5A) of 7\u0026ndash;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\u0026ndash;140 species and its spectrum appeared superimposable with that of the full-length protein (Fig. 5B). At pH 7.5, 7\u0026ndash;140 displays random secondary structure, according to previous data\u003csup\u003e17\u003c/sup\u003e. Interestingly, native MS (Fig. 5C) revealed differences in the conformational ensemble of Syn and 7\u0026ndash;140. The truncated variant principally populated the most compact conformer (59 % for 7\u0026ndash;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\u0026ndash;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\u0026ndash;140 Syn, effectively biasing occupancy towards the more compact states. Finally, cells exposed to the monomeric form of 7\u0026ndash;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).\u003c/p\u003e\n\u003cp\u003eSyn is known to undergo a disorder-to-helix transition upon binding to highly curved, negatively charged lipid vesicles\u003csup\u003e47\u003c/sup\u003e. To investigate the role of the first six N-terminal residues in this process, we compared full-length Syn with the truncated variant 7\u0026ndash;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. 6 A, B). Quantitative analysis (Fig. 6 C) showed that the binding affinities were comparable (Syn: Kd = 18 \u0026plusmn; 20 \u0026micro;M; 7\u0026ndash;140: Kd = 42 \u0026plusmn; 8 \u0026micro;M), indicating that N-terminal truncation does not substantially impair membrane association. However, the overall helicity was reduced in 7\u0026ndash;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\u0026ndash;140 did not in the examined timeframe (4 hours). SUV fusion is accompanied by a loss in helical structure (Fig. S4B).\u003c/p\u003e\n\u003cp\u003eHDX\u0026ndash;MS measurements provided higher-resolution insights into this difference (Fig. 6 D, 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\u003csup\u003e48,49\u003c/sup\u003e. The mean deuterium uptake for these regions showed that the N-terminal region (residues 1\u0026ndash;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\u0026ndash;140 (7\u0026ndash;39) was destabilized, reaching full deuteration within 1 min. Protection in the central region (residues 40\u0026ndash;98) was also reduced in 7\u0026ndash;140, indicating that stable helix formation at the extreme N-terminus contributes to cooperative stabilization along the sequence. The C-terminal region (residues 99\u0026ndash;140) was unaffected, remaining unstructured in both proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of the first six residues affects both fibril kinetics and morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, we examined how the removal of the first six N-terminal residues affects aggregation, and how this truncated form may co-aggregate with the full-length protein. Under aggregation conditions, Syn, 7\u0026ndash;140 Syn and the mix of the two proteins (3:1 ratio) exhibited distinct behaviors (Fig. 7A,B; Fig. S6A, Table S2). This ratio was selected to mimic the abundance of full-length Syn compared to truncated Syn species found in LB (~10\u0026ndash;30 %)\u003csup\u003e13\u003c/sup\u003e. Syn displayed a lag phase of 43 \u0026plusmn; 7 h and a half-time (t₁\u003csub\u003e/\u003c/sub\u003e₂) of 63 \u0026plusmn; 16 h, with the highest overall fluorescence intensity. 7\u0026ndash;140 Syn showed slower kinetics, with a lag phase of 50 \u0026plusmn; 2 h, a t₁\u003csub\u003e/\u003c/sub\u003e₂\u0026nbsp;of 100 \u0026plusmn; 1 h, and a fluorescence yield at plateau of approximately 60 % of that of Syn. The mixed sample exhibited intermediate behavior, with a lag phase of 47 \u0026plusmn; 6 h, t\u003csub\u003e1/2\u0026nbsp;\u003c/sub\u003eof 67 \u0026plusmn; 2 h, and a fluorescence plateau ~80 % of that of Syn. After aggregation reached a plateau, TEM micrographs were acquired for each sample and the fibril width distribution measured (Fig. 7D, Fig. S6B). Interestingly, Syn fibrils appear with a smooth surface, whereas many of the 7\u0026ndash;140 Syn and some of the mixed fibrils display a rugged surface. Additionally, Syn fibrils were the widest with an average width of 17.4 \u0026plusmn; 4.0 nm, while 7\u0026ndash;140 Syn were the thinnest (12.6 \u0026plusmn; 3.2 nm). Fibrils from the mixed sample showed an intermediate width (15.0 \u0026plusmn; 4.1 nm).\u003c/p\u003e\n\u003cp\u003eSuccessively, samples were ultracentrifuged to separate insoluble protein fraction. The resulting pellets were resuspended in 7 M Gnd\u0026ndash;HCl, and UV spectra were recorded to assess the amount of insoluble protein (Fig. S7A). The three conditions exhibited different extent of light scattering after resuspension: 7\u0026ndash;140 Syn showed the lowest scattering contribution, Syn an intermediate level, and the mixed sample the highest. After baseline correction to remove scattering contribution (Fig. S7B), the insoluble fractions accounted for ~49 % of the total protein for both Syn and 7\u0026ndash;140 Syn, and ~44 % for the mixed sample. To determine the composition of the insoluble fractions, the resuspended pellets were analyzed by RP\u0026ndash;HPLC (Fig. 7C), and peak identities were confirmed by MS (not shown). In the Syn and 7\u0026ndash;140 Syn samples, the main chromatographic peaks corresponded to the expected species. Minor additional peaks were attributed to secondary fragmentation probably generated during prolonged incubation, agitation and sample handling. Notably, in the mixed sample (blue line), the peak eluting at ~33 min was confirmed by MS to contain the 7\u0026ndash;140 species. Despite the 3:1 ratio of the mixed sample, quantitative analysis of the chromatogram peaks revealed an approximately 1:1 ratio of Syn and 7\u0026ndash;140 Syn.