An orphan dermaseptin from frog skin reversibly assembles to amyloid-like aggregates in a pH-dependent fashion Ruth Go ¨ ßler-Scho ¨ fberger 1 ,Gu ¨ nter Hesser 2 , Martin Muik 3 , Christian Wechselberger 4 and Alexander Jilek 1 1 Institute of Organic Chemistry, Johannes Kepler University Linz, Austria 2 CSNA Center for Surface- and Nanoanalytics, Johannes Kepler University Linz, Austria 3 Institute of Biophysics, Johannes Kepler University Linz, Austria 4 Center for Biomedical Nanotechnology, Upper Austrian Research, Linz, Austria Keywords amphibian skin; amyloid; bioactive peptide; cytotoxicity; self-assembly Correspondence A. Jilek, Institute of Organic Chemistry, Johannes Kepler University Linz, Altenbergerstrasse 69, A-4040 Linz, Austria Fax: +43 732 2468 8747 Tel: +43 732 2468 9498 E-mail: alex.jilek@gmx.at (Received 6 April 2008, revised 5 August 2009, accepted 7 August 2009) doi:10.1111/j.1742-4658.2009.07266.x Dermaseptin PD-3-7 (aDrs) from frog skin contains three aspartic acid resi- dues resulting in a negative net charge at neutral pH, as opposed to numer- ous other dermaseptins which are cationic helical antimicrobial peptides. Still, this peptide can be fitted into an amphipathic a helix by an Edmundson wheel projection. However, folding to the proposed helix was induced to only a low extent by zwitterionic vesicles or even detergents. Furthermore, no evidence of antibacterial or cytotoxic activity from soluble aDrs could be obtained. The peptide has an inherent propensity to an extended conforma- tion in aqueous solution and self-assembles into amyloid fibrils in a reversible pH-controlled fashion, which was studied in some detail; above pH 5, the amyloid fibrils disassemble in a cooperative manner. This is probably caused by deprotonation of both side chain and terminal carboxyl groups, which results in intermolecular electrostatic repulsion. At neutral pH, this process proceeds instantaneously to the soluble form. Within the transition interval (pH 5–6.5), however, ‘backward’ granular aggregates, 10–500 nm in size, are formed. Such metastable amorphous aggregates, which are quickly released from an amyloid depot by a shift in pH, can mediate a strong cytotoxic effect. This activity does not involve lysis or interference with the cellular redox status, but apparently acts via an as yet unidentified mechanism. In this study, we present a new member of an emerging class of self-assembling frog skin peptides with extraordinary self-aggregation properties, which may potentially be relevant for biological processes. Structured digital abstract l MINT-7256467: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind ( MI:0407)bycircular dichroism (MI:0016) l MINT-7255686: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind ( MI:0407)bybiophysical (MI:0013) l MINT-7256439: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind ( MI:0407)byfluorescence microscopy (MI:0416) l MINT-7256449: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind ( MI:0407)byelectron microscopy (MI:0040) l MINT-7256430: Dermaseptin (uniprotkb:O93455) and Dermaseptin (uniprotkb:O93455) bind ( MI:0407)byfluorescence technologies (MI:0051) Abbreviations aDrs, dermaseptin PD-3-7; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; SUV, small unilamellar vesicles; TEM, transmission electron microscopy; ThT, thioflavin T. FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5849 Introduction Amphibian skin glands are known to contain a vast variety of biologically active peptides, such as neuro- peptides, peptide hormones, opioid peptides and pep- tide antibiotics [1,2]. Many of these may help the individuals to interact with their environment, i.e. they are targeted exogenously [3]. In the skin of Pachymedu- sa dacnicolor, a tree frog from southern Mexico, the opioid peptides [Ile2]-deltorphin and dermorphin [4], tryptophyllins [5] and five heterogenous defence-related peptides have been detected [6]. The latter group are termed dermaseptins, because their members share a conserved pre-pro-region, whereas the mature peptides can have very different structural characteristics [7]. The majority of these dermaseptins are cationic linear anti- microbial peptides able to adopt an amphipathic a helix [8]. These properties enable the peptides to intercalate into phospholipid bilayers in a detergent-like manner [9,10]. However, some skin peptides derived from pre- cursors with the characteristic pre-pro-region do not share these structural features. For example, the derma- septin PD-3-7 (DMS5_PACDA) of this species, which in principle could