Eosinophil Cationic Protein Aggregation - American Chemical Society

Biomacromolecules 2010, 11, 1983–1990

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Eosinophil Cationic Protein Aggregation: Identification of an N-Terminus Amyloid Prone Region Marc Torrent,* Francesco Odorizzi,† M. Victo`ria Nogue´s, and Ester Boix Departament de Bioquı´mica i Biologia Molecular, Facultat de Biocie`ncies, Universitat Auto`noma de Barcelona, 08193-Bellaterra, Spain Received March 26, 2010; Revised Manuscript Received June 10, 2010

Eosinophil cationic protein (ECP) is an antimicrobial protein belonging to the superfamily of RNase A. ECP exhibits a broad spectrum of action against bacteria and, at higher concentrations, displays cytotoxic activity to eukaryotic cells. Recently, a powerful aggregation activity for lipid vesicles and for the gram-negative E. coli specie has also been related to the protein toxicity. Here we present the amyloid-like aggregation capacity of ECP. This is the first report of amyloid aggregation in a native nonengineered ribonuclease. The ECP aggregates are able to bind the amyloid-diagnostic dyes Thioflavin T and Congo Red and display a protofibril morphology when observed under electronic microscopy. We have also identified an N-terminus hydrophobic patch (residues 8-16) that is required for the amyloid aggregation process. A single substitution, I13A, breaks the aggregation prone sequence and abolishes the amyloid aggregation ability. Moreover, the corresponding R1N19 peptide is able to reproduce the protein amyloid-like aggregation behavior. The results may provide new clues on the protein antimicrobial mechanism and its toxicity to the host tissues in inflammation processes.

Introduction Antimicrobial proteins and peptides (AMPPs) are ubiquitously found in nature. These proteins and peptides usually share common characteristics: (a) a net positive charge, (b) a high content of hydrophobic amino acids (about 50%), (c) a high sequence variability, and (d) the adoption of a defined secondary structure upon membrane interaction.1,2 In general, AMPPs present a broad antimicrobial activity against both gram-negative and gram-positive bacteria, and some of them also display antimicrobial activity against fungi and parasites.3,4 Different AMPPs exert their cytotoxicity by a similar mechanism that involves the binding and permeation of the cell membrane.5,6 In addition to their action on the cell membrane, the mechanism by which AMPPs exert their activity seems to be modulated by other properties of the pathogen, such as the bacteria wall composition and the presence of some intracellular targets.7 Recently, it has been described that some AMPPs, such as dermaseptins, temporins, and lactoferrin, can undergo a process of amyloid fiber formation.8-10 Moreover, lysozyme has also been largely characterized and used as an amyloid protein model.11,12 Amyloid aggregates are reported to be toxic to eukaryotic cells; the oligomeric intermediates of mature amyloid fibrils being the principal toxic agent.13,14 No amyloid-like aggregates have been described up to date for any native ribonuclease (RNase). However, a characteristic 3D domain swapped oligomerization behavior has been thoroughly described for RNase A.15-17 Moreover, these domainswapped dimers and oligomers display unique biological and enzymatic activities.18 Experimental results on an engineered variant of RNase A demonstrate that domain-swapped structures can serve as a good scaffold for amyloid-like structures.19 * To whom correspondence should be addressed. Phone: 34 93 5811707. Fax: 34 93 5811264. E-mail: [email protected]. † Present address: Department of Biotechnology and Biosciences, University of Milan-Bicocca, 20126 Milan, Italy.