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNeurodegenerative disorders such as PD are often correlated with neuroinflammatory states. Here, we propose a previously unrecognized interaction between Syn and Thb possibly linking inflammatory protease activity and protein misfolding in PD. Importantly, Thb has been previously implicated in PD pathology by contributing to neuronal cell death, activating microglia and contributing to oxidative stress\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur data show that, when mixed in pathological mimicking conditions, Thb cleaves Syn and generates 7\u0026ndash;140 Syn, as the predominant proteolytic species, selecting Lys6 despite the presence of 15 lysine residues in the protein sequence. The presence of a unique (main) site of proteolysis, the Lys6\u0026ndash;Gly7 peptide bond, focussed our attention on both the specificity of Thb and the cleavage susceptibility of Syn. Thb has a preferential consensus cleavage site with Arg in position P1. After Arg the other residue recognized albeit with less preference is Lys\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Importantly, position P1\u0026rsquo; is always a Gly and/or other hydrophilic not acidic residues. This aligns well with our observed binding site between Lys6 and Gly7. This also explains the further cleavages observed to a much lower extent (between Lys10\u0026ndash;Ala11 and Lys32\u0026ndash;Thr33) due to enzyme preference. Interestingly, Plasmin -another serine protease with similar specificity to Thb albeit less stringent \u0026ndash; has been reported to generate the same fragments we observed (i.e. 11\u0026ndash;140 and 33\u0026ndash;140) without ever generating the 7\u0026ndash;140 species\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eImportantly, we also show that NaCl has a critical role on the reaction influencing the 7\u0026ndash;140 Syn production yield. Na⁺ binding to Thb allosterically stabilizes its catalytically active conformation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In parallel, it is known that NaCl accelerates Syn aggregation by stabilizing more extended conformations with an exposed NAC region\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Despite these notions, we observed slower Thb-mediated Syn proteolysis in the presence of NaCl. To further explore the interplay between structure and enzymatic processing, the conformational ensemble of Syn familiar mutants (A30P, E46K, H50Q, A53T) was examined. We found that mutants that preferentially choose a more extended Syn conformer like A53T give high proteolysis yields, while E46K which generally is more compact has lower yields. This is in line with what is typically expected for proteolysis, where a more flexible extended substrate is processed faster\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The effect of NaCl on Syn proteolysis by Thb is therefore multifaceted. While Na⁺ enhances Thb catalytic activity by stabilizing its \u0026ldquo;fast\u0026rdquo; conformational state and promoting more extended conformations of Syn, the concomitant presence of Cl⁻ ions likely shields the positively charged N-terminal region of Syn, resulting in an overall slower proteolysis reaction. Hence, Syn proteolysis is a complex reaction that depends on conformational flexibility and electrostatics. In our assay, the presence of 20 mM Na⁺ from phosphate buffer already saturates the Na⁺-binding site of Thb, so adding 150 mM NaCl primarily alters ionic strength rather than allosteric activation. This condition may shift the electrostatic landscape without significantly changing the intrinsic catalytic efficiency of the enzyme.\u003c/p\u003e\u003cp\u003eUnder neuroinflammatory conditions, Thb levels increase within CNS especially due to BBB breakdown\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, even reaching up to 25 nM, sufficient to act on neuronal substrates\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. While Syn is largely depicted as cytoplasmic protein an increasing number of studies report the presence of both monomeric and oligomeric forms in the extracellular space, including synaptic clefts and EVs are increasing\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The simultaneous extracellular presence of Thb and Syn raises the possibility of protease-mediated Syn processing, potentially affecting synaptic function and neurodegeneration. This interaction is likely compartment-specific (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Intracellularly, Syn is abundant whereas Thb is largely absent, limiting direct proteolysis; conversely, in the extracellular milieu, Thb concentrations are high while Syn levels remain relatively low. Such compartmental asymmetry suggests that Thb-mediated Syn processing may occur in restricted microdomains where both proteins transiently co-localize and accumulate, such as sites of neuron membrane leakage, vesicle release, synaptic clefts, or regions of BBB disruption. Notably, NaCl concentrations differ markedly between intracellular (~\u0026thinsp;10 mM) and extracellular (~\u0026thinsp;150 mM) environments\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Such ionic differences could attenuate Thb-mediated Syn proteolysis in the extracellular space, implying that sustained or repeated Thb exposure may be required for significant cleavage to occur in vivo. These localized interactions could influence the aggregation state, clearance, intercellular transmission and functions of Syn, thereby contributing to the propagation of synucleinopathies and associated neuroinflammatory processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe removal of the first six residues of Syn would have a drastic impact on its characteristics and functions. On the one hand, 7\u0026ndash;140 Syn shows a reduced helical stabilization upon membrane binding. Mechanistically, this may derive from a missing salt bridge between D2 and K6 identified by Fusco et al\u003csup\u003e56\u003c/sup\u003e to be important for helix nucleation. Cholak et al\u003csup\u003e57\u003c/sup\u003e even showed that the removal of residues 2\u0026ndash;14 reduced protein membrane localization, confirming the N-terminal anchor has an impact in vivo. These differences would manifest in lacking membrane remodeling capabilities which facilitate vesicle fusion and neurotransmitter release\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. On the other hand, 7\u0026ndash;140 Syn lacks 2 Met residues implicated in the scavenging of lipid hydroperoxides\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Missing reconversion of the lipids to their natural form would result in accumulation of oxidative damage in brains of PD patients. As a final remark, the removal of the first six N-terminal residues markedly slows aggregation kinetics consistent with a more compact starting conformation that is recruited by fibril ends with reduced efficiency. Consequently, the truncated species may persist longer in soluble oligomeric states and act as seeding competent intermediates. Hence, these species might contribute to a more chronic form of Syn\u0026ndash;mediated toxicity. Fully understanding the structural and toxic properties of these intermediates will be essential to assess whether neuroinflammation enhances Syn toxicity through proteolytic processing.