ARTICLE https://doi.org/10.1038/s41467-021-24039-2 OPEN α-Helical peptidic scaffolds to target α-synuclein toxic species with nanomolar affinity Jaime Santos 1, Pablo Gracia2, Susanna Navarro 1, Samuel Peña-Díaz1, Jordi Pujols1, Nunilo Cremades 2 ✉, Irantzu Pallarès 1 ✉ & Salvador Ventura 1 ✉ α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson’s disease and 1234567890():,; related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species. 1 Institutde Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. 2 Institute for Biocomputation and Physics of Complex Systems (BIFI)-Joint Unit BIFI-IQFR (CSIC), University of Zaragoza, Zaragoza, Spain. ✉email: ncc@unizar.es; irantzu.pallares@uab.cat; salvador.ventura@uab.es NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 α -Synuclein (αS) is a 140 amino acid protein whose aggre- Results gation into amyloid fibrils in a subset of neuronal and glial Identification of an αS species-specific peptide ligand. We cells lies behind the onset of a group of progressive and, rationalized that the particular properties of the four main αS ultimately, fatal neurodegenerative disorders, including Parkin- conformers identified during αS amyloid aggregation, namely son’s disease (PD)1–4, that are collectively referred to as synu- monomers, non-toxic (type A/A*) oligomers, toxic (type B/B*) cleinopathies. A causative link between αS and disease is oligomers and fibrils, could be exploited to identify a selective supported by the discoveries that multiplications and missense ligand for the main species responsible for induction and pro- mutations in SNCA, the αS gene, cause dominantly-inherited pagation of toxicity, which are currently believed to be type B-like familial forms of PD5. oligomers and amyloid fibrils, respectively17. In Fig. 1a, we Interfering with αS amyloid formation and abrogating the illustrate the dissection of the differential traits of αS species (for a associated toxicity is considered a promising therapeutic strategy more detailed morphological, size and structural characterization for synucleinopathies6–8. However, the design of molecular of the different αS species in isolated preparations see Supple- entities that target specific αS toxic assemblies is challenging mentary Fig. 1). Type B-like oligomers and amyloid fibrils share because of the heterogeneous, dynamic, and transient nature of two features: (i) they expose relatively large lipophilic clusters to these species. High-throughput screening initiatives have ren- the solvent (Supplementary Fig. 1e). These hydrophobic surfaces dered promising αS aggregation inhibitors9–11. However, these induce cellular toxicity and drive subsequent fibrillation16,18–20. selection procedures are blind to the ligand mechanism of action. (ii) They possess a high anionic character at neutral pH, as a In the absence of a structure-activity relationship, it is difficult to result of the stacking of αS monomers (net charge −9). In αS, the evolve the affinity and specificity of the identified hits to generate negative charge is concentrated at the C-terminal region (residues drugs that can reach the clinics. The lack of specific and sensitive 95–140), which clusters 15 E/D amino acids. This αS segment molecules to detect the pathogenic forms of αS also constrains the remains disordered, and solvent exposed in both oligomers and early diagnosis of these diseases. fibrils13,21–23, being thus accessible to putative ligands. The in vitro aggregation of αS displays a sigmoidal growth While the solvent exposure of hydrophobic surfaces seems to profile, suggesting that it follows a nucleation-polymerization be a general feature of toxic pre-fibrillar oligomers13,24, the mechanism12, where soluble αS undergoes a nucleation process combination of highly exposed hydrophobicity and negative that produces oligomers able to grow through further monomer charge is likely unique to these two toxic αS assemblies. Thus, we addition to form insoluble amyloid fibrils. Oligomeric forms of αS hypothesized that hydrophobic patches embedded in an anionic have been detected in the brains and other tissues of patients environment might delineate a diffuse, but physicochemically- suffering from PD, and growing evidence suggests that they defined, binding surface in these two types of αS aggregates for a constitute the primary cytotoxic agents accounting for the gain- complementary molecule; ideally an amphipathic and cationic of-toxicity associated with αS aggregation, whereas both oligo- entity. A short α-helical peptide might provide a structurally mers and fibrils would be responsible for pathology dissemination stable scaffold to merge both features. in the brain2,13–15. We have recently identified the sequential We identified a naturally occurring peptide bearing a short, occurrence of two conformationally distinct types of oligomers stable, amphipathic, and cationic helical fold. PSMα3 is a 22- during αS in vitro fibrillation. The initial non-toxic disordered residue bacterial extracellular peptide that has been shown to oligomers, named as type A oligomers, undergo a structural remain in an α-helical conformation for weeks25. It has a net reorganization to form more stable and compact β sheet-enri- charge of +2, a mean hydrophobicity (H) of 0.54, an α-helical ched, and proteinase K-resistant species that exhibit intrinsic hydrophobic moment (μH) of 0.56, and the helical wheel plot cytotoxicity, named as type B oligomers16. Stable, trapped ana- evidences its amphipathic character (Fig. 1b, c). The far-UV logues of these two well-defined types of transient oligomers circular dichroism (CD) spectrum of PSMα3 confirms that it (referred to as type A* and type B* oligomers, where the star folds into an α-helix under our assay conditions (Fig. 1d). Thus, refers to the kinetically trapped nature of these isolated oligo- according to our hypothesis, this peptide fulfills all the meric forms) have been isolated and characterized in detail13,16 requirements to bind specifically to type B-like αS oligomers and, therefore, constitute important tools for the development of and amyloid fibrils. specific therapeutic and diagnostic strategies. We engineered PSMα3 to obtain a negative control peptide in In this work, we exploit our recent advances in the under- which the formation of an α-helix is strongly disfavored. This will standing of the structural determinants of toxicity of αS oligomers disrupt the peptide amphipathic character and, theoretically, to rationally identify peptide molecules able to target αS toxic abolish binding to αS type B* oligomers and amyloid fibrils. After species. By using a time-resolved single-particle fluorescence a computational proline scanning of PSMα3 using the AGADIR approach, we demonstrate that short, amphipathic, and cationic algorithm26 (Supplementary Fig. 2a), we selected the K9P and α-helical peptides do not interact with the functional monomeric F11P mutations, as they have a significant impact in helical αS, but they bind toxic oligomers and fibrils with nanomolar propensity and map to opposite faces of the α-helix (Supple- affinity, resulting in the substoichiometric inhibition of αS mentary Fig. 2b, c). The characterization of the secondary aggregation and abrogation of oligomer-induced damage in structure of the K9P-F11P PSMα3 peptide (further referred to as neuronal cell models. We then use a protein engineering disrupted PSMα3 or dPSMα3) in solution by CD confirmed the approach to dissect the molecular determinants accounting for disruption of the α-helix fold (Supplementary Fig. 2d). Thus, this interaction, which allow us to identify a human peptide, dPSMα3 constitutes a suitable negative control for further studies, constitutively expressed in the brain and gastrointestinal tract, as it keeps a sequence identity of 91% with PSMα3, but lacks its that binds with low nanomolar affinity to αS toxic assemblies, amphipathic character, a feature that we propose is key for the thus suppressing the aggregation cascade and its associated species-specific binding to the αS toxic assemblies. neurotoxicity. Thus, we describe here the rational identification and characterization of a family of highly potent peptidic ligands able to bind to αS toxic species and abrogate their detrimental Selective interaction of PSMα3 with αS toxic species. We then effects in neuronal cells. This discovery may open previously addressed the interaction of PSMα3 and dPSMα3 with the above unexplored avenues for the diagnosis and/or therapeutics of PD described four αS species. As multiple peptides are expected to and related disorders. bind multiple αS molecules in the aggregated states, the binding 2 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE Fig. 1 Rational identification of a peptide ligand for αS toxic species. a Main molecular features of the four isolated α-synuclein (αS) species. Values with a dagger (†) represent extrapolations based on the average number of monomers in each species. In the upper schemes of αS oligomeric and fibrillar species, the acidic C-terminal region is not depicted since it has been described to be in a disordered and conformationally flexible state21–23. b Helical wheel projection of PSMα3 sequence (red, hydrophobic residues; blue pallet, hydrophilic residues depending on their character). c Surface representation of the three-dimensional structure of PSMα3 with hydrophobic residues in red and hydrophilic residues in blue. d Far-UV circular