form an amphipathic a helix, differs from classical cationic antimicrobial peptides because it has a negative net charge [6]. Because of this we hence- forth call this peptide ‘anionic dermaseptin’ (aDrs). In this study, we tested whether aDrs has any anti- bacterial or cytotoxic activity. We discovered that aDrs can form amyloid-like aggregates at acidic pH. We tested the antibacterial or cytotoxic activity of these aggregates with the aim of obtaining insight into the functional relevance of self-assembly and their mode of action. Furthermore, we also investigated the aggrega- tion behaviour of aDrs homologues with a neutral or positive net charge. Results Secondary structure induction by vesicles and detergents Induction of secondary structure, an amphipathic helix in particular, by phospholipid bilayers is a good indica- tor of whether a peptide is membrane active. We used 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) unilamellar vesicles and dodecylphosphocholine deter- gent micelles to mimic the outer leaflet of eukaryotic cell membranes, which is rich in phospholipids with the zwitterionic phosphocholine headgroup. By contrast, SDS micelles favour an initial interaction of cationic antibacterial peptides by electrostatic attraction in a similar manner to negatively charged bacterial cell membranes [9,11]. The CD spectrum of soluble aDrs in buffer is that of a random coil, with 30% extended conformation and 5% turn structures independent of pH (Fig. S1). Addition of POPC small unilamellar vesi- cles (SUVs) resulted in an increase in the helical struc- ture up to 2% (7% at pH 8), although the b-sheet fraction remained largely unchanged. The presence of SDS or dodecylphosphocholine may have led to an over- estimated value for the helical fraction of up to 20% (Fig. 1). Antibacterial and cytotoxic activity of soluble aDrs We tested the antibacterial activity of aDrs against the gram-positive Bacillus subtilis and the gram-negative Escherichia coli using an inhibition zone assay. Mono- meric aDrs displayed no antibiotic action in the entire concentration range up to 32 lm. Cytotoxic activity against eukaryotic Spodoptera frugiperda (Sf9) insect cells and NIH-3T3 mouse fibroblast cells was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo- lium bromide (MTT) assay, which is sensitive to the redox potential of the cells. We detected an insignificantly low effect of aDrs on these cell lines (< 10% reduction compared with control at 16 lm; data not shown). Characterization of aDrs aggregates aDrs aggregates after prolonged incubation at acidic pH. The amyloid-like (in a strict sense) nature of Fig. 1. Induction of secondary structure in soluble aDrs. CD spectra of 100 l M aDrs (solid line) and after addition of POPC SUVs (dashed line) and dodecylphosphocholine detergent micelles (dotted line). [h] MRW , mean residue molar ellipticity. Self-assembly of anionic dermaseptin R. Go ¨ ßler-Scho ¨ fberger et al. 5850 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS these aggregates grown in vitro at pH 2 was probed using several well-established methods [12] (Fig. 2). First, we tested the binding of the amyloid-specific dyes Congo Red and thioflavin T (ThT), which are commonly used in amyloid diagnostics. Staining the aggregates with Congo Red yielded green birefrin- gence, whereas incubation with ThT gave green fluor- escence. Second, a high degree of b-sheet secondary structure was detected using FTIR spectroscopy. Investigation of the second derivative of the amide I¢ region demonstrated that maxima at 1619 and 1635 cm )1 were most likely. These bands can be attributed to b-sheet structures. A band at  1685 cm )1 , which is often observed in b-sheet-rich proteins, is almost absent (< 1%) [13,14]. Bands at 1609 and 1659 cm )1 were most likely to be contribu- tions from side chain amides [15]. Other maxima are present at 1648 and 1671 cm )1 , which can probably be assigned to random coils and sterically restricted amide bonds such as those present in turns, respec- tively. Fitting the Gauss peaks onto the band posi- tions revealed a total contribution of b-sheet structures of  80%. Third, transmission electron microscopy (TEM) investigations revealed long (> 1 lm) fibre bundles. Single fibres (protofilaments) were found to have a uniform thickness of 5 nm. Fibril formation is a reversible process dependent on pH When exposed to pH 8, the fibrils instantaneously underwent a phase transition to the predominantly monomeric random coil form of the peptide because this could pass freely through the membrane (3.5 kDa