\u003c/p\u003e\u003cp\u003eImportantly, even fibril morphology seems altered in fibrils formed by the fragment alone and by the co-aggregated species at physiological ratios. This contributes to existing literature describing different fibril morphology of Syn species lacking increasingly larger parts of the N-terminal fragment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Of note, Dewison et al\u003csup\u003e61\u003c/sup\u003e studied a recombinant Syn construct missing residues 2\u0026ndash;6, effectively differing only in the Met1 residue from the Thb-generated 7\u0026ndash;140 species. In contrast to our work, they report no differences in membrane binding and helix formation, suggesting either the importance of the first Met residues and/or of the SUV lipid composition in this regard. In accordance with our data, they observe a slower fibril assembly, highlighting the critical sensitivity of fibril growth on the precise sequence of the N-terminal region. In conclusion, our work further strengthens the notion that the N-terminal residues have a stark impact on Syn biology in PD.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTogether, these findings expand our understanding of how Thb interacts with Syn and establish a functional connection between Thb activity, proteolytic processing, ionic homeostasis, and Syn structural flexibility. Our data show that Thb-mediated proteolysis of Syn is a multifactorial process shaped by protein conformation, electrostatic interactions, and the surrounding molecular environment, each potentially contributing to PD pathology. Our findings provide mechanistic insight into the generation and significance of N-terminal truncations of Syn and may be extended to related serine proteases in vivo especially during states of chronic neuroinflammation. Defining the local, temporal and structural determinants of these truncations will be essential for understanding their contribution to disease progression and may direct the development of targeted therapeutic strategies. In particular, our results highlight the critical influence of ionic homeostasis on Thb\u0026ndash;Syn interactions. Variations in Na⁺ and Cl⁻ levels, such as those occurring during neuroinflammation or impaired ion regulation in PD, could therefore differentially affect Thb activity and Syn susceptibility to proteolysis. This suggests that ionic imbalance within the CNS may not only alter Syn aggregation pathways but also tune its enzymatic processing, ultimately influencing the course and heterogeneity of synucleinopathies.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cb\u003eMaterial.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrombin, trypsin, PPACK, (D)-Phe-Pip-Arg-pNA (S-2238), and other reagents were provided by Merck (Darmstadt, Germany). L-alpha-Phosphatidylcholine (egg, PC) and L-alpha-Phosphatidylserine (bovine liver, PS) were purchased from Avanti Lipid (Alabaster, Alabama, USA).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eExpression and purification of recombinant α-synuclein and its mutants\u003c/h2\u003e\u003cp\u003eThe 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\u0026deg;C to an A\u003csub\u003e600nm\u003c/sub\u003e 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 described\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Protein identity and integrity were assessed by mass spectrometry (MS).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProteolysis of α-synuclein\u003c/h3\u003e\n\u003cp\u003eProteolysis was carried out in 20 mM sodium phosphate buffer, pH 7.4, using Thb at an enzyme to substrate (E/S) ratio of 1:100 (mol/mol) at 22\u0026deg;C and 1:400 (mol/mol) at 37\u0026deg;C, in the presence and in the absence of 150 mM NaCl or the Thb inhibitor PPACK (E/I 1:1). Proteolysis by trypsin was conducted at an E/S of 1:1000 (by weight). All reactions were quenched at specific time by acidification with TFA in water (0.2%, by volume) and analysed by RP-HPLC. RP-HPLC analyses were carried out on a 1200 series Agilent Technologies chromatographer (Santa Clara, CA, USA), using a Jupiter C18 column (4.6 mm \u0026times; 250 mm, 5.0 \u0026micro;m; Phenomenex, CA, USA). The elution was performed by a gradient of acetonitrile/0.085% TFA and water/0.1% TFA (5%\u0026ndash;25% in 5 min, 25%\u0026ndash;28% in 13 min, 28%-39% in 3 min, 39\u0026ndash;45% in 21 min), recording the eluate absorbance at 226 nm.\u003c/p\u003e\n\u003ch3\u003eThrombin activity\u003c/h3\u003e\n\u003cp\u003eThe activity of Thb was tested by monitoring the release of p-nitroaniline (pNA) from (D)-Phe-Pip-Arg-pNA (S-2238) spectrophotometrically at 405 nm over time. The assay was conducted in 20 mM sodium phosphate, pH 7.4, in the presence and in the absence of 150 mm NaCl, at 37\u0026deg;C, using an E/S 1:200000. The extinction coefficient of pNA is 8270 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eChemical-physical characterization\u003c/h2\u003e\u003cp\u003eProtein 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\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, as evaluated from their amino acid composition by the method of Gill and von Hippel\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSize exclusion chromatography (SEC) was performed by a Superdex TM 200 Increase (10/300 GL) column (Amersham Biosciences, Uppsala, Sweden), using an \u0026Auml;KTA FPLC system (Amersham Biosciences, Uppsala, Sweden). Sample aliquots (200 \u0026micro;g) 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).\u003c/p\u003e\u003cp\u003eThe 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\u0026thinsp;\u0026minus;\u0026thinsp;190 nm, collecting data with high-tension voltage\u0026thinsp;\u0026lt;\u0026thinsp;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 cm\u003csup\u003e2\u003c/sup\u003e dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was calculated from the formula [θ] (θ\u003csub\u003eobs\u003c/sub\u003e/10) (MRW/lc), where θ\u003csub\u003eobs\u003c/sub\u003e 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 \u0026micro;mol) and POPG (10 \u0026micro;mol) 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 \u0026micro;M 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.