dichroism spectra of PSMα3. process can only be well described if both the stoichiometry of the of the coverslips, the total αS concentration of each aggregate complex and the affinity of the peptide for the αS molecules in a sample was adjusted between 1 and 5 nM (in mass concentration) particular conformer is known. In order to obtain good estimates so that the frequency of events in the measurements was very of both parameters, we exploited the power of dual-color fluor- similar between the various aggregated samples. It is important to escence cross-correlation spectroscopy (dcFCCS), a time-resolved note that, for αS aggregated species, consisting of several tens of fluorescence fluctuation technique that allows the direct obser- monomers, the species concentrations are in the picomolar range vation of co-diffusing fluorescent species arising from interactions and, as further explained in the “Methods”, single-particle between differently labeled molecules or assemblies in conditions are ensured throughout the experiments. Under these solution27,28. To this end, αS species were cystein-labeled with conditions, a marginal cross-correlation amplitude was observed maleimide-AlexaFluor488 (AF488), with each αS molecule of the for the type A* oligomers (Fig. 2b), whereas a clear cross- different species containing one fluorophore at position 122, and correlation was obtained for the interaction of the peptide with the peptides were cystein-labeled with maleimide-Atto647N at both type B* oligomers and fibrils (Fig. 2c and d, respectively), the N-terminus (see “Methods” for details). Simultaneously, we already indicating a stark difference in the binding ability of the assessed complex formation by single-particle fluorescence spec- peptide to the different aggregates. Consistently, single-particle troscopy (SPF) analysis, including Förster Resonance Energy burst-wise analysis revealed a high number of FRET events, thus Transfer (FRET) and donor/acceptor stoichiometry (S), in order validating the direct interaction of the peptide with these two αS to validate and complement the dcFCCS approach. These species (Supplementary Fig. 5a, b). In contrast, the same analysis approaches allow us to monitor distinct individual species yielded either a statistically insignificant number of FRET events simultaneously by avoiding measurements of ensemble averages or none at all for the interaction between PSMα3 and αS type A* (Supplementary Fig. 3) and have been previously used for the oligomers and monomers, respectively (Supplementary Fig. 5c). characterization of αS aggregation pathways and the study of αS These results offer a single-particle understanding of a complex interactors29–31. binding scenario and further reinforce the observations derived We first assessed the binding of PSMα3 to monomeric αS and from dcFCCS experiments. found by dcFCCS analysis that these molecules were unable to We next performed a titration experiment for analyzing the interact when mixed in an equimolar ratio (even at concentra- binding of the peptide to the three αS aggregated species. For this, tions as high as 15 nM of each molecule) (Fig. 2a), as reflected by we developed a model-independent saturation binding curve, a flat cross-correlation curve comparable to that of the negative based on the theoretical framework previously developed by control of cross-correlation (Supplementary Fig. 4). We then Kruger and coworkers32. This analysis allowed us to quantify the analyzed the interaction of PSMα3 with the different αS number of peptide molecules bound to each αS species (NP) as a aggregated species by means of dcFCCS at approximately function of the concentration of unbound peptide (Cp,free) equimolar ratios of peptide and αS molecules. Due to the (Fig. 2e). Using a simplistic Langmuir isotherm model, different stabilities of the various aggregated species upon single- we estimated the single-state dissociation constant (KD) of the molecule dilution and their differential adsorption to the surface interactions and the average maximum number of peptide NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 Fig. 2 Interaction of PSMα3 with different αS species by dcFCCS. a–d Representative auto-correlation curves for α-synuclein (αS) (blue line) and PSMα3 (red line) and cross-correlation curves for interacting molecules (purple line). The amplitude (G) error is shown as faint colored area for the corresponding correlation curves. Samples contained (a) ~15 nM αS monomer and ~15 nM PSMα3, (b) 1 nM type A* and ~5 nM PSMα3, (c) 1 nM type B* oligomers and ~5 nM PSMα3 or (d) ~5 nM sonicated fibrils and ~5 nM PSMα3. e Titration binding curves for the interaction of PSMα3 with type A* oligomers (red circles), type B* oligomers (blue circles) or sonicated fibrils (gray circles) obtained by dcFCCS, showing their corresponding analysis assuming a model of n identical and independent binding sites (referred in equation 7 as Nmax) per αS aggregated species (solid lines). NP represents the number of bound peptides per aggregate. f Auto-correlation curves (αS in blue, PSMα3 peptide in red) and cross-correlation curve for the interacting molecules (in purple) obtained in samples containing ~1 nM αS type B* oligomers and ~2 nM PSMα3 in the absence (solid lines) or presence (dashed lines) of a 500-molar excess of unlabeled monomer with respect to the particle concentration of oligomers. The inset shows the number of bound peptides (NP) per aggregate in both conditions. For αS aggregated species, each consisting of several tens of monomers, the species concentrations are in the picomolar range and, as further explained above, single-particle conditions are ensured throughout the experiments. binding sites (Nmax) in each type of αS species. Interestingly, matches the average number of αS molecules per aggregate while the KD values for the peptide-αS interaction obtained for species (19, 33, and 107 for type A*, type B* oligomers, and type A* oligomers, type B* oligomers and fibrils are very similar, fibrils, respectively, estimated by comparing the molecular in all cases in the very low nM range (3.07 nM, 6.67 nM, and 7.8 brightness of the aggregated species to that of the αS monomer), nM, respectively), the average maximum number of peptides per while for the type A* oligomers represents only one sixth of the aggregate (NP) varies remarkably, being 3, 30, and 120, average αS molecules in this type of aggregate. This data is in respectively. This indicates that the main difference between the agreement with the single-particle fluorescence analysis obtained three aggregated species in terms of PSMα3 interaction is the for the different complexes, where decreasing fluorescence number of binding sites per aggregate rather than the affinity of stoichiometry values are found for the interacting pairs with the peptide for them. Of note, the average maximum number of increasing PSMα3 concentrations (Supplementary Fig. 6) yielding binding sites obtained for the type B* oligomers and fibrils nearly a binding curve remarkably similar to that obtained by dcFCCS 4 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE (Supplementary Fig. 7). In addition, only very few FRET events neuroprotective activity in Caenorhabditis elegans models of PD were observed for the binding of PSMα3 to type A* oligomers, in that we have recently discovered9. contrast to the numerous FRET events with a defined FRET To gain further information on the inhibitory mechanism and efficiency (E) distribution observed for the binding to type B* pinpoint the αS species targeted along the complex pathway of oligomers and fibrils (Supplementary Fig. 5). Together, the aggregation, we isolated the low molecular weight species dcFCCS and single-particle fluorescence spectroscopy data generated at the early stages of aggregation (see “Methods”). demonstrate that PSMα3 is a high affinity ligand of αS toxic Electron microscopy analysis revealed that in control aggregation species, with affinities in the low nanomolar range, and with a reactions, after 12 h of incubation, αS mainly populates small high avidity for the toxic αS species, namely the type B-like fibrillar species and round prefibrillar aggregates (average oligomers and fibrils. Note that a 500-fold molar excess (in diameter between 20 and 40 nm) (Fig. 3d and Supplementary monomer equivalents) of unlabeled monomeric αS does not Fig. 11a–b). In contrast, at the same time point, samples interfere with the binding of PSMα3 to type B* oligomers incubated with PSMα3 contained a large fraction of small (Fig. 2f), which indicates a high specificity towards toxic oligomers of annular shape with diameters between 9 and 14 nm, aggregated species and negligible monomer binding, a feature morphologically similar to type B* oligomers (Fig. 3d and difficult to find in previously reported αS ligands. Supplementary Fig. 11c) and other annular oligomers previously It might be important to indicate that PSMα3 could present a described in the literature24,33,34. Together with the time-resolved certain degree of oligomerization that results in a slower diffusion fluorescence spectroscopy data, this evidence strongly suggests than expected for a peptide monomer (14.6 ± 3.6 µm2 s−1, Fig. 2). that PSMα3 could be preventing or retarding the conversion of Despite this, our dcFCCS-derived binding curves indicate that the type B-like oligomers into fibrillar species. Notably, this result monomeric form of the peptide can effectively