cut-off) during equilibrium dialysis. We henceforth refer to this state as the ‘soluble’ form of the peptide. However, this process was found to be reversible. After a change to pH 2, an amyloidous, pH-responsive gel was again formed after a lag phase of 45 h (Fig. 3). We exposed aDrs fibrils to various pH values in order to investigate their pH-dependent stability. The resulting profile revealed a sharp transition at pH 5 and a sigmoidal decrease in ThT fluorescence in the transition interval towards higher pH values (Fig. 3). Structural rearrangement of aggregates During fibril breakdown at pH 6, we observed the for- mation of metastable granular aggregates of different size and morphology (Fig. 4). Aggregate size ranged from 10 to 500 nm. These aggregates retained some ThT-binding ability and caused the sigmoidal shape of the transition profile in Fig. 3, which was measured A B C D Fig. 2. Amyloid-like characteristics of aDrs aggregates. (A) Light micrograph of Congo Red bound to aDrs reveals ‘apple-green’ birefringence between crossed polarisators. Scale bar, 50 lm. (B) Fluorescence micro- scopy image of ThT bound to aDrs reveals green fluorescence. Scale bar, 50 lm. (C) FTIR spectrum of the amide I’ region and second derivative. (D) Negative stain elec- tron-micrographs of aDrs fibrils formed at acidic pH. Scale bar, 200 nm. R. Go ¨ ßler-Scho ¨ fberger et al. Self-assembly of anionic dermaseptin FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5851 1 h after the addition of buffer. This may indicate that, in a pH interval close to the transition point, a certain degree of stacked b-sheet structure is temporarily preserved. After incubation at pH 6, TEM inspection of aliquots of the sample revealed that the number of amorphous aggregates was drastically reduced after 5 days. After 14 days, such aggregates were apparently undetectable (data not shown). However, equilibrium dialysis, independent of time, yielded 20 lm of soluble aDrs (12–14 kDa membrane cut-off). Thus, only a A B Fig. 3. Phase behaviour of aDrs as a function of pH. (A) Response of aDrs to repeated pH shifts (arrows). The kinetics of 200 l M aDrs was measured by recording the change in fluorescence of ThT at 482 nm over time. After a lag period of 45 h, fluorescence increased linearly upon a decrease in pH to pH 2. (B) Effect of pH on aDrs amyloid-like aggregates. Fluorescence of ThT bound to aDrs aggregates derived from fibrils recorded at 482 nm as a func- tion of pH after incubation for 1 h. A B C D Fig. 4. Characterization of metastable amorphous aggregates of aDrs. (A) Negative stain electron micrographs of aggregates formed from fibrils at pH 6.0 show large metastable amorphous aggregates after 30 min. Scale bar, 100 nm. (B) Time dependency of the disas- sembly of aDrs fibrils after a pH increase to pH 6.0 followed by ThT fluorescence. (C) CD spectra of fibrils (dashed line), aggregates formed from fibrils at pH 6.0 after 5 min (solid line) and soluble aDrs (dotted line). [h] MRW , mean residue molar ellipticity. (D) FTIR spectra of the amide I’ region of fibrils (dashed line), aggregates formed from fibrils at pH 6.0 after 5 min (solid line) and soluble aDrs (dotted line). All spectra contain a contribution of  20% from a band at 1635 cm )1 (thin line) which probably stems from b sheets. Self-assembly of anionic dermaseptin R. Go ¨ ßler-Scho ¨ fberger et al. 5852 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS small fraction of total aDrs had passed through the membrane. We therefore suggest that, at pH 6, at the final state stable oligomers (n ‡ 5) are formed, which could not be characterized in detail because of the detection limits of negative-stain TEM and light scat- tering. Nevertheless, the putatively aggregated character was found not to hinder retransition to the amyloidous state upon a decrease in pH (data not shown). In the FTIR spectra recorded 5 min after the pH- shift to 6.0, a minor peak ( 10%) at 1621 cm )1 was clearly seen (Fig. 4). This suggested that a significant number of interchain hydrogen bonds are still present in the aggregates. Interestingly, this fraction remained unchanged over the following hour, whereas ThT fluorescence decreased further by  40% over this time (Fig. 4). Within this interval, fibrils are no longer detectable by TEM. In FTIR spectra of the soluble peptide, the 1621 cm )1 band finally disappeared in favour of band contributions at 1645 cm )1 (random coil) and  1658 cm )1 . The latter band is within the range of contributions from a helices. CD spectra, however, do not support an a-helical