\u003csup\u003e63\u003c/sup\u003e:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\theta\\:}_{i}^{222}=\\:{\\theta\\:}_{coil}^{222}+\\:\\frac{{\\theta\\:}_{helix}^{222}-{\\theta\\:}_{coil}^{222}}{2}\\:\\left[1+\\:\\frac{X}{n}+\\frac{1}{n{k}_{B}{P}_{t}}-\\sqrt{{\\left(1+\\frac{X}{n}+\\frac{1}{n{k}_{B}{P}_{t}}\\right)}^{2}-\\frac{4X}{n}}\\right]$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{i}^{222}\\)\u003c/span\u003e\u003c/span\u003e is the ellipticity at each time point, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{coil}^{222}\\)\u003c/span\u003e\u003c/span\u003e is the ellipticity at the beginning of the titration, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{helix}^{222}\\)\u003c/span\u003e\u003c/span\u003e is the ellipticity at saturating conditions, X is the protein to phospholipids molar ratio, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:n\\)\u003c/span\u003e\u003c/span\u003e is the number of lipids interacting, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{k}_{B}\\)\u003c/span\u003e\u003c/span\u003e is the binding constant and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{P}_{t}\\)\u003c/span\u003e\u003c/span\u003e is the initial protein concentration. Percentage of alpha-helix content was estimated with following Eq.\u0026nbsp;6\u003csup\u003e4\u003c/sup\u003e:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Percentage\\:helicity=100\\:x\\:\\frac{\\left({\\theta\\:}_{222}^{exp}-{\\theta\\:}_{222}^{u}\\right)}{\\left({\\theta\\:}_{222}^{h}-{\\theta\\:}_{222}^{u}\\right)}$$\u003c/div\u003e\u003c/div\u003e ;\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{222}^{exp}\\)\u003c/span\u003e\u003c/span\u003e is the ellipticity at 222 nm under saturating conditions, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{222}^{h}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\theta\\:}_{222}^{u}\\)\u003c/span\u003e\u003c/span\u003e correspond to the ellipticity at 222 nm of a protein with 100% and 0% of helical content (estimated to be -39500 deg cm\u003csup\u003e2\u003c/sup\u003e dmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and \u0026minus;\u0026thinsp;3000 deg cm\u003csup\u003e2\u003c/sup\u003e dmol\u003csup\u003e\u0026minus;\u0026thinsp;\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, respectively).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eMass spectrometry measurements\u003c/h2\u003e\u003cp\u003eMass spectrometry analyses were performed on a Xevo\u0026reg; G2-XS ESI-Q-TOF mass spectrometer (Waters Corporation, Milford, Massachusetts, USA). Measurements were carried out in positive mode at 1.2\u0026ndash;1.6 kV capillary voltage and 30\u0026ndash;40 V cone voltage. The source temperature was set at 30\u0026deg;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 \u0026micro;M) 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 \u0026micro;L 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.\u003c/p\u003e\u003cp\u003eFor 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 \u0026micro;M 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\u0026deg;C. For the H/D exchange reaction, an aliquot (5 \u0026micro;l) of each sample was diluted 10-fold in deuterated buffer (20 mM sodium phosphate, pD 7.4, in 99.9% D\u003csub\u003e2\u003c/sub\u003eO) and was then allowed to exchange for either 10, 30, 60 sec or 10 min, at 4\u0026deg;C. H/D exchange was quenched at 0\u0026deg;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 from\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Deuterium incorporation was determined according to the following equation: %D = (mt \u0026ndash; m0)/ (m100\u0026thinsp;\u0026minus;\u0026thinsp;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.\u003csup\u003e65\u003c/sup\u003e. 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\u003csup\u003e+\u003c/sup\u003e 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\u0026thinsp;\u0026lt;\u0026thinsp;4 or \u0026gt;\u0026thinsp;33 amino acids were excluded, due to identification ambiguity and poor sequence localization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eAggregation study\u003c/h2\u003e\u003cp\u003eFreshly resuspended proteins were centrifuged at 12.000 rpm for 15 min and filtered through 22 \u0026micro;m PVDF filter to obtain monomeric Syn and 7\u0026ndash;140 Syn for the aggregation assays. Total protein concentration in each aggregation reaction was 70 \u0026micro;M. Three conditions were tested: i) Syn, ii) 7\u0026ndash;140 Syn and iii) a mixture of Syn and 7\u0026ndash;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\u0026deg; 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 \u0026micro;M in 20 mM sodium phosphate buffer (pH 6.0) containing 25 \u0026micro;M 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 Amylofit\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, 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\u0026ndash;solubilized pellets were analyzed by RP-HPLC with the same gradient described above. The identity of the peaks was confirmed by ESI-MS.\u003c/p\u003e\u003cp\u003eTransmission electron microscopy (TEM) was performed by negative staining method, in which 5.0 \u0026micro;L of sample (0.5 mg/ml) was dried on the carbon grid and subsequently stained with 10 \u0026micro;L 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 \u0026gt;\u0026thinsp;100 singular measurements were made for each sample. Only well-defined fibrils with high contrast were considered.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCell culture and viability test\u003c/h2\u003e\u003cp\u003eThe cytotoxic effects of Syn and 7\u0026ndash;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 \u0026micro;g/mL streptomycin. Cells were maintained in a humidified incubator at 37\u0026deg;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 \u0026micro;M Syn or 7\u0026ndash;140Syn for 24 or 48 hours. Following treatment, 20 \u0026micro;L of MTS reagent (CellTiter 96\u0026reg;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\u0026deg;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\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation from 3 independent experiments, each one performed in sextuplicate.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests.\u003c/h2\u003e\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding.\u003c/h2\u003e\u003cp\u003eThis project was supported by a competitive grant from University of Padova (Progetti di Ateneo, 2022, #BIRD222840); by the Italian Ministry of Education, University and Research (MUR) Progetti di Ricerca di Rilevante Interesse Nazionale (PRIN 2022, #2022FPWYA8); and by AFM (Association contre les Myopathies, research grant #28892).