Eosinophil cationic protein (ECP) is a member of the RNase A superfamily. The protein is stored in the secondary granules of eosinophils and has been shown to possess antimicrobial activity against bacteria, parasites, and fungi and also has moderate activity against the syncitial respiratory virus (SRV).20,21 Its antimicrobial capacities have been related to its ability to interact and disrupt negatively charged membranes through a carpet-like mechanism,22 triggering lipid vesicles aggregation before inducing their content release.23 It has also been suggested that ECP’s antibacterial action against the gramnegative bacteria E. coli is modulated by its outer membrane binding capacity and high ability to aggregate cells.24 This aggregation behavior has been related to a hydrophobic patch present at the surface of the molecule.25 Eosinophils are involved in several diseases, like idiopathic hypereosinophilic syndrome (HIS)26 and eosinophilic meningitis,27 and can induce, when injected in animal models, the cerebellar Purkinje cell degeneration, known as Gordon phenomena.28 Increased persisting levels of blood circulating eosinophils may cause cerebral infarction, axonal neuropathy, and dementia,29 and these symptoms have been related to eosinophil cytotoxic proteins.30 Additionally, recent studies on ECP effects on eukaryotic cell lines have described how the protein induces apoptosis after its binding and aggregation to the cell surface.31 Interestingly, dementia and amyloid deposits have been linked to bacterial infections. Aβ deposition and tau phosphorylation processes are promoted by bacteria debris or lipopolysaccharide (LPS) presence, inducing brain barrier permeabilization and atrophy.32 Moreover, eosinophil infiltration is present in Alzheimer’s disease33 and amylin modulates the inflammatory activity of eosinophils.34 In the present work, we report the ability of ECP to form amyloid protofibrils under specific conditions and a key region that is required for amyloid fibril formation has been identified.

10.1021/bm100334u  2010 American Chemical Society Published on Web 07/12/2010

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Biomacromolecules, Vol. 11, No. 8, 2010

Experimental Section Materials. Thioflavin T (Th-T), Congo Red, and RNase A type XII-A were purchased from Sigma-Aldrich (St. Louis, MO). Protein and Peptide Synthesis and Purification. R1N19 peptide, corresponding to the first 19 residues of ECP sequence, was purchased from NEOmps (Strasbourg, France) prepared using solid-phase peptide synthesis and purified by HPLC using a 0.1% (v/v) HCl/acetonitrile gradient. I13A ECP mutation was incorporated using the Quick-change mutagenesis kit from Invitrogen (Carslbad, CA). Recombinant ECP, I13A mutant, and EDN were obtained as previously described.35,36 I13A ECP mutant was sequenced before expression and protein identity was checked by MALDI-TOF mass spectrometry. Fibril Formation. Protein and peptide fibrils were formed after incubation of 50 µL of 10 mg/mL protein solution or peptide solution in 100 mM glycine-HCl, pH 3 buffer, for 2 weeks at 37 °C. To test the pH effect on amyloid fibril formation, two additional buffers were used under the same conditions: 100 mM sodium acetate, pH 5, and 100 mM sodium phosphate, pH 7. Binding to Amyloid-Diagnostic Dyes. Th-T binding assay was carried out using a Varian Cary Eclipse fluorescence spectrophotometer (Palo Alto, CA). A total of 5 µL of 10 mg/mL protein solution or peptide solution were diluted in 45 µL of 10 mM sodium phosphate, 100 mM NaCl, and pH 7.5 buffer containing 40 µM Th-T. Fluorescence data were collected after 5 min to ensure that thermal equilibrium had been achieved. Excitation wavelength was set to 440 nm with a 2.5 nm slit width and the fluorescence emission spectra were recorded from 460 to 570 nm with a 5 nm slit width. Th-T binding kinetics was followed for 2 weeks, and the data corresponding to an emission wavelength of 485 nm were fitted to the following equation37 using the Origin software (Northampton, MA).

f)

F{exp[(1 + F)kt] - 1} {1 + F exp[(1 + F)kt]}

In this equation, f represents the fraction of fibrillar material in the system, k ) kea (where ke represents the elongation rate and a is the concentration of the monomer), and F is the dimensionless value that describes the ratio between the seeding rate (kn) and k. Binding of ECP to Congo red was tested using a Varian CARY100 spectrophotometer (Palo Alto, CA) to perform the spectroscopic band-shift assay.38 A total of 5 µL of 10 mg/mL protein or peptide solution was diluted in 45 µL of 5 mM sodium phosphate, 150 mM NaCl, and pH 7.0 buffer containing 15 µM Congo red. Samples were maintained 5 min at 25 °C before analysis. Absorption spectra for negative control solutions of dye in the absence of protein and of protein in the absence of dye were collected to subtract the scattering contribution to the spectra. Transmission Electron Microscopy (TEM). Samples were prepared as described in the fibril formation section, diluted 10-fold with the corresponding buffer, and, for each sample, 5-10 µL were placed on carbon-coated copper grids. After 5 min, the grids were washed with the corresponding buffer and stained using 2% (w/v) uranyl acetate for 5 min before analysis using a JEOL JEM 2011 (Tokio, Japan) transmission electron microscope. Circular Dichroism. CD experiments were performed with a Jasco J-715 spectropolarimeter (Maryland, MD). Measurements of the farUV CD spectra (190-260 nm) were made with a 2 mm path-length quartz cuvette. Samples (10 µL) obtained following the fibril formation procedure described above were diluted to a final volume of 500 µL in 5 mM sodium phosphate, pH 7. Spectra were recorded at 25 °C. Nontreated protein and peptide samples were analyzed under the same conditions. In each case, the resulting spectrum was the average of 20 scans. Percentage of secondary structure was estimated using the Jasco software, according to the method described by Yang et al.39 and by Selcon software.40