bind the αS endorses the use of the kinetically stabilized type B* oligomers as aggregated species with NP values as low as 1 (further information mimicries of the toxic oligomers that populate αS aggregation is provided in the “Methods”). reactions. Interestingly, when we analyzed the binding of the PSMα3 analogous, but disordered peptide, dPSMα3, we could not detect any interaction with any of the four αS species (Supplementary PSMα3 protects cells from αS oligomer-induced cell damage. Fig. 8), indicating that an amphipathic distribution of the peptide As it occurs with toxic oligomers from other amyloidogenic residues, achieved through an α-helical conformation, is a systems, the toxicity of type B* oligomers relies on their ability to requirement for the interaction. interact and disrupt cellular membranes13. In αS this activity is Overall, our dcFCCS and single-particle fluorescence encoded in two of their characteristic structural elements13: (i) an spectroscopy-derived binding analysis indicates that PSMα3 exposed N-terminal region that acts as the initial anchor to the binds with low nanomolar affinity to αS aggregated species. The membrane surface, similarly as with the monomeric functional degree of binding is limited by the number of available interaction form of the protein, and (ii) a β-sheet core, composed primarily sites, which is likely associated with the extent of solvent-exposed by the central region of the protein, with significant hydrophobic hydrophobic surface per aggregate, which depends on both the surface exposed to the solvent that then inserts into the lipid size and the lipophilicity of the aggregate, in agreement with our bilayer causing major perturbations. The highly negatively initial reasoning. charged C-terminal region of the protein remains disordered without significant interactions with the membrane. We hypothesized that the binding of PSMα3 to type B* PSMα3 inhibits αS amyloid aggregation. We hypothesized that oligomers, mediated in part by the solvent-exposed hydrophobic the high affinity and number of binding sites of type B* oligomers regions of the β-sheet core, would block their exposed lipophilic for PSMα3 might result in the partial or full coverage of the elements, thus decreasing its ability to insert into and perturb the surface of these assemblies, as well as their structurally homo- membrane bilayer and induce cellular toxicity. We treated human logous type B oligomers, thus preventing their progression to SH-SY5Y neuroblastoma cells with 10 μM of oligomers and fibrils during the αS amyloid aggregation process. To assess if this observed that, as previously reported35, they possess a high was the case, we set up in vitro αS aggregation reactions in the affinity for cellular membranes (Fig. 4a–b). When the oligomers absence and presence of an equimolar concentration of PSMα3 were preincubated with an equimolar concentration of PSMα3, (70 μM) and followed its progression by monitoring the increase we observed a ∼60% reduction in the amount of αS bound to cells in thioflavin-T (Th-T) fluorescence. After 32 h of incubation, a relative to untreated oligomers, indicating that PSMα3 binding to ∼90% decrease in Th-T fluorescence emission, relative to the type B* oligomers directly affects the binding of the oligomers to untreated sample, was observed in the presence of PSMα3, sug- cellular membranes. As expected, pretreatment of the oligomers gesting that the peptide acts as a potent inhibitor of αS amyloid with dPSMα3 did not interfere with their interaction with cells. aggregation (Fig. 3a). Inhibition was orthogonally confirmed by One of the earliest effects of type B* oligomer-mediated quantifying the fraction of αS that remains soluble at the end- membrane perturbation is the substantial increase in the levels of point of the reaction spectroscopically and by SDS-PAGE (Sup- intracellular reactive oxygen species (ROS)24, which in turn elicits plementary Fig. 9a, b). The inhibitory activity of PSMα3 was mitochondrial dysfunction36. We assessed if the blockage of the concentration-dependent and significant inhibition was observed oligomer regions involved in membrane perturbation by PSMα3 even at a substoichiometric 20:1 ratio (αS:PSMα3) (Fig. 3b). binding could protect membrane integrity and therefore prevent Transmission electron microscopy (TEM) images confirmed that its associated increase in intracellular ROS levels. Treatment of samples incubated with PSMα3 contained very few fibrils per field, neuroblastoma cells with 10 μM of oligomers induced a drastic in comparison to untreated samples (Fig. 3c). The observation that increase in ROS levels (Fig. 4c–d). However, when these dPSMα3 exhibited a negligible anti-aggregative activity (Supple- oligomers were preincubated with equimolar (1:1) and substoi- mentary Fig. 10) reinforces the connection between the binding of chiometric (1:0.2) concentrations of PSMα3, the ROS levels of the amphipathic (hydrophobic/cationic) helical peptide to αS treated cells approached those of healthy, untreated, cells, oligomers and its potent amyloid inhibition activity. indicating that PSMα3 protects against oligomers-induced PSMα3 is a better inhibitor of αS amyloid aggregation than damage. This detoxifying activity seems to be associated with SynuClean-D (Fig. 3a), a small molecule with high the particular structural and physicochemical properties of this NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 Fig. 3 Effect of PSMα3 on in vitro αS amyloid fibrillation. a Inhibition of α-synuclein (αS) amyloid aggregation as measured by Th-T fluorescence after 32 h incubation in the presence of equimolar concentrations of PSMα3, SynuClean-D (SC-D), and dPSMα3. **** p < 0.0001 relative to untreated αS (unpaired two-tailed t tests (Welch-corrected)). Data were expressed as mean ± s.e.m (n = 6 and 3 independent experiments for untreated and treated conditions respectively). NS no significant, p = 0.23. b Aggregation kinetics of 70 μM αS and titration of the inhibitory activity of PSMα3 at different concentrations: 35 μM (green), 14 μM (orange), 7 μM (blue), 3.5 μM (gray) and in the absence of PSMα3 (black). Data were expressed as mean ± s.e.m (n = 9 independent experiments). c Representative TEM micrographs of αS aggregated for 32 h in the absence (left) and presence of an equimolar concentration of PSMα3 (right) that came from two independent replicates. d Representative TEM micrographs illustrating the morphological differences between low- molecular weight aggregates of αS after 12 h of incubation in absence (left) and presence of PSMα3 (right). Results are consistent in two independent replicates. Inset shows an annular oligomer at high magnification. peptide since treatment with equimolar concentrations of predictions are displayed in Supplementary Table 1. Then, we dPSMα3 failed to exert any protective effect. evaluated the anti-aggregative properties of these molecules, under the assumption that the inhibitory capacity is connected Dissection of PSMα3 aggregation-inhibitory determinants. To with the oligomer-peptide interaction affinity. this point, we have assigned the αS binding, anti-aggregation and A first requirement for binding is a continuous hydrophobic face cytoprotective properties of PSMα3 to its helical, amphipathic to interact with the surface of oligomers or fibrils. In our view, the and cationic character. To confirm that this is the case, we specific sequence of this helical side would be irrelevant, as long as it reverse-engineered PSMα3 into a non-natural peptide scaffold keeps its lipophilic character. To demonstrate that this assumption with low sequence complexity that keeps its critical properties. is correct, we mutated all the residues in the hydrophobic face of the We employed a set of bioinformatics tools to predict the helical PSMα3 α-helix to leucine (All_Leu), generating an amphipathic propensity, helical hydrophobic moment, and thermodynamic peptide devoid of any sequence diversity in this side. Simulta- stability of our successive designs using AGADIR26, neously, we designed a variant of All-Leu devoid of the last three C- HELIQUEST37, and FOLDX38, respectively. Data regarding those terminal residues (All_Leu19) since they are not part of the α-helix, 6 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE and thus they are not expected to contribute significantly to the ionizable residues. We designed a new variant (Scaffold_19) with binding. Both redesigned peptides folded into α-helices and retained only four different amino acids (Leu, Asp, Glu, and Lys) by the inhibitory activity, with a potency that approaches that of introducing two point-mutations (A5E_G16K) in the All_Leu19 PSMα3 (Fig. 5a–b). Thus, we concluded that it is the generic peptide. We decided to maintain the peptide net charge by hydrophobic character of the helical face and not its sequence or introducing residues with opposed charges. Thus, Scaffold_19 only composition that is relevant for the binding. has Leu in the hydrophobic face and charged residues in the Next, to further reduce the peptide sequence complexity, we hydrophilic one. This variant folded into an α-helix and showed the redesigned the hydrophilic face in such a way that it only contained same anti-aggregation activity than the parental variant (Fig. 5c). NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 Fig. 4 Suppression of the αS oligomers-induced damage in neuroblastoma cells. a Representative confocal images showing the α-synuclein (αS) load per cell after the treatment with 10 μM of type B* oligomers pretreated with an equimolar concentration of PSMα3 or dPSMα3. Scale bar represents 30 μm. b Quantification of the αS load per cell. ****p < 0.0001 relative to untreated cells. oooop < 0.0001 relative to cells treated with αS type B* oligomers. Unpaired two-tailed t tests (Welch-corrected). 