content. These spectra are virtually indistinguishable from those of the soluble form. Antibacterial and cytotoxic activity of disassembling fibrils Next, we were interested to see whether aggregated forms of aDrs would be biologically active. Because the inhibition zone assay is not applicable for nondif- fusable aggregates, we preincubated bacteria with aDrs fibrils (up to 16 lm equivalent) at pH 5.0 in a liquid growth inhibition assay. This yielded colony numbers equal to the control experiment. Furthermore, mea- surement of D indicated that cells not had been lysed (data not shown). By contrast, when aDrs fibrils were co-incubated with insect cells at pH 6.0, their ability to reduce MTT was significantly reduced compared with controls (Fig. 5). We suspected that these aggregates might have a transient character, therefore we also tested the effect of fibrils preincubated in culture med- ium at pH 6 (‘backward’ aggregates) for different time spans, as well as potential ‘forward’ oligomers which might be formed from soluble aDrs at pH 6 (incubated for 7 days) (Fig. 5). In these experiments, the toxicity of aDrs ‘backward’ aggregates decreased with incubation time, with an apparent typical half- life for the toxic species of 5 days. By contrast, the soluble form did not develop any noteworthy toxic activity upon incubation in culture medium at pH 6 (Fig. 5). Cellular effects on Sf9 cells Localization studies may provide a clue to the poten- tial target organelle and thus the mode of cytotoxic action. A fluorophor was attached via a thioether link- age to an additional Cys residue which was appended to the C-terminus. BODIPY C1–Fl was chosen for this purpose because of its moderate hydrophobicity and its suitability for laser techniques. The labelled peptide (aDrs*) was shown to have similar aggregation and cytotoxic properties as unlabelled aDrs (Fig. S2). Con- focal fluorescence microscopy revealed that both aggre- gated and soluble aDrs* were found at the plasma membrane in the early stages (1 h) and after 12 h were intracellularly distributed within Sf9 target cells, but did not enter the nucleus (Fig. S3). With the setup used, we observed no autofluorescence of the cells (data not shown). Leakage of cytoplasmic material (blobs) was not observed at any time. In order to investigate the progression of the poison- ing effect of aDrs aggregates (12 lm) we also varied A B Fig. 5. Cytotoxic effect of disassembling fibrillar aDrs aggregates on Sf9 insect cells investigated by the MTT assay. (A) Cells were treated with lyophilized fibrils resuspended in medium at pH 6.0 at the indicated peptide concentration for 24 h. (B) Cells were treated with ‘backward’ aggregates (15 l M) after preincubation in medium at pH 6.0 for the indicated intervals. ‘F’, soluble aDrs (15 l M) was incubated in medium at pH 6.0 for 1 week. S, soluble aDrs (15 l M). (A,B) Reported values are relative to control cells without peptide. R. Go ¨ ßler-Scho ¨ fberger et al. Self-assembly of anionic dermaseptin FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5853 the incubation time with the cells. MTT reduction levels were detectably affected after as early as 3 h of incubation (Fig. S3). Furthermore, replacement of the culture medium 5 min after the application of aggre- gates did not reduce the toxic effect, which indicated a rapid interaction with the cells (data not shown). The release of intracellular material can be shown by determining the cytoplasmic enzyme lactate dehydro- genase (LDH) in the culture medium. Within the first 12 h after application of the aggregates (12 lm), only a minute fraction of the total LDH levels present in the cells could be detected in the supernatant (Fig. S3). We also investigated the redox status in cells treated with aggregates at 15 lm. The results show no detect- able increase in reactive oxygen species following expo- sure for 3 h compared with the control (Fig. S4). Variants and pH-stable aggregates aDrs variants were designed to confirm the role of the carboxyl side chains in the pH effect (Table 1). There- fore, in peptide N4 we replaced all three aspartic acids by asparagines. Peptide N4 formed fibrils at pH 2 with a delayed kinetics. However, when the solution was brought to pH 6.0, the fibrils rearranged to form stable amorphous aggregates with some ThT-binding capabil- ity, similar to the metastable aggregates of aDrs. Inter- estingly, the same behaviour was observed for a C-terminally amidated aDrs (aDrsa) (data not shown). However, C-terminal amidation of peptide N4 (N4a) fully stabilized the fibrils at