Torrent et al. Fourier Transformed Infrared Spectroscopy (FTIR). For FTIR measurements, lyophilized proteins or peptides were dissolved in 10 mM HEPES and 100 mM NaCl pH 7.4 buffer. Each spectrum was obtained from 100 independent scans, with a resolution of 2 cm-1 within the 1800-1400 cm-1 range. The data were acquired using the a Bruker Tensor 27 FT-IR spectrometer (Ettlingen, Germany) with a Golden Gate MKII ATR accessory and normalized using the OPUS MIR Tensor 27 software. The spectra were deconvoluted using an FHHH of 15 cm-1 and a k factor of 1.8. To measure the relative areas of band components, deconvoluted spectra were curve-fitted by means of a least-squares iterative program. Fluorescence Microscopy. Samples containing fibrils were centrifuged at 13000 × g, the pellet was incubated 5 min with 50 µL of 10 mM sodium phosphate, 100 mM NaCl, and pH 7.5 buffer containing 40 µM Th-T, and then washed two times with the same buffer without Th-T to discard nonbound soluble dye. Samples were observed under a Leica DMBR fluorescence microscope (Wetzler, Germany) using a FITC/GFP filter using a 40× objective.

Results Analysis of the Aggregation Pattern of RNases. ECP and EDN are two eosinophil proteins belonging to the RNase A superfamily. Both RNases, together with RNase A, the reference family member, have been analyzed using the Aggrescan server41 (Figure 1A). Aggrescan is based on an amino acid aggregation-propensity scale derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm can identify protein segments involved in the aggregation of disease-related proteins and predict the effect of amino acid mutations. Aggrescan prediction results show an aggregation prone region in the ECP N-terminus, comprising the residues 8-16, which is not shared with either EDN or RNase A. The protein surface chemical properties of ECP have also been analyzed (Figure 1B) showing an exposed hydrophobic patch, which corresponds to the amyloid prone region identified by Aggrescan. On the contrary, EDN and RNase A do not show any surface exposed aggregation patch. The other regions detected by the Aggrescan software correspond to the highly buried folding core of the protein and therefore are not accessible to direct fibril formation. ECP Amyloid-Like Aggregation. ECP and EDN samples (10 mg/mL in 100 mM glycine buffer, pH 3) were maintained at 37 °C and assayed for amyloid aggregates detection at different time intervals. The results obtained (Figure 2A) for 2 weeks treated-ECP using Th-T binding show a 6-fold increase in the fluorescence emission at 482 nm with respect to the untreated sample. The aggregation kinetics was also followed using the Th-T dye. Figure 2B shows that amyloid aggregation has a lag time of 3.1 ( 0.3 days, a half aggregation time (t1/2) of 4.5 ( 0.4 days, and the aggregation process was almost complete in 6.2 ( 0.3 days. These values have been calculated by fitting the data to the model described by Sabate´ et al.37 (Figure 2B). Only minor, nonsignificant changes in the emission spectrum were detected when Th-T was added to treated EDN or RNase A at the same conditions, and no amyloid aggregation has been detected even at the end of the assay (see Supporting Information, Figure S1). We analyzed the secondary structure pattern of the protein samples using far-UV circular dichroism spectroscopy (CD; Figure 2C). Freshly dissolved ECP displayed the expected CD spectraforatypicalribonucleasefolding,asreportedpreviously.42,43 After 2 weeks of incubation, a significant increase (7%) in β-sheet structure has been detected in the ECP sample. No