76, 72, and 67 cells, (respectively, for αS oligomers, PSMα3 1:1, PSMα3 1:02 and dPSMα3) were analyzed from two independent experiments. c Quantification of the levels of intracellular ROS of SH-SY5Y cells incubated with 10 μM of type B* oligomers preincubated with different concentrations of PSMα3 or dPSMα3. oooop < 0.0001 relative to cells treated with αS type B* oligomers. (Unpaired two-tailed t tests (Welch-corrected)). 233, 230, 240, 212, and 100 cells, (respectively, for untreated, αS oligomers, PSMα3 1:1, PSMα3 1:02 and dPSMα3) were analyzed from two independent experiments d Representative confocal images of the analysis of panel (c). Scale bar represents 30 μM. In (b) and (c) data are represented as box and whiskers plots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (where IQR is the inter-quartile range) and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. Fig. 5 Redesign of PSMα3 variants to dissect the molecular determinants of the anti-aggregative activity. a–d Helical wheel (red, hydrophobic residues; blue pallet, hydrophilic residues) (up), circular dichroism spectra (mid) and titration of the inhibitory activity of PSMα3 variants at different concentrations (down): 35 μM (green), 14 μM (orange), 7 μM (blue) and in the absence of PSMα3 variants (black). Variants: All_Leu (a), All_Leu19 (b), Scaffold_19 (c), and Anionic_scaffold (d). Data were expressed as mean ± s.e.m (n = 9 independent experiments). The simplicity of Scaffold_19 allowed us to redesign the net be encoded in natural human peptides. First, we screened the charge of the peptide, to validate the other physicochemical EROP-Moscow oligopeptide database39 for human cationic property theoretically contributing to binding: a net positive peptides longer than 10 residues (≥3 helical turns), obtaining 287 charge. We generated a peptide with a net charge of −2 by hits. Next, we run AGADIR on them to exclude peptides with a introducing two charge-reversing mutations (K6E_K12E). This low helical propensity, which resulted in 25 peptides, from which anionic peptide (Anionic_scaffold) folds into an α-helix and has a only 9 peptides were predicted to have at least a partial amphi- helical hydrophobic moment (μH) of 0.65, indicative of an pathic character, according to their helical hydrophobic moment amphipathic nature, but does not inhibit αS amyloid aggregation, (μH) (Supplementary Table 2). Then we screened the literature for confirming that a cationic character in the hydrophilic face is a candidates whose tissue distribution overlapped with that of αS requirement for binding (Fig. 5d). and selected LL-37, the only human member of the cathelicidin Overall, we succeeded in dissecting the peptide features family of antimicrobial peptides, for its further characterization. responsible for aggregation inhibition. In the process, we LL-37 is a 37-residue peptide resulting from a post-translational generated, Scaffold_19, a short peptide with low sequence cleavage at the C-terminus of cathelicidin hCAP1840. This pep- complexity whose inhibitory activity does not stem from the tide is constitutively expressed in the brain and the gastro- primary sequence, but instead from a defined spatial distribution intestinal tract; its presence in both tissues is engaging, as the of two physicochemical traits. brain-gut axis connection is gaining momentum in PD41–43. First, we confirmed that LL-37 adopts an α-helical conforma- tion under our assay conditions (Fig. 6a, b). With α-helical LL-37 inhibits αS aggregation and oligomers-induced cell hydrophobic moment (μH) of 0.52, the helical-wheel projection damage. Once we elucidated the determinants of this mechanism and the available 3D-structures37 indicate that this α-helix would of αS amyloid inhibition, we wondered if this activity could also be both cationic and amphipathic (Fig. 6a, b). Then, we titrated 8 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE the anti-aggregative activity of LL-37, confirming that it and spFRET (Fig. 6d, f and Supplementary Fig. 12) reported a suppresses αS amyloid formation at substoichiometric concentra- strong binding to type B* oligomers and fibrils, a weak tions (Fig. 6c). Sedimentation analysis provides orthogonal interaction with type A* oligomers and the absence of any support for the αS aggregation inhibitory activity of LL-37 interaction with the αS monomer, indicating that LL-37 and (Supplementary Fig. 9). Next, we labeled LL-37 with maleimide- PSMα3 share a very similar binding mechanism (Fig. 6g). In this Atto647N at a single engineered cysteine at the N-terminus, and case, LL-37 displays slightly higher affinities than PSMα3 (KD = we performed time-resolved dual-color fluorescence spectroscopy 3.62 nM for type B* oligomers, KD = 5.14 nM for sonicated fibrils experiments as described previously for PSMα3. Both dcFCCS and KD = 1.92 nM for type A* oligomers), and a significantly NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 Fig. 6 Characterization of the interaction of LL-37 with the αS toxic species. a Helical wheel projection of LL-37 sequence (red, hydrophobic residues; blue pallet, hydrophilic residues; green, proline). b Far-UV circular dichroism spectra of LL-37 in PBS pH 7.4. c Aggregation kinetics of 70 μM α-synuclein (αS) and titration of the inhibitory activity of LL-37 at different concentrations: 35 μM (green), 14 μM (orange), 7 μM (blue), 3.5 μM (gray) and in the absence of peptide (black). Data were expressed as mean ± s.e.m (n = 9 independent experiments). Representative auto-correlation curves for αS and LL- 37 peptide and cross-correlation curves for interacting molecules are shown in blue, red and purple lines, respectively. The amplitude (G) error is shown as faint colored area for the corresponding correlation curves. Samples contained (d) ~15 nM αS monomers and ~15 nM LL-37, (e) 1 nM type B* oligomers and ~5 nM LL-37 or (f) ~5 nM sonicated fibrils and ~5 nM PSMα3. g Titration binding curves for the interaction of LL-37 with type A* oligomers (red circles), type B* oligomers (blue circles) or sonicated fibrils (gray circles) obtained by dcFCCS, showing their corresponding analysis assuming a model of n independent binding sites per αS aggregated species (solid lines). NP represents the number of bound peptides per aggregate. h Representative confocal images of SH-SY5Y cells treated with 10 μM of type B* oligomers in the presence of an equimolar concentration of LL-37. Scale bar represents 30 μm. i Quantification of the intracellular ROS of the experiment displayed in panel (h). ****p < 0.0001 relative to untreated cells. oooop < 0.0001 relative to cells treated with αS type B* oligomers. Unpaired two-tailed t tests (Welch-corrected). A total of 233, 230, and 199 cells, (respectively, for untreated αS oligomer and LL-37 1:1) were analyzed from two independent experiments. For αS aggregated species, consisting of several tens of monomers, the species concentrations in (d–g) are in the picomolar range and, as further explained above, single-particle conditions are ensured throughout the experiments. In (i) data are represented as box and whiskers plots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (where IQR is the inter-quartile range) and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge. higher number of binding sites in type B* oligomers (Nmax = 64) Many small bioactive peptides are derived from larger precursors and sonicated fibrils (Nmax = 181), while remain the same as and generated after proteolytic cleavage44. In some cases, these PSMα3 for the number of binding sites in the type A* oligomers, peptides are encrypted inside globular proteins, and their processing which was in any case marginal (Nmax = 3). Consistent with the results in the manifestation of a new biological function. LL-37 is a LL-37 ability to bind type B* oligomers with high affinity, the cathelicidin-derived peptide constitutively expressed in the human preincubation of these toxic species with the human peptide at an brain45. Here, we show that LL-37 is a tight binder of αS toxic equimolar concentration completely abolished the production of assemblies, with anti-aggregation and cytoprotective properties. LL- ROS in neuroblastoma cells (Fig. 6h, i). LL-37 is not related in 37 has been reported to inhibit the amyloid aggregation of two sequence to PSMα3 or Scaffold_19, but the three peptides share other disease-linked peptides, Aβ-4246 and IAPP47. However, the the same structural and physicochemical traits. This confirms that mechanism behind this activity is different from the one we a linear combination of these properties suffices to identify, and describe here since it relies on a certain degree of sequence potentially design, potent inhibitors of αS aggregation. homology between short linear stretches in these molecules, with Whether LL-37 is actually involved or not in the pathogenesis LL-37 binding to both the monomers and the aggregated of PD remains unexplored. However, it is tempting to speculate species46,47. The KDs for the binding of LL-37 to freshly resus- that small peptides able