\u003c/p\u003e\u003ch2\u003eAuthor Contribution.\u003c/h2\u003e\u003cp\u003eConceptualization: P.T., B.F., L.A., P.P.d.L. Methodology: P.T., B.F., V.S., A.S., M.S. Data analysis: P.T., B.F., V.S., A.S. Supervision: L.A., D.S., P.P.d.L. Writing: P.T., L.A., P.P.d.L. All authors reviewed, edited and approved.\u003c/p\u003e\u003ch2\u003eAcknowledgements.\u003c/h2\u003e\u003cp\u003eWe are thankful to the Mass Spectrometry Facility of Department of Pharmaceutical and Pharmacological Science, and Imaging Facility of Department of Biology, at University of Padova, for technical support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKalia, L. V. \u0026amp; Lang, A. E. Parkinson\u0026rsquo;s disease. \u003cem\u003eThe Lancet\u003c/em\u003e \u003cstrong\u003e386\u003c/strong\u003e, 896\u0026ndash;912 (2015).\u003c/li\u003e\n\u003cli\u003eConway, K. A. \u003cem\u003eet al.\u003c/em\u003e Acceleration of oligomerization, not fibrillization, is a shared property of both \u0026alpha;-synuclein mutations linked to early-onset Parkinson\u0026rsquo;s disease: Implications for pathogenesis and therapy. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 571\u0026ndash;576 (2000).\u003c/li\u003e\n\u003cli\u003eFlagmeier, P. \u003cem\u003eet al.\u003c/em\u003e Mutations associated with familial Parkinson\u0026rsquo;s disease alter the initiation and amplification steps of \u0026alpha;-synuclein aggregation. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e113\u003c/strong\u003e, 10328\u0026ndash;10333 (2016).\u003c/li\u003e\n\u003cli\u003eMeade, R. M., Fairlie, D. P. \u0026amp; Mason, J. M. Alpha-synuclein structure and Parkinson\u0026rsquo;s disease \u0026ndash; lessons and emerging principles. \u003cem\u003eMolecular Neurodegeneration\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 29 (2019).\u003c/li\u003e\n\u003cli\u003eWinner, B. \u003cem\u003eet al.\u003c/em\u003e In vivo demonstration that \u0026alpha;-synuclein oligomers are toxic. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 4194\u0026ndash;4199 (2011).\u003c/li\u003e\n\u003cli\u003eEliezer, D., Kutluay, E., Bussell, R. \u0026amp; Browne, G. Conformational properties of \u0026alpha;-synuclein in its free and lipid-associated states1. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e307\u003c/strong\u003e, 1061\u0026ndash;1073 (2001).\u003c/li\u003e\n\u003cli\u003eJao, C. C., Hegde, B. G., Chen, J., Haworth, I. S. \u0026amp; Langen, R. Structure of membrane-bound \u0026alpha;-synuclein from site-directed spin labeling and computational refinement. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 19666\u0026ndash;19671 (2008).\u003c/li\u003e\n\u003cli\u003eWeinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. \u0026amp; Lansbury, P. T. NACP, A Protein Implicated in Alzheimer\u0026rsquo;s Disease and Learning, Is Natively Unfolded. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 13709\u0026ndash;13715 (1996).\u003c/li\u003e\n\u003cli\u003eMahul-Mellier, A.-L. \u003cem\u003eet al.\u003c/em\u003e Differential role of C-terminal truncations on alpha-synuclein pathology and Lewy body formation. \u003cem\u003enpj Parkinsons Dis.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 261 (2025).\u003c/li\u003e\n\u003cli\u003eZarranz, J. J. \u003cem\u003eet al.\u003c/em\u003e The new mutation, E46K, of \u0026alpha;-synuclein causes parkinson and Lewy body dementia. \u003cem\u003eAnnals of Neurology\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 164\u0026ndash;173 (2004).\u003c/li\u003e\n\u003cli\u003eGreenbaum, E. A. \u003cem\u003eet al.\u003c/em\u003e The E46K Mutation in \u0026alpha;-Synuclein Increases Amyloid Fibril Formation*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 7800\u0026ndash;7807 (2005).\u003c/li\u003e\n\u003cli\u003eMachiya, Y. \u003cem\u003eet al.\u003c/em\u003e Phosphorylated \u0026alpha;-Synuclein at Ser-129 Is Targeted to the Proteasome Pathway in a Ubiquitin-independent Manner*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 40732\u0026ndash;40744 (2010).\u003c/li\u003e\n\u003cli\u003eMa, L. \u003cem\u003eet al.\u003c/em\u003e C-terminal truncation exacerbates the aggregation and cytotoxicity of \u0026alpha;-Synuclein: A vicious cycle in Parkinson\u0026rsquo;s disease. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Molecular Basis of Disease\u003c/em\u003e \u003cstrong\u003e1864\u003c/strong\u003e, 3714\u0026ndash;3725 (2018).\u003c/li\u003e\n\u003cli\u003eSorrentino, Z. A. \u003cem\u003eet al.\u003c/em\u003e Physiological C-terminal truncation of \u0026alpha;-synuclein potentiates the prion-like formation of pathological inclusions. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e293\u003c/strong\u003e, 18914\u0026ndash;18932 (2018).\u003c/li\u003e\n\u003cli\u003eMurray, I. V. J. \u003cem\u003eet al.\u003c/em\u003e Role of \u0026alpha;-Synuclein Carboxy-Terminus on Fibril Formation in Vitro. \u003cem\u003eBiochemistry\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 8530\u0026ndash;8540 (2003).\u003c/li\u003e\n\u003cli\u003eLiu, C.-W. \u003cem\u003eet al.\u003c/em\u003e A Precipitating Role for Truncated \u0026alpha;-Synuclein and the Proteasome in \u0026alpha;-Synuclein Aggregation: IMPLICATIONS FOR PATHOGENESIS OF PARKINSON DISEASE*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 22670\u0026ndash;22678 (2005).\u003c/li\u003e\n\u003cli\u003eThrush, R. J., Vadukul, D. M. \u0026amp; Aprile, F. A. A Facile Method to Produce N-Terminally Truncated \u0026alpha;-Synuclein. \u003cem\u003eFront. Neurosci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, (2022).\u003c/li\u003e\n\u003cli\u003eThrush, R. J., Vadukul, D. M., Allerton, S. C., Storch, M. \u0026amp; Aprile, F. A. Surface Wetting Is a Key Determinant of \u0026alpha;-Synuclein Condensate Maturation. 2024.06.06.597302 Preprint at https://doi.org/10.1101/2024.06.06.597302 (2025).\u003c/li\u003e\n\u003cli\u003eVamvaca, K., Volles, M. J. \u0026amp; Lansbury, P. T. The First N-terminal Amino Acids of \u0026alpha;-Synuclein Are Essential for \u0026alpha;-Helical Structure Formation \u003cem\u003eIn Vitro\u003c/em\u003e and Membrane Binding in Yeast. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e389\u003c/strong\u003e, 413\u0026ndash;424 (2009).\u003c/li\u003e\n\u003cli\u003eSantos, J. \u003cem\u003eet al.\u003c/em\u003e A Targetable N-Terminal Motif Orchestrates \u0026alpha;-Synuclein Oligomer-to-Fibril Conversion. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, 12702\u0026ndash;12711 (2024).