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Figure 1. Analysis of ECP, EDN, and RNase A primary and three-dimensional structure. (A) Results of Aggrescan prediction for RNase A (green), EDN (purple), and ECP (red). NHSA values represent the Normalized Hot-Spot Area per residue.41 (B) Sequence alignment of ECP, EDN and RNase A. Secondary structure elements of RNase A are depicted at the top and the “poly-serine” loop is marked in blue. The sequence alignment was performed using the ESPript program. Molecular representations were drawn using Pymol (DeLano Scientific). Ribbon and molecular surface representation of the three-dimensional structures of ECP (1DYT55), EDN (1GQV56), and RNase A (7RSA57) are shown. Ribbons are colored from the N- to the C-terminus and the ribonuclease catalytic site is marked by a green circle. In the molecular surface representation, hydrophobic residues are labeled in gray, cationic residues in blue, anionic residues in red, cysteine residues in yellow, proline residues in orange and noncharged polar residues in cyan.

appreciable changes have been detected in the EDN and RNase A samples (data not shown). We have further investigated the properties of the aggregated ECP sample by measuring its binding to Congo Red. Congo Red is another amyloid-diagnostic dye, which has an absorbance maximum at 490 nm that shifts to red upon binding to amyloidlike material. In accordance to the results obtained by Th-T binding, no shift in the Congo Red spectrum has been detected after the

addition of incubated EDN or RNase A to the dye, thus, reporting that no amyloid fibrils were formed in both cases (see Supporting Information, Figure S1). However, ECP showed a detectable shift (6 nm between maxima) in the Congo Red spectrum, displaying also the typical shoulder on the right part of the spectrum, indicating the presence of amyloid-like aggregates (Figure 2D). The same protein solutions were analyzed by transmission electron microscopy (TEM) (Figure 2E). In agreement with

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Figure 2. Characterization of amyloid aggregation process of ECP. (A) Fluorescence spectra of Th-T in absence (squares) and in presence (circles) of treated-ECP (10 mg/mL protein solution in 100 mM glycine buffer, pH 3, maintained at 37 °C for 2 weeks). (B) Th-T binding kinetics for ECP. The process has been monitored during 2 weeks, the fluorescence at 485 nm was recorded and plotted as a function of time. (C) Circular dichroism spectrum of ECP at t ) 0 (continuous line) and ECP at t ) 15 days (dotted line) at 0.20 mg/mL concentration in 5 mM sodium phosphate buffer, pH 7. (D) Spectrum of Congo Red binding assay in absence (continuous line) and in presence (dotted line) of treated-ECP. See Experimental Section for additional technical information. (E) TEM micrograph of ECP aggregates; scale bar is 1 µm. A magnification (10×) of the aggregates is shown on the top right corner. (F) Fluorescence micrograph taken from ECP aggregates stained with Th-T, on a Leica DMBR fluorescence microscope using a FITC/GFP filter and a 40× objective.

the dye-binding results, fibrillar-like material has been detected in the ECP sample. In this case, short, thin protofibrils of about 150 nm in length and 10 nm in width have been observed. This type of assembly remained without apparent change for weeks, and no higher-order association into fibrils was observed in these conditions. The reduced increase in Congo Red binding and β-sheet structure may be explained by the failure of ECP to form large mature fibrils. On the contrary, no fibrillar material has been detected in the EDN or RNase A samples, nor in freshly prepared ECP samples (data not shown). The formation of amyloid aggregates was also checked by fluorescence microscopy to ensure that Th-T binding was specific for ECP protofibrils (Figure 2F). The results show that Th-T binds specifically on ECP aggregates giving fluorescence. No fluorescence could be observed for EDN or RNase A samples (data not shown).

These results confirm that ECP can undergo, under specific conditions, an amyloid-like aggregation process that is not shared by the other homologous ribonucleases EDN or RNase A. Effect of pH on ECP Protofibril Formation. To study the pH effect on amyloid-like aggregation, ECP was incubated at two additional pH values: 5 and 7. It can be observed that ECP is able to form protofibrils only at pH 3. Also, it is interesting to note that, around pH 7, ECP forms amorphous aggregates that do not bind Th-T nor Congo Red and appear unstructured under microscope observation (Figure 3). The pH 7 aggregates begin to precipitate almost instantly after sample preparation, thus, pointing out that at these conditions disordered and unstructured precipitates are formed at high aggregation rate. To further explain this behavior, both the protonation state of amino acid residues and the pH dependence of protein stability must be taken into account. H15 is the only amino acid



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Figure 3. pH effect on ECP amyloid protofibril formation. ECP samples at pH 3, 5, and 7 were maintained for 2 weeks at 37 °C in the corresponding buffer as indicated in the Experimental Section and analyzed by TEM (scale bar 0.2 µm) and by Th-T binding (circles). Th-T control samples are represented by squares. Note that in each graphic the best scale on the ordenate is used.