to interact actively with αS aggregated pended and 24-days-incubated Aβ-42 peptide are 13.30 μM and species might cohabitate with this protein in tissues relevant to 20.30 μM, respectively46; thus, several orders of magnitude weaker the disease. These human peptides may open an unexplored than the ones we report here for αS. Irrespective of the affinity and avenue for PD treatment, i.e., by stimulating their endogenous species selectivity, it is tempting to suggest that endogenous pep- expression. tides, similar to those described here, could compose a regulatory system where they act as silent guardians of the proteome by tar- Discussion geting aggregation-prone proteins. Because of its involvement in PD and other synucleinopathies, αS Apart from potential future therapeutic implications, the ability aggregation remains a promising target for therapeutic inter- of the amphipathic cationic helical peptides to bind to αS toxic vention. Herein, we propose a strategy for targeting the αS species species with high affinity might find a direct application in behind the onset of these neurodegenerative diseases selectively. diagnosis. The presence of αS aggregates in biofluids is considered By binding to αS toxic oligomers and fibrils, the described col- a biomarker for PD and other synucleopathies48,49. However, lection of peptides inhibits the progression of αS aggregation, current detection methods are not specific and sensitive enough while suppressing oligomer mediated cell damage. Importantly, for their clinical implementation. For instance, ELISA approaches because the binding determinants are structurally encoded, these based on the so-called conformation-specific antibodies, perform peptidic molecules do not recognize monomeric αS. Furthermore, better when the detection is normalized relative to the total levels the avidity of these peptides for early non-toxic oligomers is more of αS or when the same epitope-blocking antibody is used for than one order of magnitude lower than the one for type-B* both capture and detection50. This indicates that a major lim- oligomers and fibrils, indicating that they are very selective for itation of these methods is the unwanted cross-reaction of the these toxic species. antibodies with the large excess of αS monomer in the fluid. The We describe here, non antibody-related biomolecules targeting peptides we describe here do not interact with monomeric αS, αS aggregated species, which have been rationally predicted, and the presence of up to 500-fold molar excess of monomeric αS identified, and engineered. PSMα3 is a first-in-class hit molecule does not interfere with the detection of nanomolar amounts of that sets the ground for the future advancement of a generation of toxic oligomers. This property, together with their close-to- leads for disease modification in PD and other synucleinopathies. antibody affinities, may turn useful for diagnostic purposes. We The requisites for a high peptide binding affinity and αS toxic envision that a strategy which combines sequence-specific αS species selectivity are relatively simple: hydrophobic and posi- ligands (i.e., available antibodies) with our species-specific pep- tively charged surfaces with opposed orientations in space. This is tides might succeed in the selective and sensitive detection of best exemplified by Scaffold_19, a short and low complexity toxic αS species in biological fluids. peptide that fulfills those conditions. This defined binding mode Overall, the molecular entities we describe in this work may should help in the development and diversification of ligands help to develop therapeutic and diagnostic strategies for the with increased activities. synucleinopathies. 10 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE Methods aggregation kinetics methodology section. The non-reacted protein and small non- αS expression and purification. Escherichia coli BL21 (DE3) cells containing a fibrillar species that could be formed during the aggregation reaction were removed pET21a plasmid encoding the αS gene were grown in LB medium supplemented from the sample by 3 consecutive steps of centrifugation and resuspension of the with 100 μM/mL ampicillin. Protein expression was induced at an optical density precipitated fraction in PBS buffer at pH 7.4. Fibrils were then sonicated 1 min, 50% of 0.8 (600 nm) with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 4 h. cycles, 80% amplitude in a Vibra-Cell VC130 Ultrasonic Processor (Sonics, Newton, Cells were harvested by centrifugation and washed up by resuspension and cen- USA) to generate fibrillar samples with a relatively homogeneous size distribution of trifugation in PBS pH 7.4. Next, pellets were resuspended in 50 mL per culture liter small fibrils. The concentration of the AF488-labeled fibrillar samples was deter- in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1 μg/mL pepstatin, 20 μg/mL mined by subtracting the absorbance of the monomer after centrifugation at 495 nm aprotinin, 1 mM benzamidine, 1 mM PMSF, 1 mM EDTA, and 0.25 mg/mL lyso- using an extinction coefficient of 72,000 M−1 cm−1, with respect to the total soluble zyme) and sonicated using a LabSonic®U sonicator (B. Braun Biotech International, protein at time 0. For type A* oligomers, the concentration was adjusted in situ for Melsungen, Germany). Samples were boiled during 10 min at 95 °C and cen- each experiment so that a suitable and consistent burst-rate was reached. Thus, an trifugated at 20,000 g at 4 °C for 40 min. The soluble fraction was treated with 136 interference of EGCG in quantifying the sample was avoided. μL/mL of 10% w/v streptomycin sulfate and 228 μL/mL of pure acetic acid. Upon centrifugation, soluble extracts were fractionated by adding 1:1 of saturated ammonium sulfate and resuspending the insoluble fraction with 50% ammonium Far circular dichroism analysis. Far-UV CD spectra of the different peptide sulfate. The pellet was resuspended in 100 mM pH 8 ammonium acetate (5 mL per solutions were recorded on a Jasco J-815 CD spectrometer (Halifax, Canada) culture liter) and pure EtOH 1:1 (v/v) and harvested by centrifugation. The Software- Jasco spectra manager v2 at 25 °C using samples of 15 μM peptide final insoluble fraction was resuspended in Tris 20 mM pH 8, filtered with a 0.22 μm concentration in Milli-Q water. CD signal was measured from 260 nm to 190 nm at filter and loaded into an anion exchange column HiTrap Q HP (GE Healthcare, 0.2 nm intervals, 1 nm bandwidth, 1 sec of response time and a scan speed of 100 Chicago, USA) coupled to an ÄKTA purifier high performance liquid chromato- nm/min on a 0.1 cm quartz cell. Ten accumulations were recorded and averaged graphy system (GE Healthcare, Chicago, USA). Tris 20 mM pH 8 and Tris 20 mM for each measurement. For LL-37 peptide samples, CD spectra were recorded in pH 8, NaCl 1 M were used as buffer A and buffer B. αS was eluted using a using a PBS pH 7.4, because of structural differences of this peptide in water and saline step gradient: Step 1: 0%–20% buffer B, 5 cv; Step 2: 20%–45% buffer B, 11 cv; Step solvents. 3: 100% buffer B, 5 cv. Purified αS was dialyzed against 5 L ammonium acetate 50 mM in two steps; 4 h and overnight. Finally, protein purity was addressed using 15% SDS-PAGE. The purest fractions were lyophilized and stored at −80 °C. For Time-resolved fluorescence spectroscopy. Dual-Color Time-Resolved Fluores- the experiments, αS lyophilized aliquots were resuspended to a final concentration cence Spectroscopy experiments were performed on a commercial MT200 (Pico- of 210 μM using PBS pH 7.4 and filtered using 0.22 μm filters. αS concentration was Quant, Berlin, Germany) time-resolved fluorescence confocal microscope with a determined measuring the absorbance at 280 nm and using the extinction coeffi- Time-Correlated Single Photon Counting (TCSPC) unit. Laser diode heads were cient 5960 M−1 cm−1. used in Pulsed Interleaved Excitation (PIE), and the beams were coupled through a single-mode waveguide and adjusted to laser powers of 6 µW (481 nm) and 5 µW (637 nm) measured after the dichroic mirror for optimal count rates while avoiding Peptide preparation. PSMα3, dPSMα3, All_Leu, All_Leu_19, Scaffold_19, Anio- photobleaching and saturation. The coverslip was placed directly on the immersion nic Scaffold and LL-37 were purchased from Synpeptide (Shanghai, China) with a water on top of a Super Apochromat 60x NA 1.2 objective with a correction collar purity >95%. Single cysteine containing variants were purchased from Genscript (Olympus Life Sciences, Waltham, USA). A dichroic mirror of 488/640 nm (Piscataway, USA) with a purity >95%. LL-37 was diluted in Milli-Q sterilized (Semrock, Lake Forest, IL, USA) was used as the main beam splitter. Out-of-focus water, divided into aliquots and lyophilized. Cysteine containing peptides were emission light was blocked by a 50 µm pinhole and the in-focus emission light was resuspended in PBS pH 7.4, 5 mM TCEP and subsequently labeled with the cor- then split by a 50/50 beamsplitter into two detection paths. Bandpass emission responding fluorophore. PSMα3, dPSMα3, All_Leu, All_Leu_19, Scaffold_19 and filters (Semrock, Lake Forest, IL, USA) of 520/35 for the green dye (AF488) and Anionic Scaffold were dissolved in a 1:1 mixture of trifluoroacetic acid and hex- 690/70 for the red dye (Atto647N) were used before the detectors. Single Photon afluoroisopropanol and sonicated for 10 min. Stock solutions were divided into