\u003c/li\u003e\n\u003cli\u003eL\u0026ouml;\u0026ouml;v, C., Scherzer, C. R., Hyman, B. T., Breakefield, X. O. \u0026amp; Ingelsson, M. \u0026alpha;-Synuclein in Extracellular Vesicles: Functional Implications and Diagnostic Opportunities. \u003cem\u003eCell Mol Neurobiol\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 437\u0026ndash;448 (2016).\u003c/li\u003e\n\u003cli\u003eLee, H.-J., Bae, E.-J. \u0026amp; Lee, S.-J. Extracellular \u0026alpha;-synuclein\u0026mdash;a novel and crucial factor in Lewy body diseases. \u003cem\u003eNat Rev Neurol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 92\u0026ndash;98 (2014).\u003c/li\u003e\n\u003cli\u003eKlegeris, A. \u003cem\u003eet al.\u003c/em\u003e Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells. \u003cem\u003eThe FASEB Journal\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 2000\u0026ndash;2008 (2006).\u003c/li\u003e\n\u003cli\u003eLee, H.-J. \u003cem\u003eet al.\u003c/em\u003e Direct Transfer of \u0026alpha;-Synuclein from Neuron to Astroglia Causes Inflammatory Responses in Synucleinopathies*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 9262\u0026ndash;9272 (2010).\u003c/li\u003e\n\u003cli\u003eRoodveldt, C., Christodoulou, J. \u0026amp; Dobson, C. M. Immunological features of \u0026alpha;-synuclein in Parkinson\u0026rsquo;s disease. \u003cem\u003eJournal of Cellular and Molecular Medicine\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1820\u0026ndash;1829 (2008).\u003c/li\u003e\n\u003cli\u003eDavie, E. W. \u0026amp; Kulman, J. D. An Overview of the Structure and Function of Thrombin. \u003cem\u003eSemin Thromb Hemost\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 3\u0026ndash;15 (2006).\u003c/li\u003e\n\u003cli\u003eDi Cera, E. Thrombin. \u003cem\u003eMolecular Aspects of Medicine\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 203\u0026ndash;254 (2008).\u003c/li\u003e\n\u003cli\u003eArai, T., Miklossy, J., Klegeris, A., Guo, J.-P. \u0026amp; McGeer, P. L. Thrombin and Prothrombin Are Expressed by Neurons and Glial Cells and Accumulate in Neurofibrillary Tangles in Alzheimer Disease Brain. \u003cem\u003eJ Neuropathol Exp Neurol\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 19\u0026ndash;25 (2006).\u003c/li\u003e\n\u003cli\u003eKrenzlin, H., Lorenz, V., Danckwardt, S., Kempski, O. \u0026amp; Alessandri, B. The Importance of Thrombin in Cerebral Injury and Disease. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 84 (2016).\u003c/li\u003e\n\u003cli\u003eShlobin, N. A., Har-Even, M., Itsekson-Hayosh, Z., Harnof, S. \u0026amp; Pick, C. G. Role of Thrombin in Central Nervous System Injury and Disease. \u003cem\u003eBiomolecules\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 562 (2021).\u003c/li\u003e\n\u003cli\u003eRajput, P. S. \u003cem\u003eet al.\u003c/em\u003e Neuron-generated thrombin induces a protective astrocyte response via protease activated receptors. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 246\u0026ndash;262 (2020).\u003c/li\u003e\n\u003cli\u003eChen, X. \u003cem\u003eet al.\u003c/em\u003e Thrombin induces morphological and inflammatory astrocytic responses via activation of PAR1 receptor. \u003cem\u003eCell Death Discov.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 189 (2022).\u003c/li\u003e\n\u003cli\u003eYe, F., Garton, H. J. L., Hua, Y., Keep, R. F. \u0026amp; Xi, G. The Role of Thrombin in Brain Injury After Hemorrhagic and Ischemic Stroke. \u003cem\u003eTransl. Stroke Res.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 496\u0026ndash;511 (2021).\u003c/li\u003e\n\u003cli\u003eTurgeon, V. L. \u0026amp; Houenou, L. J. The role of thrombin-like (serine) proteases in the development, plasticity and pathology of the nervous system. \u003cem\u003eBrain Research Reviews\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 85\u0026ndash;95 (1997).\u003c/li\u003e\n\u003cli\u003eVaughan, P. J., Su, J., Cotman, C. W. \u0026amp; Cunningham, D. D. Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer\u0026rsquo;s disease. \u003cem\u003eBrain Research\u003c/em\u003e \u003cstrong\u003e668\u003c/strong\u003e, 160\u0026ndash;170 (1994).\u003c/li\u003e\n\u003cli\u003eJohnson, S. L., Iannucci, J., Seeram, N. P. \u0026amp; Grammas, P. Inhibiting thrombin improves motor function and decreases oxidative stress in the LRRK2 transgenic Drosophila melanogaster model of Parkinson\u0026rsquo;s disease. \u003cem\u003eBiochemical and Biophysical Research Communications\u003c/em\u003e \u003cstrong\u003e527\u003c/strong\u003e, 532\u0026ndash;538 (2020).\u003c/li\u003e\n\u003cli\u003eRamis, R., Ortega-Castro, J., Vilanova, B., Adrover, M. \u0026amp; Frau, J. Unraveling the NaCl Concentration Effect on the First Stages of \u0026alpha;-Synuclein Aggregation. \u003cem\u003eBiomacromolecules\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 5200\u0026ndash;5212 (2020).\u003c/li\u003e\n\u003cli\u003ePelc, L. A., Koester, S. K., Kukla, C. R., Chen, Z. \u0026amp; Di Cera, E. The active site region plays a critical role in Na+ binding to thrombin. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e298\u003c/strong\u003e, 101458 (2022).\u003c/li\u003e\n\u003cli\u003eAyala, Y. \u0026amp; Cera, E. D. Molecular Recognition by Thrombin. Role of the Slows\u0026rarr;Fast Transition, Site-specific Ion Binding Energetics and Thermodynamic Mapping of Structural Components. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e235\u003c/strong\u003e, 733\u0026ndash;746 (1994).\u003c/li\u003e\n\u003cli\u003eEgidi, I., Guidi, M. \u0026amp; Giove, F. Compartmentalization of sodium in the human brain: a mini-review of 23Na-MRI methods. \u003cem\u003eFront. Phys.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, (2025).\u003c/li\u003e\n\u003cli\u003eDang, O. D., Vindigni, A. \u0026amp; Di Cera, E. An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e92\u003c/strong\u003e, 5977\u0026ndash;5981 (1995).\u003c/li\u003e\n\u003cli\u003eStojanovski, B. M., Pelc, L. A. \u0026amp; Di Cera, E. Thrombin has dual trypsin-like and chymotrypsin-like specificity. \u003cem\u003eJournal of Thrombosis and Haemostasis\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1009\u0026ndash;1015 (2024).\u003c/li\u003e\n\u003cli\u003eHubbard, S. J. The structural aspects of limited proteolysis of native proteins. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology\u003c/em\u003e \u003cstrong\u003e1382\u003c/strong\u003e, 191\u0026ndash;206 (1998).