neutral pH (Fig. S5). Dis- assembly of amyloid-like aDrs is thus likely caused by a cumulative repulsive effect of negative charges. Nota- bly, the modifications in N4a also confer a potent cytotoxic activity (R. Go ¨ ßler-Scho ¨ fberger and A. Jilek, unpublished observations) which was not characterized in detail, to the monomeric form. Discussion Most cationic antimicrobial peptides are unstructured in solution. Folding into the active amphipathic con- formation is triggered by binding to the target mem- brane. In this study, we used vesicles and micelles to see whether aDrs would also fold to the proposed amphipathic helix. The CD spectra indicated a high propensity for an extended conformation in solution and only a low increase in helical secondary structure upon addition of zwitterionic vesicles or even deter- gents. This may not suffice for a lytic activity (Fig. 1). Indeed, we could not detect any antibacterial or significant cytotoxic activity of the monomer, sug- gesting a function divergent from classical peptide antibiotics. Reversible self-assembly of aDrs is controlled by pH At acidic pH, after a lag period, aDrs forms amyloid- like b-sheet aggregates (Fig. 2). Such delayed kinetics are frequently observed during the formation of amy- loid and result from the obligatory formation of an energetically unfavourable intermediate, which acts as a nucleus for fibre growth [16,17]. Interestingly, the peptide does not contain aromatic residues, which in other cases are thought to contribute significantly to the propensity of a polypeptide to form amyloid [18,19]. Three aspartic acids as well as the unprotected C-terminus were shown to be responsible for the disso- ciation of the b-sheet stacks above pH 5.0, when the side chain and terminal carboxyl groups are deproto- nated (Fig. 3). Such a highly cooperative reversible unfolding transition in a narrow pH interval is known for small globular proteins and has recently also been described for small model peptides, which underwent sharp pH-dependent ‘melting’-like phase transitions. [20]. The high speed of the fibril-to-monomer transi- tion compared with the kinetics of their formation, however, may be indicative of different pathways. The refolding of crucial parts of the polypeptide dependent on pH changes in organelles or the pH gradients in glands may control its function in many biological processes. The pH therefore serves as an activity switch or positional marker. In frog skin, the granular glands, in which the secretory peptides are formed, undergo various morphological changes during their de novo formation or regeneration which most likely also include a decrease in pH [21]. Just like mamma- lian skin, which has a pH of 4.5–5.5, frog skin also has a pH which may be essential for several key defence functions [22]. As a consequence, maturation of the secretory granules might be accompanied by aggregation of aDrs. In turn, environmental pH would trigger the dissociation of these aggregates after release of the gland contents. Table 1. Peptide sequences of aDrs and variants in single letter code. C* denotes fluorophor coupled to cysteine via a thioether linkage. 5101520 aDrs LLGDLLGQTSKLVNDLTDTVGSIV-OH aDrs* C*-OH aDrs a NH 2 N4 N N N N4a N N N NH 2 Self-assembly of anionic dermaseptin R. Go ¨ ßler-Scho ¨ fberger et al. 5854 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS Metastable granular aggregates are formed during disassembly Within the transition interval (pH 5–6.5), metastable amorphous ‘backward’ aggregates were transiently formed during melting of the amyloid-like fibrils (Fig. 4). The clearance or rearrangement of these gran- ules apparently comprised two observable overlapping processes which reflect their dynamic character: (a) dis- ruption of the remaining longitudinal ridges which are formed by the aligned b sheets and are thought to con- stitute the ThT-binding sites (Fig. 4) [23]; and, with a different kinetics, (b) breaking of residual interchain hydrogen bonds which are still present in the granules, yet to a lesser extent when compared with the amyloid form of the peptide (Fig. 4). Otherwise, the structure of the aggregated peptide seems to resemble that of the soluble form because, according to CD and FTIR spectra, it consists of comparable contributions from b hairpin and randomly ordered segments. The half-life of these aggregates, which were found to be cytotoxic to cultured insect cells, was  5 days at pH 6 (Fig. 5). Presumably, within the transition interval the final state is nontoxic oligomeric within the range 5–25 mers, which