Avalanche Diodes (SPADs) (Micro Photon Devices, Bolzano, Italy) served as aliquots and vacuum dried with a SpeedVac (Thermo Fisher Scientific, Waltham, detectors. Each measurement had an acquisition time of 1–3 min. USA) and stored at −80 °C until assayed. Peptide aliquots were resuspended in For FCS experiments, the effective focal volume of the green channel and its pure Milli-Q water prior their use. structural parameters in our system were determined using a 1 nM solution of Atto488 (ATTO-TEC GmbH, Siegen, Germany) yielding Veff, g = 0.51 fL and κg = αS and peptide labeling. Site-specific labeling of αS was performed in an αS 3.97. Positive and negative cross-correlation controls were performed with a dual- variant with a single engineered cysteine at position 122 (αS N122C). This variant labeled dsDNA (10 nM) and an equimolar mixture (15 nM each) of AF488- and was expressed and purified as described above but including 5 mM DTT in all Atto647N-labeled monomeric αS (Supplementary Fig. 4). The positive control was purification steps. The protein was labeled with maleimide-modified Alexa Fluor also used for the determination of the red and dual-color effective focal volume and 488 (AF488) (Invitrogen, Carlsbad, USA) for 15–20 h at 4 °C in the dark. After their structural parameter, yielding Veff,r = 0.1 fL, Veff,gr = 0.091 fL, κr = 2.78, and quenching the reaction with 10 mM DTT, free unreacted dye in the protein κgr = 2.67, respectively. solution was subsequently separated using a P10 desalting column (GE Healthcare, AF488-labeled aggregated αS samples were diluted in PBS pH 7.4 to a final Waukesha, USA), and the labeled protein solution was flash frozen with liquid protein concentration of ~1-5 nM in a 50 μL droplet which was spotted directly nitrogen and stored at −80 °C. The different peptides, PSMα3, dPSMα3, and LL- onto a cover glass (Corning, Corning, USA) previously coated with a 1 mg/mL BSA 37, were labeled at a single engineered cysteine at the N-terminus with maleimide- solution. Atto 647N-labeled peptides were titrated into the droplet and the peptide modified Atto647N (ATTO-TEC, Siegen, Germany). The same labeling and pur- concentration was measured individually for each experiment by autocorrelation ification strategy were followed as for αS, although in this case the unreacted free analysis of the red dye. No significant changes in correlation amplitudes were dye was removed from the protein solution using a polyacrylamide desalting col- observed over time after equilibrating the samples for 2 min. Experiments were umn (Thermo Fisher Scientific, Waltham, USA). Two cleaning steps were required performed at 20 °C and samples were covered to avoid evaporation. It is important to remove completely the free dye from the labeled peptide solution. to note that, for αS aggregated species, consisting of several tens of monomers, the species concentrations are in the picomolar range and, as further explained below, single-particle conditions are ensured throughout the experiments. The aggregated Preparation of the different isolated αS aggregates samples. For the isolation species coexist with a certain amount of monomeric αS due to the stark sample of type B* oligomers purified αS was dialyzed against Milli-Q water and lyophilized dilution employed in the experiments and, therefore, the donor auto-correlation for 48 h in aliquots of 6 mg. The aliquots were resuspended in 500 μL of PBS pH 7.4 curves in Figs. 2, 6 and Supplementary Figs. 8, 12, 13, show both the diffusion to a final concentration of ca. 800 μM, filtered through 0.22 μm filters and incubated component of the monomer and the aggregate. A similar behavior is observed for at 37 °C without agitation for 20–24 h. The sample was then ultracentrifuged at peptides PSMα3 and LL-37, which can exist as oligomerized species. For obtaining 288,000 g in a SW55Ti Beckman rotor, in order to remove any possible fibrillar the diffusion coefficients of the different aggregates the diffusion component of the species formed during the incubation, and later filtered by four consecutive cycles of monomeric species in the samples was filtered out by intensity-filtered dcFCCS filtration through 100 kDa centrifuge filters (Merck, Darmstadt, Germany) in order analysis as explained below. The diffusion coefficient (Dg or Dr) fitted to data for αS to remove the great excess of monomeric protein from the oligomeric solution. Type species are 103 ± 16 µm2 s−1, 4 ± 0.9 µm2 s−1, 3.46 ± 1.2 µm2 s−1 and 0.81 ± 0.12 A* oligomers were generated by incubating 210 μM of αS in PBS pH 7.4 with ten µm2 s−1 for the monomer, type A*oligomers, type B* oligomers and fibrils, molar equivalents of (-)-epigallocatechin-3- gallate (EGCG) (Merck, Darmstadt, respectively, in very good agreement with the diffusion coefficients expected Germany) for 48 h at 37 °C. The excess of compound and unreacted monomeric according to their corresponding sizes as determined by AFM and DLS (see protein were then removed by six consecutive cycles of filtration through 100 kDa Supplementary Fig. 1) and as reported before13,19. In addition, such intensity centrifuge filters (Merck, Darmstadt, Germany). The concentration of the final thresholding yields a confocal volume mean occupancy (N) well below 1 for all oligomeric solutions was determined measuring the absorbance at 280 nm and fluorescent species involved, with N = 0.019, N = 0.043, and N = 0.053 for type using an extinction coefficient of 5960 M−1 cm−1 or absorbance at 495 nm and an A*oligomers, type B* oligomers, and fibrils, respectively. Therefore, in terms of extinction coefficient of 72,000 M−1 cm−1 for AF488-labeled oligomers. In all cases, burst selection for the PIE-FRET and fluorescence stoichiometry analysis, where the oligomers were kept at room temperature and were used within 3 days after the same intensity threshold is applied, the experiments were conducted under their production. The fibrillar samples were produced as explained in the single-particle conditions. This becomes even more evident when looking at the NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 raw data in the form of intensity time traces of, for instance, the PSMα3 - type B* efficiency and stoichiometry were calculated as given by oligomer interaction experiments (Supplementary Fig. 14). F A;IE Dr values of 14.6 ± 3.6, 19.2 ± 4.2 µm2 s−1 and 108 ± 18 µm2 s−1 were calculated E¼ ð5Þ for PSMα3, LL-37, and dPSMα3, respectively, with N = 0.053 and N = 0.11, for F D þ F A;SE PSMα3 and LL-37, respectively. These data indicate that PSMα3 and LL-37 exhibit a certain degree of oligomerization, despite no aggregates were detected in TEM F D þ F A;IE images of peptides alone (not shown), and the data indicates that they bind to their S¼ ð6Þ F D þ F A;IE þ F A;DE targets in the monomeric form (Figs. 2, 6 and Supplementary Fig. 7). Both data acquisition and analysis were performed on the commercially where FD is the fluorescence intensity in the donor (green) channel, FA,IE is the available software SymphoTime64 version 2.3 (PicoQuant, Berlin, Germany). For fluorescence intensity in the acceptor (red) channel through indirect excitation and the oligomeric and fibrillar samples, a lower intensity threshold of 27 photons in FA,DE is the fluorescence intensity in the acceptor (red) channel after direct the green dye autocorrelation analysis was applied to filter out the low intensity excitation by PIE pulse. signal arising from the monomeric αS events generated upon dilution-induced Stoichiometry values were corrected for the difference in mean intensity disaggregation of the aggregated samples. This threshold was calculated as three between the monomeric αS and peptide bursts, obtained from monomeric αS-only times the mean intensity of monomeric αS obtained from the analysis of a sample and peptide-only measurements; the obtained mean intensity ratio Imean,αS: of pure αS monomers. In addition, an upper intensity threshold was applied to Imean,peptide was found to be 0.77. Stoichiometry distributions were fitted to a log- auto-correlation and cross-correlation analysis to filter out any possible artifacts normal distribution to obtain the mean stoichiometry value for each measurement. such as dust particles or aggregate clusters (even though these events were very The number of bound peptides per aggregate (NP) was then estimated by scarce): 500 photons for monomer, type A* and type B* oligomers and 1500 multiplying the mean stoichiometry value previously obtained by the mean number photons for sonicated fibrils. Data on the red channel corresponding to the peptide of αS monomers present on each aggregate as calculated empirically from the fluorescence signal was intensity-filtered with a lower intensity threshold in analogy molecular brightness in FCCS experiments. The free peptide concentration (CP, to the green channel owing to the fact that the peptide can also exist as self- Free) and NP obtained by either FCCS or single-burst stoichiometry analysis were assembled species. The reference signal was that of the monomer-only used for calculating the binding curves as described by Kruger and coworkers32. To dPSMα3 sample. The PIE excitation scheme together with the TSCPC acquisition obtain the dissociation constant KD and the maximum specific binding sites Nmax, enabled the application of a lifetime-weighted filter, which aided removal of the resulting binding curves were fitted to the following specific binding model with background