Figure 4. Plot of H15 titration curve and pH dependent protein stability. Titration curve (squares) was constructed using the H++ server44 using the three-dimensional structure 1DYT.55 Data of protein stability (triangles) was obtained from Nikolovski et al.43

belonging to the proposed amyloid region that shows side chain protonation changes depending on the pH. Figure 4 shows the plot of the pH protein stability dependence43 versus the H15 protonated state.44 We can observe that the two curves cross near pH 3, the most favorable pH for protein aggregation. Thus, the protonation state of H15 may be critical during the partial unfolding of the protein at acidic pH, which precedes the aggregation. Interestingly, the computed pKa of the EDN H15 and RNase A H12 reveals values of 6.5 and 6.2, respectively, in contrast with the 4.0 value for ECP. These results show that the high hydrophobic environment of this residue in ECP decreases drastically its pKa value. H15 protonation would lead to aggregation by losing its positive charge and thus reducing the electrostatic repulsion that would inhibit the aggregation process at higher pH values (Figure 3). Analysis of the Amyloid Prone Region. Analysis of the protein sequence using Aggrescan identified a single amino acid (Ile13) at ECP N-terminus, whose substitution to alanine disrupts

the 8-16 aggregation prone region. The involvement of the protein N-terminus in the amyloid aggregation process was confirmed by site directed mutagenesis and peptide synthesis. I13A ECP mutant has been constructed to evaluate the role of the 8-16 hydrophobic patch in the ECP protofibril formation. I13A ECP mutant (10 mg/mL in 100 mM glycine buffer, pH 3) maintained at 37 °C for 2 weeks showed no Th-T nor Congo Red binding (Figure 5). These results indicate that the Nterminus region detected by Aggrescan is crucial for the ECP aggregation behavior, as alteration of the region structure abolishes the protofibril formation capacity of ECP. In addition, we have analyzed the aggregation properties of the ECP N-terminal fragment R1N19. Peptide R1N19 (10 mg/ mL in 100 mM glycine buffer, pH 3), maintained at 37 °C for 2 weeks, is able to bind both Th-T and Congo Red dyes (Figures 6A,D). Treated peptide increases the Th-T fluorescence by more than 20-fold with respect to the nonincubated peptide sample, and it shows a high red-shifted Congo Red spectrum (9 nm between maxima) with respect to the freshly solved sample. Peptide R1N19 aggregation was almost complete in 1.3 ( 0.2 days, with a half aggregation time of 0.5 ( 0.4 days and a reduced lag-time of only 1 ( 0.5 h (Figure 6B). The peptide is partially structured in solution, as described previously,42 and shows a drastic conversion to β-sheet structure upon incubation (from 30 to 63%), losing most of the R-helix and random structure (Figure 6C). The presence of the β-sheet structure has also been observed in ATR-FTIR analysis (see Supporting Information, Figure S2). R1N19 is also able to form amyloid fibrils that can be observed by TEM. In contrast to ECP, peptide fibrils can form an extensive network, with rod shaped fibers of more than 10 µm length and about 10 nm width (Figure 6E) that bind Th-T specifically (Figure 6F). However, R1N19 does not form

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Figure 5. Ile13 to Ala mutation effect in amyloid protofibril formation on ECP. (A) Fluorescence spectra of Th-T in the absence (squares) and in the presence (circles) of incubated I13A ECP mutant. (B) Congo Red binding assay in the absence (continuous line) and in the presence (dotted line) of incubated I13A ECP mutant. See Experimental Section for additional technical information.

From these results we conclude that the identified N-terminus region, required for the ECP amyloid-like aggregation, can reproduce by itself the aggregation process.