could not be visualized by TEM. Thus a critical oligomer size or their metastable char- acter may be required for cytotoxicity. By contrast, at pH 8, a predominantly monomeric state is instanta- neously formed. Granular aggregates cause neither cytolysis nor generation of reactive oxygen species It is widely accepted that the aggregation state of antimicrobial peptides in solution is an important parameter determining their activity against eukaryo- tic cells [24,25]. Some antimicrobial peptides such as dermaseptin S4 aggregate to cytotoxic amorphous structures [24,26]. Cytolytic activity, however, is mediated by the folded amphipathic monomer, which is localized at the plasma membrane, where high effective concentrations can be reached at the sites of impact of the aggregates. We were interested in deli- neating the fundamental biochemical mechanism through which aDrs granular intermediates are cyto- toxic to eukaryotic cells. Apparently, the peptide quickly interacted with the cells and penetrated into the cytoplasm independent of the aggregation state (Fig. S3). Cellular damage, however, was induced only by the aggregated form and became detectable after 3 h. During these stages, the integrity of the cell membrane remained largely intact because no LDH (M r , 140 kDa; diameter,  8 nm) was released from the cells [27]. These results rule out a cytolytic mechanism. In some instances, antimicrobial peptides such as apidaecin [28] are directed against intracellular targets rather than against the cell membrane. However, protofibrils, the prefibrillar ‘forward’ oligomers, are suspected of being the effectors of cellular damage associated with numerous protein deposition diseases [29], including neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease [30], and amyloi- doses [31,32]. The inherent cytotoxicity of protofibrils is characterized by the intracellular localization of the polypeptides, the intactness of the cell membrane, the onset of apoptosis and mitochondrial damage [33,34]. Although the action of aDrs fulfils the first two criteria, we did not detect the generation of reactive oxygen species, which appear during apoptosis and accompany protofibril action. Therefore, our results indicate that aDrs aggregates act via an independent, as yet unidentified mechanism. In contrast to protofibrils, amyloid fibrils are not inherently cytotoxic. Nevertheless, the cytotoxicity of certain Ab-amyloid species may depend on structural parameters [34,35]. Furthermore, cytotoxicity can be induced by binding secondary components such as GM1 ganglioside [36]. A correlation of amyloid with innate immune defence A few cases of diverse functional roles for amyloid in biology have been shown to date [37]. Whenever amy- loid participates in physiological processes, its forma- tion appears to be tightly regulated or the prolonged presence of toxic polymerization intermediates is avoided. In strong contrast, it has been suggested that protofibrils and oligomeric states of antimicrobial pep- tides share common features of cytotoxic action [38,39]. This hypothesis may hold for aDrs. Further studies are needed in order to fully understand the processes lead- ing to membrane penetration and ultimately to cellular damage. Very recently, another dermaseptin, derma- septin S9, has been found to form amyloid in vitro. Interestingly, soluble, weakly self-associated forms of dermaseptin S9 were shown to mediate the chemotaxis of human leukocytes, whereas spherical aggregates of the same peptide exerted antimicrobial activity [40]. In conclusion, there is now emerging evidence that amyloid-like aggregates and their intermediate states could be an integrative component of the innate immune system, for which amphibian skin is an approved model system [41–43]. Remarkably, the noncytotoxic aDrs from this source exhibits a reversible R. Go ¨ ßler-Scho ¨ fberger et al. Self-assembly of anionic dermaseptin FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5855 pH-dependent self-assembly to amyloid in vitro.A natural defence strategy could involve such an amyloid deposit, from which a temporarily cytotoxic agent could be quickly formed triggered by an increase in pH. Experimental procedures Peptide synthesis and purification All peptides were prepared using solid-phase peptide synth- esis and were provided by Peptide Specialty Laboratories (Heidelberg, Germany). BODIPY C1-FLÒ was from Invi- trogen (Carlsbad, CA, USA). The peptides were purified by RP-HPLC using a 0.1%(v ⁄ v) HCl ⁄ acetonitrile gradient solvent system. SUVs A solution of 20 mgÆmL )1 