and spectral cross-talk. n identical and independent binding sites: The corrected auto-correlations of the green and the red channel (Gi) were N max X given by Y¼ ð7Þ ðK D þ XÞ hF i ðt ÞF i ðt þ τ Þi The binding curves and binding parameters obtained from either FCCS or G i ðτ Þ ¼ 1 ð1Þ Fi2 single-burst stoichiometry analysis were compared (Supplementary Fig. 7) and found to be remarkably similar, which validates the analysis. OriginPro9.1 software where Fi (t) denotes the fluorescence intensity either the green or the red channel, τ was used for graphical data representation and statistical analysis. is the correlation time and the angled brackets indicate a time average over the acquisition time. The cross-correlation (Gx) between the green and the red channel was given by Aggregation kinetics. αS amyloid aggregation was monitored in a 96 wells plate (non-treated) (Sarstedt, Germany) containing Teflon polyballs (1/8′′ diameter) hF g ðt ÞF r ðt þ τ Þi (Polysciences Europe GmbH, Eppelheim, Germany) as described by Pujols and Gx ðτ Þ ¼ 1 ð2Þ coworkers9. Each well contained 150 μL solutions of 70 μM αS in PBS buffer with 40 hF g ihF r i μM thioflavin-T and the corresponding concentration of peptide. Plates were incu- Auto-correlation curves for both the green and red channel were fitted with a 2 bated at 37 °C, 100 rpm in an orbital culture shaker Max-Q 4000 (Thermo Fisher diffusion-component model accounting for residual monomeric αS and bound and Scientific, Waltham, USA). Aggregation was analyzed every 2 h using a Victor3.0 unbound peptide, respectively, using the following equation: Multilabel Reader Software-PerkinElmer 2030. (PerkinElmer, Waltham, USA). End- point measurements were performed after 32 h of incubation. Fluorescence intensity f f was measured in triplicate by exciting with a 430–450 nm filter and collecting the Gi ðτ Þ ¼ G0i   i;1 ffiþ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi   i;2 ffi; qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi emission with a 480–510 nm filter. The resulting kinetics were normalized to the τ τ τ ð3Þ 1þτ 1 þ κ2 xτ 1þτ 1 þ κ2 xττ maximum fluorescence of the αS control (untreated). Di;1 Di;1 Di;2 Di;2 whereG0i is the correlation amplitude at correlation time 0, fi,1 and fi,2 denote the Atomic force microscopy. αS samples were diluted to a protein concentration of fractional amplitudes of the monomeric and aggregated αS for the green channel 0.1–0.5 µM and deposited on cleaved Muscovite Mica V-5 (Electron Microscopy (where i = g) and the bound and unbound peptide for the red channel (where i = Sciences; Hatfield, Pensilvania, USA). Slides were washed with double distilled r) and κ2 is the structure parameter of the focal volume. The same applies for the water and allowed to dry before imaging acquisition on a Bruker Multimode 8 diffusion terms τ Di;1 and τ Di;2 . No correlated blinking is expected when multiple (Bruker; Billerica, USA) using a FMG01 gold probe (NT-MDT Spectrum Instru- dyes are present on one particle as it is our case and therefore a blinking term was ments Ltd., Russia) in intermittent-contact mode in air. Images were processed not included. using Gwyddion (version 2.48) and the width measurements were corrected for the Cross-correlation amplitudes were fitted with a 1-component simple diffusion tip shape and size (10 nm). model since only one diffusion coefficient is expected for the interacting species (Supplementary Fig. 13) using the following equation: Polyacrylamide gel electrophoresis. For monomeric and fibrillar αS species, 5 μg 1 protein in denaturing loading buffer were loaded onto a 15% acrylamide SDS- Gx ðτ Þ ¼ G0x  qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi : PAGE. For type A* and type B*, 2 μg protein in non-denaturing buffer were loaded ð4Þ 1 þ ττ 1 þ κ2 xττ onto a 15% native-PAGE. The only difference between the denaturing and non- D;x D;x denaturing gel electrophoresis was the absence of SDS in the sample, gel and buffer With the corrected green dye autocorrelation function and the mean intensity of the native PAGE. No boiling step was included in either case. Unprocessed scans of monomeric αS, the average aggregate particle number (NAg) for each αS of the gels are presented in the Source Data file. aggregated sample was estimated as N Ag ¼ G10 . The average particle number for the peptide was calculated in g Dynamic light scattering. Estimations of the hydrodynamic radius of αS species analogy to that of αS. The peptide concentration was calculated as were made on a DynaPro NanoStar (Wyatt, USA) equipped with a Peltier tem- CP ¼ V NxN r ;where Nr is the average number of particles in the red confocal perature control. Protein samples were prepared at a 25 µM concentration in fil- eff ;r A tered PBS (0.22 μm cellulose acetate syringe filters). DLS measurements were volume, Veff,r is the red focal volume and NA is the Avogadro number. The cross- performed at 25 °C at a fixed angle of 90 °. Twenty acquisitions per measurement correlation amplitudes, dual-laser focal volume, Veff, x, and peptide concentrations, were collected using a 2 s acquisition time. An average of 10 measurements were CP, were used for calculating the number of peptides bound to each αS species (NP) performed for the statistical size analysis. Data was analyzed using the Dynamics and the free peptide concentration (CP, Free) as described by Kruger and coworkers32. software (version 6.12.03). For single-burst FRET and stoichiometry analysis, an acceptor (red dye) direct excitation lower threshold based on the mean intensity of the time trace (IA,mean + Fourier-Transform infrared (FT-IR) spectroscopy. αS aggregates species were 2 × σ) was used to filter out those events without an active acceptor molecule. To transferred to deuterated buffer, by either centrifugation/resuspension or filtering further select those events arising from αS aggregates, a burst selection intensity cycles, to a final protein concentration of ca. 4 mg/ml. Samples were then deposited threshold of 100 photons was used. In the FRET analysis, experimentally between two CaF2 polished windows separated by a PTFE Spacer (Harrick Sci- determined correction factors were applied: spectral cross-talk α was 0.004, direct entific Products Inc., USA). Spectra were collected in transmission mode at room excitation β was 0.0305 and detection efficiency γ was 0.517. Burst-wise FRET temperature using a VERTEX 70 FTIR Spectrometer (Bruker, USA) equipped with 12 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24039-2 ARTICLE a cryogenic MCT detector cooled in liquid nitrogen. IR spectra were processed and stained using Hoescht 33342 at a concentration of 0.5 μg/mL for 5 min. Images of analyzed using standard routines in OPUS version 6.5 (Bruker, USA), RAMOPN intracellular αS were obtained under UV light using double excitation at 488 nm (NRC, National Research Council of Canada) and Spectra-Calc-Arithmetic© ver- and 350 nm lasers, for AF488 and Hoescht, and the emission was collected at sion A2.21 (Galactic Inc., USA). 515–560 nm and 405 nm, respectively. Images were processed and analyzed with Image J (version 1.52p) A dot blot assay was performed as a control to discard epitope-masking artifacts ANS fluorescence spectroscopy. 10 μM of each αS sample was incubated with caused by the potential primary or secondary antibodies binding to the peptide. 2 500 μM 8-anilo-1-naphtalene-sulfonic acid (ANS) in PBS for 45 min before μL of type B* oligomers untreated and treated for 15 min with an equimolar recording the spectra. The extinction coefficient of ANS at 350 nm was assumed to concentration of PSMα3 were spotted onto a nitrocellulose membrane and allowed be 5000 cm−1 M−1. In order to monitor ANS binding to the each αS species, to dry. Antibody incubations were performed as described for the cellular assay. No samples were excited at 350 nm and their emission spectra were recorded from 400 significant differences in the signals of oligomers assayed in the presence or the to 650 nm in 1-nm steps. Spectra were collected at room temperature in a Cary absence of PSMα3 were detected (data not shown). Eclipse Fluorescence Spectrophotometer (Varian, Palo Alto, California, United States) with slit-widths of 5/5 nm. An averaging time of 100 ms was used. Redesign of PSMα3 variants. To guide and assist the design of PSMα3 peptide variants some computational tools were employed. Briefly, AGADIR was used to Transmission electron microscopy. For electron microscopy analyses, end-point predict the helical propensity of the peptide variants based on the helix/coil aggregated samples were sonicated for 5 min at minimum intensity in an ultrasonic transition theory26. FoldX allows a rapid evaluation of the effect of mutations on bath (VWR ultrasonic cleaner) and placed onto carbon-coated copper grids and the stability, folding and dynamics of proteins38. We exploited it to evaluate if the allowed to adsorb for 5 min. The grids were then washed with distilled water and designed mutations may compromise the stability of the α-helix specially regarding negative stained with 2% (w/v) uranyl acetate for 1 min. Finally, the excess of extensive redesign or those involving electrostatic repulsions. The peptides mean uranyl acetate was absorbed using ashless filter paper and the grids were left to air- hydrophobicity (H), and their helical hydrophobic moment (μH), a measure of the dry for 15 min. A TEM JEM-1400 Software-Gatan Digital Micrograph 1.8 (JEOL, amphiphilicity of a helix, were calculated according to Eisenberg and coworkers52. Peabody, USA) microscope was used operating at an accelerating voltage of 120 kV. The more representative images of each grid were selected. Images were processed and analyzed with Image J (version 1.52p) Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Sedimentation assay. αS aggregation was performed as previously described. End- point samples were subjected to ultracentrifugation at 100,000 g for 30 min at 20 °C Data availability in a SW55Ti Beckman rotor in order to fractionate soluble and fibrillar species. αS All the data presented in this study are available in the paper or in the Supplementary concentration in the soluble fraction was determined by measuring the absorbance Information. Further raw data (i.e., Time traces of the time-resolved fluorescence at 280 nm (ε = 5960 M−1 cm−1). Soluble fractions in denaturing loading buffer spectroscopy) supporting the findings of this study are available from the corresponding were boiled for 5 min and loaded onto a 15% acrylamide SDS-PAGE. Proteins were author upon reasonable request. The screened database of human peptides was obtained revealed with BlueSafe (NZYTech, Portugal). Unprocessed scans of the gels are from the EROP-Moscow oligopeptide database (http://erop.inbi.ras.ru/)39. Source data presented in the Source Data file. are provided with this paper. Isolation of low molecular weight aggregates generated during αS in vitro aggregation. αS aggregation was performed as previously described in absence and Received: 5 June 2020; Accepted: 27 May 2021; presence of PSMα3. Aliquots at the analyzed time point were taken and flash frozen in liquid nitrogen and stored at −80 °C until assayed. To fractionate our sample into insoluble species, low-molecular weight aggregates and monomers, we adapted the centrifugation-based protocol developed by Kumar and coworkers51. αS pre- parations were subjected to ultracentrifugation at 100,000 g for 30 min at 20 °C in a SW55Ti Beckman rotor in order to isolate larger fibrillar species. The soluble References fraction (100 μl) containing low molecular weight aggregates and monomeric αS 1. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. & was then filtrated through 100 kDa centrifuge filters (Merck, Darmstadt, Germany) Goedert, M. Alpha-synuclein in Lewy bodies. Nature 388, 839–840 (1997). in order to fractionate these two species. The filtrated samples contain monomeric 2. Winner, B. et al. In vivo demonstration that alpha-synuclein oligomers are or -theoretically- dimeric αS. The excess of monomeric species retained in the filter toxic. Proc. Natl Acad. Sci. 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Structural characteristics of alpha-synuclein oligomers. Int Rev. Cell Mol. Biol. 329, 79–143 (2017). 25. Marinelli, P., Pallares, I., Navarro, S. & Ventura, S. Dissecting the contribution Acknowledgements of Staphylococcus aureus alpha-phenol-soluble modulins to biofilm amyloid This work was funded by the Spanish Ministry of Economy and Competitiveness structure. Sci. Rep. 6, 34552 (2016). (MINECO) BIO2016-78310-R and BIO2017-91475-EXP to S.V, by the Ministry of Sci- 26. Munoz, V. & Serrano, L. Elucidating the folding problem of helical peptides ence and Innovation (MICINN) PID2019-105017RB-I00 to S.V, by ICREA, ICREA- using empirical parameters. Nat. Struct. Biol. 1, 399–409 (1994). Academia 2015 to S.V. N.C. was supported by MINECO RYC-2012-12068, MINECO/ 27. Bacia, K. & Schwille, P. Practical guidelines for dual-color fluorescence cross- FEDER, EU BFU2015-64119-P and MICIU/FEDER, EU PGC2018-096335-B-100. J.S. correlation spectroscopy. Nat. Protoc. 2, 2842–2856 (2007). was supported by MICINN via a doctoral grant (FPU17/01157). We thank the members 28. Schwille, P., Meyer-Almes, F. J. & Rigler, R. Dual-color fluorescence cross- of the Microscopy Services of the UAB for their assistance, Dr. Salvador Bartolomé from correlation spectroscopy for multicomponent diffusional analysis in solution. the Laboratori de Luminiscència i Espectroscòpia de Biomolècules, UAB, and Dr. Biophys. J. 72, 1878–1886 (1997). Evangelos Sisamakis, PicoQuant, for insightful discussions. 29. Tosatto, L. et al. Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants. Sci. Rep. 5, Author contributions 16696 (2015). S.V. conceived the project. J.S., I.P., and S.V. designed all the peptide sequences. J.S., S.P- 30. Nath, S., Meuvis, J., Hendrix, J., Carl, S. A. & Engelborghs, Y. Early D., and J.P. performed in vitro experiments. J.S. and S.N. designed, conducted and aggregation steps in alpha-synuclein as measured by FCS and FRET: evidence analyzed cellular experiments. P.G. and N.C. performed and analyzed time-resolved for a contagious conformational change. Biophys. J. 98, 1302–1311 (2010). fluorescence spectroscopy experiments. J.S., I.P., and S.V. analyzed the data. J.S., P.G., N. 31. Whiten, D. R. et al. Single-molecule characterization of the interactions C., I.P., and S.V. prepared the paper with contributions from all the authors. between extracellular chaperones and toxic alpha-synuclein oligomers. Cell Rep. 23, 3492–3500 (2018). 32. Kruger, D., Ebenhan, J., Werner, S. & Bacia, K. Measuring protein binding to Competing interests lipid vesicles by fluorescence cross-correlation spectroscopy. Biophys. J. 113, Authors have submitted a patent application on the bases of the here presented research. 1311–1320 (2017). It is protected the use of LL-37 for therapy and PSMα3 for therapy and diagnosis. 33. Lashuel, H. A., Hartley, D., Petre, B. M., Walz, T. & Lansbury, P. T. Jr. Inventors: S.V., I.P., J.S., N.C., P.J.G. Title: inhibitors of alpha synuclein aggregation and Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature uses thereof. Property: Universidad Autónoma de Barcelona. Universidad de Zaragoza. 418, 291 (2002). Request number: EP20382658. Priority date and Country: 22-07-2020, EU. All other 34. Lashuel, H. A. et al. Alpha-synuclein, especially the Parkinson’s disease- authors declare no competing interests. associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089–1102 (2002). 35. Perni, M. et al. Multistep inhibition of alpha-synuclein aggregation and Additional information Supplementary information The online version contains supplementary material toxicity in vitro and in vivo by trodusquemine. ACS Chem. Biol. 13, available at https://doi.org/10.1038/s41467-021-24039-2. 2308–2319 (2018). 36. Ludtmann, M. H. R. et al. alpha-synuclein oligomers interact with ATP Correspondence and requests for materials should be addressed to N.C., I.P. or S.V. synthase and open the permeability transition pore in Parkinson’s disease. Nat. Commun. 9, 2293 (2018). Peer review information nature communications thanks Hilal Lashuel and other, 37. Stothard, P. The sequence manipulation suite: JavaScript programs for anonymous, reviewers for their contributions to the peer review of this work. Peer review analyzing and formatting protein and DNA sequences. Biotechniques 28, reports are available. 1102–1104 (2000). 38. Schymkowitz, J. et al. The FoldX web server: an online force field. Nucleic Reprints and permission information is available at http://www.nature.com/reprints Acids Res 33, W382–W388 (2005). 39. Zamyatnin, A. A., Borchikov, A. S., Vladimirov, M. G. & Voronina, O. L. The Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in EROP-Moscow oligopeptide database. Nucleic Acids Res. 34, D261–D266 (2006). published maps and institutional affiliations. 40. Durr, U. H., Sudheendra, U. S. & Ramamoorthy, A. L. L.-37 the only human member of the cathelicidin family of antimicrobial peptides. Biochim Biophys. Acta 1758, 1408–1425 (2006). 41. Brundin, P. & Melki, R. Prying into the prion hypothesis for Parkinson’s Open Access This article is licensed under a Creative Commons disease. J. Neurosci. 37, 9808–9818 (2017). Attribution 4.0 International License, which permits use, sharing, 42. Braak, H., Rub, U., Gai, W. P., Del & Tredici, K. Idiopathic Parkinson’s adaptation, distribution and reproduction in any medium or format, as long as you give disease: possible routes by which vulnerable neuronal types may be subject to appropriate credit to the original author(s) and the source, provide a link to the Creative neuroinvasion by an unknown pathogen. J. Neural Transm. (Vienna) 110, Commons license, and indicate if changes were made. The images or other third party 517–536 (2003). material in this article are included in the article’s Creative Commons license, unless 43. Lionnet, A. et al. Does Parkinson’s disease start in the gut? Acta Neuropathol. indicated otherwise in a credit line to the material. If material is not included in the 135, 1–12 (2018). article’s Creative Commons license and your intended use is not permitted by statutory 44. Kastin AJ. Handbook of biologically active peptides. (Elsevier, 2013). regulation or exceeds the permitted use, you will need to obtain permission directly from 45. Burton, M. F. & Steel, P. G. The chemistry and biology of LL-37. Nat. Prod. the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Rep. 26, 1572–1584 (2009). licenses/by/4.0/. 46. De Lorenzi, E. et al. Evidence that the human innate immune peptide LL-37 may be a binding partner of amyloid-beta and inhibitor of fibril assembly. J. Alzheimers Dis. 59, 1213–1226 (2017). © The Author(s) 2021 14 NATURE COMMUNICATIONS | (2021)12:3752 | https://doi.org/10.1038/s41467-021-24039-2 | www.nature.com/naturecommunications