Discussion

Figure 6. Characterization of amyloid aggregation process of peptide R1N19. (A) Fluorescence spectra of Th-T in the absence (squares) and in the presence (circles) of treated peptide (10 mg/mL peptide solution in 100 mM glycine buffer, pH 3, maintained at 37 °C for 2 weeks). (B) Th-T binding kinetics for R1N19 peptide. The process was monitored over a period of 2 weeks; the fluorescence at 485 nm was recorded and plotted as a function of time. (C) Circular dichroism spectrum of recently dissolved R1N19 (continuous line) and treated R1N19 peptide (dotted line) at 0.20 mg/mL concentration in 5 mM sodium phosphate buffer, pH 7. (D) Congo Red binding assay in the absence (continuous line) and in the presence (dotted line) of treated R1N19 peptide. See Experimental Section for additional technical information. (E) TEM micrograph of R1N19 peptide aggregates, scale bar is 0.2 µm. (F) Fluorescence micrograph taken from R1N19 fibrils stained with Th-T, on a Leica DMBR fluorescence microscope using a FITC/GFP filter and a 40× objective.

amorphous aggregates at pH 7 (data not shown). This observation suggests that some other region in the protein may be involved in its aggregation propensity.

Amyloid aggregation is a topic of extensive research because of its importance in some neurodegenerative diseases such as Alzheimer, Parkinson, and Hungtinton diseases, where amyloid deposits are described to share common features.45 An amyloid formation process has also become an essential clue for the description of protein folding and misfolding processes. Although native protein conformation represents an energy minimum in the folding pathway, some other structures have been described to be more stable rather than the native one. In fact, amyloid fibrils display highly ordered suprastructures of extraordinary stability.46 Besides, the amyloid-like fibrils propensity has been correlated to both antipathogen and immunomodulating abilities.9 Some AMPPs, such as lyzozyme and lactoferrin,8,11 have been described to undergo an amyloid formation process. Recently, it has been reported that the amyloid deposits propensity may be related to infection.32 In the present work, we have demonstrated that ECP, an eosinophil ribonuclease involved in the innate immune response, is able to form amyloid aggregates. The amyloid protofibrils formed by ECP bind amyloid-diagnostic dyes as Th-T and Congo Red, present a fibrilar structure under electron microscopy and their percentage of β-sheet in relation to the native ECP is significantly increased. ECP represents the first report of a native RNase member of the RNase A superfamily, that undergoes an amyloid-type aggregation. A previous study described that an engineered variant of RNase A, containing a poly-glutamine region added in the hinge loop 112-115, can undergo an amyloid fibril formation process by using the well-known RNase A domainswapping capacity.19 Using this model, the authors have proposed an assembly model of domain-swapped proteins around an amyloid fiber backbone, where the inserted loop interacts with itself. Although domain-swapped structures can form oligomers that are stable under various conditions, we were not able to detect RNase A amyloid fibrils in the tested conditions. It has been described that incubated samples of RNase A at high protein concentration (up to 1 M), are composed mainly of tetramers and only traces of higher order oligomers (up to 14 monomers length), and no evidence of amyloid-like aggregates have been found.17,47 Nevertheless, transient oligomeric forms can eventually lead to amyloid-type aggregates, as reported for some


Figure 7. Modeling of ECP N- and C-terminal domain-swapped dimer structures using the RNase A dimer templates 1A2W58 and 1F0 V,15 respectively. (A) ECP and RNase A dimer superpositions in ribbon representation in blue and green respectively. The corresponding rmsd values for the N- and C-swapped dimers are 0.62 and 1.81 Å. (B) Domain-swapped ECP dimer structures in a cartoon representation. One monomer is colored dark blue and the other in cyan. The ECP aggregation prone sequence is colored in red. Modeling calculations were conducted using Modeler59 and molecular representations were drawn using Pymol (DeLano Scientific).