POPC (Avanti Polar Lipids, Ala- baster, AL, USA) in water was sonicated for 10 min under argon in a bath-type sonicator, resulting in a clear SUV suspension. CD spectroscopy Peptides were dissolved in 50 mm phosphate-buffer pH 7.0 to a final concentration of 100 lm. SDS or dodecylphosphocho- line was added to a final concentration of 15 or 5 mm, respec- tively. Alternatively, POPC SUVs were added to a peptide ⁄ lipid ratio of 1 : 33 (3.25 mm POPC). CD spectra were recorded on a Jasco (Easton, MD, USA) J-810 CD spec- trophotometer. Spectra were measured in a 1 mm pathlength cell at 20 °C. Five scans in the wavelength range 200–250 nm were recorded for each spectrum at a rate of 20 nmÆmin )1 . Baseline was corrected using pure buffer or buffer and POPC, respectively. Secondary structure content was estimated by linear combination of reference spectra given in [44]. ThT assay ThT stock solution (0.08% w ⁄ v ThT in 0.1 m HCl) was added to aDrs gel (1 mgÆmL )1 ) and either examined directly using fluorescence microscopy (Olympus, Tokyo, Japan, IMT-2 microscope equipped with an FITC-filter cube) or diluted in phosphate buffer of the desired pH for direct fluorescence measurement at the respective pH (excitation at 440 nm, emission 482 nm, slitwidth 5 nm). Within the pH interval of interest detection of amyloid should not be affected by hydroxylation of ThT [45]. Congo Red assay [46] Lyophilized aDrs fibrils were incubated with a filtrated Congo Red stock solution (saturated Congo Red, NaCl in 80% ethanol) [47], centrifuged and the resuspended pellet was examined for birefringence under crossed polarisators with a light microscope (Olympus) using a DPlanApo 20 objective. TEM Peptide samples were spotted on carbon-coated formvar-cov- ered copper grids (Ager Scientific, Stansted, UK), negatively stained with 2% (w ⁄ v) uranyl acetate (Electron Microscope Sciences, Hatfield, PA, USA) or NanoVan (Nanoprobes, Yaphank, NY, USA) and examined with a Jeol 2010 electron microscope (Tokyo, Japan) operated at 100 kV. FTIR Hydrogen exchange of the peptides was performed by repeated dissolution in D 2 O and lyophilization at a neutral pD. Peptide samples were dissolved in DCl ⁄ D 2 O to a final concentration of 2 mm and allowed to aggregate. All spectra were collected at room temperature on a Bruker (Billerica, MA, USA) spectrometer (model Tensor 27) as pre- viously described [48]. The amide I’ region (1600–1700 cm )1 ) of the sample spectrum was examined [49]. Buffers in D 2 O were used to measure background spectra. Prior to curve fit- ting, peaks resulting from water vapour were interactively removed and a 13-point Savitsky–Golay smoothing filter was applied. A straight baseline passing through the ordinates at 1600 and 1700 cm )1 was subtracted. Second-derivative spec- tra were calculated in order to identify peak positions. The contributions of the individual peaks were estimated by fitting Gauss peaks at the most likely band assignments. Secondary structure was correlated to band frequencies according [13]. Equilibrium dialysis Five-hundred micrlitre Micro-Equilibrium Dialyzers (Har- vard Apparatus, Holliston, MA, USA) were equipped with Spectra ⁄ Por RC Membranes, MWCO 12-14000 (Spectrum- labs, Breda, Netherlands) or Spectra ⁄ Por 3, MWCO 3500. After loading with 250 lg peptide, the chambers were rotated at 37 °C for 24 h. The amount peptide present in both chambers was analysed by RP-HPLC using a 0.1% (v ⁄ v) TFA ⁄ acetonitrile gradient solvent system. Inhibition zone assay [50] E. coli and B. subtilis were grown at 37 °Cin10mLLB medium pH 6.5 to a D 600 of 1.2. An agar plate was over- lain with 150 lL of diluted (1 : 10) bacteria stock in 20 mL of 0.7% Agarose Type I in LB medium pH 6.5. Holes were prepared and filled with peptide dilutions (0, 4, 16 and 32 lm). The plates were incubated overnight at 37 °C and inspected for clear inhibition zones. Self-assembly of anionic dermaseptin R. Go ¨ ßler-Scho ¨ fberger et al. 5856 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS Liquid growth inhibition assay [51] Preinocula of E. coli were prepared in LB medium (pH 6.5 or 5.5) at 37 °C and diluted to a D 600 of 0.0001 (1 : 10 6 ). Peptides were added to the bacteria suspension to final con- centrations of 0, 4or16lm and incubated at 37 °C for 2 h. Bacteria were plated out, incubated overnight at 37 °C and the colonies counted. Cell culture Sf9 cells were cultured in Insect Express SF9-S2 medium (PAA cell culture company, Pasching, Austria) adjusted to pH 6.0 at 27 °C. NIH-3T3 cells (mouse fibroblasts) were cultured in Dulbecco’s modified