\u003c/li\u003e\n\u003cli\u003eBorthakur, K., Sisk, T. R., Panei, F. P., Bonomi, M. \u0026amp; Robustelli, P. Determining accurate conformational ensembles of intrinsically disordered proteins at atomic resolution. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 9036 (2025).\u003c/li\u003e\n\u003cli\u003eFongaro, B., Cappelletto, E., Sosic, A., Spolaore, B. \u0026amp; Polverino de Laureto, P. 3,4-Dihydroxyphenylethanol and 3,4-dihydroxyphenylacetic acid affect the aggregation process of E46K variant of \u0026alpha;-synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect. \u003cem\u003eProtein Science\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, e4356 (2022).\u003c/li\u003e\n\u003cli\u003eHeuvel, R. H. van den \u0026amp; Heck, A. J. Native protein mass spectrometry: from intact oligomers to functional machineries. \u003cem\u003eCurrent Opinion in Chemical Biology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 519\u0026ndash;526 (2004).\u003c/li\u003e\n\u003cli\u003eDavidson, W. S., Jonas, A., Clayton, D. F. \u0026amp; George, J. M. Stabilization of \u0026alpha;-Synuclein Secondary Structure upon Binding to Synthetic Membranes*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e273\u003c/strong\u003e, 9443\u0026ndash;9449 (1998).\u003c/li\u003e\n\u003cli\u003eFusco, G. \u003cem\u003eet al.\u003c/em\u003e Direct observation of the three regions in \u0026alpha;-synuclein that determine its membrane-bound behaviour. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 3827 (2014).\u003c/li\u003e\n\u003cli\u003eRizzotto, E. \u003cem\u003eet al.\u003c/em\u003e DOPAC as a modulator of \u0026alpha;-Synuclein and E46K interactions with membrane: Insights into binding dynamics. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e \u003cstrong\u003e294\u003c/strong\u003e, 139427 (2025).\u003c/li\u003e\n\u003cli\u003eChoi, S.-H., Lee, D. Y., Kim, S. U. \u0026amp; Jin, B. K. Thrombin-Induced Oxidative Stress Contributes to the Death of Hippocampal Neurons In Vivo: Role of Microglial NADPH Oxidase. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 4082\u0026ndash;4090 (2005).\u003c/li\u003e\n\u003cli\u003eGallwitz, M., Enoksson, M., Thorpe, M. \u0026amp; Hellman, L. The Extended Cleavage Specificity of Human Thrombin. \u003cem\u003ePLOS ONE\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e31756 (2012).\u003c/li\u003e\n\u003cli\u003eUliana, F. \u003cem\u003eet al.\u003c/em\u003e Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1693 (2021).\u003c/li\u003e\n\u003cli\u003eKim, K. S. \u003cem\u003eet al.\u003c/em\u003e Proteolytic Cleavage of Extracellular \u0026alpha;-Synuclein by Plasmin: IMPLICATIONS FOR PARKINSON DISEASE*. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e \u003cstrong\u003e287\u003c/strong\u003e, 24862\u0026ndash;24872 (2012).\u003c/li\u003e\n\u003cli\u003eLeem, E., Jeong, K. H., Won, S.-Y., Shin, W.-H. \u0026amp; Kim, S. R. Prothrombin Kringle-2: A Potential Inflammatory Pathogen in the Parkinsonian Dopaminergic System. \u003cem\u003eExperimental Neurobiology\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 147\u0026ndash;155 (2016).\u003c/li\u003e\n\u003cli\u003eBen Shimon, M. \u003cem\u003eet al.\u003c/em\u003e Thrombin regulation of synaptic transmission and plasticity: implications for health and disease. \u003cem\u003eFront. Cell. Neurosci.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2015).\u003c/li\u003e\n\u003cli\u003eFusco, G. \u003cem\u003eet al.\u003c/em\u003e Structural Ensembles of Membrane-bound \u0026alpha;-Synuclein Reveal the Molecular Determinants of Synaptic Vesicle Affinity. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 27125 (2016).\u003c/li\u003e\n\u003cli\u003eCholak, E. \u003cem\u003eet al.\u003c/em\u003e Avidity within the N-terminal anchor drives \u0026alpha;-synuclein membrane interaction and insertion. \u003cem\u003eThe FASEB Journal\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 7462\u0026ndash;7482 (2020).\u003c/li\u003e\n\u003cli\u003eKhounlo, R. \u003cem\u003eet al.\u003c/em\u003e Membrane Binding of \u0026alpha;-Synuclein Stimulates Expansion of SNARE-Dependent Fusion Pore. \u003cem\u003eFront. Cell Dev. Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, (2021).\u003c/li\u003e\n\u003cli\u003eMaltsev, A. S., Chen, J., Levine, R. L. \u0026amp; Bax, A. Site-Specific Interaction between \u0026alpha;-Synuclein and Membranes Probed by NMR-Observed Methionine Oxidation Rates. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, 2943\u0026ndash;2946 (2013).\u003c/li\u003e\n\u003cli\u003eMcGlinchey, R. P., Ni, X., Shadish, J. A., Jiang, J. \u0026amp; Lee, J. C. The N terminus of \u0026alpha;-synuclein dictates fibril formation. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e118\u003c/strong\u003e, e2023487118 (2021).\u003c/li\u003e\n\u003cli\u003eDewison, K. M. \u003cem\u003eet al.\u003c/em\u003e Residues 2 to 7 of \u0026alpha;-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, e2315006121 (2024).\u003c/li\u003e\n\u003cli\u003eGill, S. C. \u0026amp; von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. \u003cem\u003eAnalytical Biochemistry\u003c/em\u003e \u003cstrong\u003e182\u003c/strong\u003e, 319\u0026ndash;326 (1989).\u003c/li\u003e\n\u003cli\u003eRovere, M. Circular Dichroism and Isothermal Titration Calorimetry to Study the Interaction of \u0026alpha;-Synuclein with Membranes. \u003cem\u003eMethods Mol Biol\u003c/em\u003e \u003cstrong\u003e1948\u003c/strong\u003e, 123\u0026ndash;143 (2019).\u003c/li\u003e\n\u003cli\u003eWei, Y., Thyparambil, A. A. \u0026amp; Latour, R. A. Protein helical structure determination using CD spectroscopy for solutions with strong background absorbance from 190 to 230 nm. \u003cem\u003eBiochimica et Biophysica Acta (BBA) - Proteins and Proteomics\u003c/em\u003e \u003cstrong\u003e1844\u003c/strong\u003e, 2331\u0026ndash;2337 (2014).\u003c/li\u003e\n\u003cli\u003ePeterle, D., Wales, T. E. \u0026amp; Engen, J. R. Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen\u0026ndash;Deuterium Exchange Mass Spectrometry Experiments. \u003cem\u003eAnal. Chem.\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 10142\u0026ndash;10150 (2022).\u003c/li\u003e\n\u003cli\u003eMeisl, G. \u003cem\u003eet al.\u003c/em\u003e Molecular mechanisms of protein aggregation from global fitting of kinetic models. \u003cem\u003eNat Protoc\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 252\u0026ndash;272 (2016).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Chemical characterization of the protein material corresponding to the main peaks of the chromatograms shown in Fig. 2. The calculated masses were obtained from the amino acid sequence of Syn.