antimicrobial peptides.10 Up to this date, no domain-swapping behavior in ECP has been described. N-Terminal domain swapping in ribonucleases is favored by the loosening of the N-terminus domain from the main body of the protein and, in the case of RNase A, dimeric and oligomeric forms are indeed promoted by partial unfolding by full protonation at an acidic pH.48 In fact, ECP amyloid-like aggregation is promoted at acidic pH. Notwithstanding, the absence of the “poly-serine” loop present in RNase A (Figure 1) points out that some differences might arise in hypothetical ECP Nterminus domain-swapped dimers and oligomers. Actually, oligomerization in ECP could be more impeded, as a shorter loop may restrain the formation of the N-terminus exchange. In addition, C-terminal dimer formation may be less favored as the high proline contents in the ECP (and also EDN) loop 115-122 may break the continuous β-sheet structure observed in the C-terminus domain-swapped of RNase A.15 To further consider both scenarios, we have modeled putative ECP dimeric structures using as a template the N- and C-domain-swapped RNase A dimer structures.49 The modeling results suggest that both domain-swapped dimers are indeed feasible, with a slightly better fit for the N-terminus exchange, which would be facilitated by the shift translation of the ECP N-terminus residues toward the RNase A loop area (Figure 7). In both ECP dimeric models, residue I13 remains highly solvent exposed and could direct from there the amyloid aggregation process. In this context, the higher hydrophobicity of Ile with respect to Ala would explain the lack of amyloid aggregation of the I13A mutant related to the wild-type ECP. One can hypothesize that dimeric structures may pile up by stacking interactions, where the exposed side of the described aggregation prone region described would play a key role in a similar fashion as the polyglutamine loop inserted in the RNase A variant described by Sambashinvan et al.19 In view of the results obtained, a detailed analysis at atomic level on ECP amyloid aggregates should be conducted to detail if any domain-swapped structures are implied in the aggregation mechanism. The ECP N-terminus peptide (R1N19) shows, in solution, an R-helix propensity, which is enhanced in a lipidic environment.42 The peptide can disrupt lipid bilayers and aggregate lipid vesicles,42 and these properties might be due to an alpha


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to beta transition triggered by the local pH reduction at the anionic phospholipid layer, as reported for other short R-helical antimicrobial peptides.50 This process would explain the welldocumented ECP activity on lipid vesicles.23 Although there is no report about ECP fibril formation in vivo, the ECP propensity to form amyloid aggregates may be modulated by its ability to interact with both bacterial and cellular membranes, leading to cell agglutination and contributing to the bacteria clearance at the infection focus, providing a new example of amyloid formation for an antimicrobial polypeptide.9 Our previous in vivo studies by confocal microscopy showed indeed how the protein-bacteria aggregates precede the cell death.25 However, aggregation and amyloid formation may not be sufficient for bactericidal activity. In fact, analysis of the ECP N-terminus contribution to the protein antimicrobial properties indicated that the peptide R1N19 can aggregate lipid vesicles but do not have a significant activity on bacteria cultures.42 ECP aggregation may also take place when eosinophils degranulate at the inflammation site and may trigger the characteristic toxic side effects on the host tissues in eosinophilia disorders.51 The protein aggregation may be avoided in normal physiological conditions to prevent toxic side effects to the host tissues. In this way, glycosilation of ECP observed in vivo52,53 might be important to prevent protein fibril formation, as described recently for some other proteins.54

Conclusion In conclusion, the results presented here show that ECP is able to form amyloid protofibrils in vitro in a pH-dependent manner. This amyloid-like aggregation ability is lost by a single mutation at I13 residue, whereas R1N19 peptide retains the ECP protofibril formation capacity. Thus, we report here the first evidence of amyloid-like aggregation for a nonengineered ribonuclease in vitro and identified a region (8-16) in the N-terminus of the protein that is required for the observed amyloid behavior. The findings presented here provide a new example of an antimicrobial host defense protein that can undergo an amyloid aggregation process with potential implication in eosinophilrelated diseases. Acknowledgment. Authors are especially thankful to Raimon Sabate´, Natalia Sańchez de Groot, and Salvador Ventura for their helpful assistance in the discussion and experimental settings. Transmision electron microscopy was performed at the Servei de Microsco`pia of the Universitat Auto`noma de Barcelona (UAB). We thank Alejandro Sańchez for his assistance with electron microscopy samples. Fluorescence microscopy and spectrofluorescence assays were performed at the Laboratori d’Ana`lisi i Fotodocumentacio´, UAB. Circular dichroism and ATR-FTIR assays were performed at the Servei d’Ana`lisi Quı´mica, UAB. EDN plasmid for recombinant protein expression was kindly provided by Richard J. Youle (NINDS, NIH, Bethesda, MD). F.O. was the recipient of an ERASMUS programme fellowship. The work was supported by the Ministerio de Educacioń y Cultura (Grant Nos. BFU2006-15543C02-01 and BFU2009-09371), by the Generalitat de Catalunya (2009 SGR 795), and by the Fundacio´ La Marato´ de TV3 (Grant No. TV3-031110). Supporting Information Available. (S1) RNase A and EDN binding to amyloid diagnostic dyes, and (S2) ATR-FTIR spectra

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of ECP (A) and peptide R1N19 (B) aggregates. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11)

(12)

(13)

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