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eRP-HPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; RT (min)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 253px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Mass (Da)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpecies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eFound\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCalculated\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e35.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e14 460.51 \u0026plusmn; 0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e14 460.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003eSyn\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e769.33 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e769.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e1\u0026ndash;6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 708.38 \u0026plusmn; 0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 708.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e7\u0026ndash;140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Chemical characterization of the protein material corresponding to the peaks of the chromatograms of the proteolysis mixtures shown in Fig. 4. The calculated masses were obtained from the amino acid sequence of Syn variants. The yield of reaction (last column) was calculated as the ratio between the area under the peak of the 7\u0026ndash;140 from each species and that of full-length protein.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eSpecies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eRP-HPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; RT (min)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 253px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Mass (Da)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eYield %\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eFound\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eCalculated\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e7\u003c/em\u003e\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e\u003cem\u003e140 Syn\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e32.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 708.38 \u0026plusmn; 0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 708.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e78.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e7\u003c/em\u003e\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e\u003cem\u003e140 A30P\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e31.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 733.90 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 734.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e75.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e7\u003c/em\u003e\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e\u003cem\u003e140 E46K\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e30.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 706.53 \u0026plusmn; 0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 707.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e18.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e7\u003c/em\u003e\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e\u003cem\u003e140 H50Q\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e34.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 698.27 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 699.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e61.42\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e7\u003c/em\u003e\u003c/strong\u003e\u0026ndash;\u003cstrong\u003e\u003cem\u003e140 A53T\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e31.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003e13 737.88 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e13 738.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 126px;\"\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"α-synuclein, thrombin, α-synuclein pathological mutants, proteolysis, truncated forms, Parkinson disease, neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-8050047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8050047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProteolytic processing of α-Synuclein (Syn) contributes to the molecular diversity and toxicity of its aggregates in Parkinson disease (PD), yet the proteases responsible for generating disease-relevant truncations remain incompletely defined. Here, we identify thrombin (Thb) \u0026mdash; a serine protease best known for its role in haemostasis \u0026mdash; as a highly selective Syn-processing enzyme with relevance to neuroinflammatory conditions. Thb predominantly cleaves Syn at Lys6\u0026ndash;Gly7, producing a 7\u0026ndash;140 fragment as the major proteolytic species. This reaction displays remarkable site specificity despite multiple lysine residues and is modulated by ionic strength and Syn conformational flexibility. The 7\u0026ndash;140 fragment adopts a slightly more compact conformational ensemble, shows reduced membrane binding, and forms fibrils with slower aggregation kinetics and altered morphology compared to the full-length protein. These properties may extend the lifetime of soluble oligomeric intermediates, potentially contributing to chronic Syn-mediated toxicity. Our findings reveal a previously unrecognized link between inflammatory Thb activity and Syn proteostasis, suggesting that neuroinflammation-associated proteases may influence PD progression through selective truncation of Syn.\u003c/p\u003e","manuscriptTitle":"Linking Thrombin to α-Synuclein truncation reveals a molecular bridge between neuroinflammation and Parkinson disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 10:21:23","doi":"10.21203/rs.3.rs-8050047/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0739addb-3cbe-4616-9b8c-f81dfa6d985e","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58023330,"name":"Biological sciences/Biochemistry/Protein folding/Protein aggregation"},{"id":58023331,"name":"Biological sciences/Biochemistry/Proteases"}],"tags":[],"updatedAt":"2026-04-17T12:41:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 10:21:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8050047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8050047","identity":"rs-8050047","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}