Eagle’s medium containing 10% bovine calf serum in a 5% CO 2 humidified atmo- sphere at 37 °C. MTT inhibition assay [52] Cells were seeded at a density of 4 · 10 3 cellsÆwell )1 on 24-well plates in 500 lL of fresh medium. Fresh monomeric aDrs or a fibril suspension were added to the cells to final concentrations of 0, 1, 4 and 16 lm. Alternatively, fibril suspensions preincubated at pH 5.5 for 0, 15 or 60 min were added to the cells to final concentrations of 15 lm. After 24 h incubation, MTT stock solution in NaCl ⁄ P i was added to each well to give a final concentration of 0.5 mgÆmL )1 and incubated for further 4 h. Five hundred microlitres of cell lysis buffer (10% SDS, 50% N,N-dimethyl- formamide) was added to each well. The samples were incu- bated overnight at 27 or 37 °C, respectively, and absorbance at 570 nm was determined with a Cary spectrophotometer. LDH release assay [27] Sf9 cells were seeded at a density of 4 · 10 3 cellsÆwell )1 on 24-well plates in 500 lL of fresh medium and fibril suspen- sion was added to the cells to a final concentration of 15 lm. The concentration of LDH in 200 lL culture supernatants was determined with a Cyto Tox 96Ò non-radioactive cyto- toxicity assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Untreated cells were used to determine background lysis, cells treated with 1% Triton X-100 were used to determine total LDH (100% lysis). Determination of reactive oxygen species The generation of intracellular reactive oxygen species in Sf9 cells was examined by oxidation of the dye 2¢,7¢-dichloro- fluorescin diacetate (Invitrogen). Cells were seeded in 24-well plates and allowed to reach 50% confluence. The cells were loaded with 5 lm 2¢,7¢-dichlorofluorescin diacetate and incubated in the presence of 15 lm aggregated peptide for differing time intervals. Reactive oxygen species levels were detected by measuring the fluorescence of the oxidized dye with a plate reader (Zenyth 3100 Multimode Spectrofluori- meter, Anthos Mikrosysteme GmbH, Krefeld, Germany) with excitation at 485 nm and emission at 535 nm. Laser scanning fluorescence microscopy Sf9 cells were seeded into an AttoFluor chamber (Invitro- gen). Fibril suspension or soluble aDrs was added to the cells to a final concentration of 15 lm. After 3 and 20 h of incubation, a QLC100 Real-Time Confocal System (Visi- Tech Int., Sunderland, UK) was used for recording fluores- cence images. A Photometrics CoolSNAPHQ monochrome camera (Roper Scientific, Sarasota, FL, USA) and a dual port adapter (dichroic: 505lp; emission filter: 535 ⁄ 50; Chroma Technology Corp., Rockingham, VT, USA) were connected in conjunction with an argon ion multiwave- length (514 nm) laser (Spectra Physics, Mountain View, CA, USA). This system was attached to an Axiovert 200M microscope (Zeiss, Oberkochen, Germany). Acknowledgements We thank Christa Mollay for helpful suggestions and proofreading the manuscript, Dr Irene Frischauf for operating the plate reader, Professor Hermann Gruber for advice concerning the preparation of SUVs, Profes- sor Christoph Romanin for providing access to the confocal microscope, Professor Reinhard Vlasak for the Sf9 cells and Professors Gu ¨ nther Kreil and Heinz Falk for critically reading the manuscript and useful comments. This work was supported by the FWF grant P19393 to A. Jilek, and by grant K-151.217 ⁄ 4-2007 ⁄ Lin of the ‘Land Obero ¨ sterreich’ to A. Jilek. References 1 Lazarus LH & Attila M (1993) The toad, ugly and venomous, wears yet a precious jewel in his skin. Prog Neurobiol 41, 473–507. 2 Kreil G (1994) Antimicrobial peptides from amphibian skin: an overview. Ciba Found Symp 186, 77–90. 3 Olivera BM (2006) Conus peptides: biodiversity-based discovery and exogenomics. J Biol Chem 281, 31173– 31177. 4 Wechselberger C, Severini C, Kreil G & Negri L (1998) A new opioid peptide predicted from cloned cDNAs from skin of Pachymedusa dacnicolor and Agalychnis annae. FEBS Lett 429, 41–43. 5 Chen T, Orr DF, O’Rourke M, McLynn C, Bjourson AJ, McClean S, Hirst D, Rao P & Shaw C (2004) Pachymedusa dacnicolor tryptophyllin-1: structural characterization, pharmacological activity and cloning of precursor cDNA. Regul Pept 117, 25–32. R. Go ¨ ßler-Scho ¨ fberger et al. Self-assembly of anionic dermaseptin FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5857 6 Wechselberger C (1998) Cloning of cDNAs encoding new peptides of the dermaseptin-family. Biochim Biophys Acta 1388, 279–283. 7 Vanhoye D, Bruston F, Nicolas P & Amiche M (2003) Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain. Eur J Biochem 270, 2068–2081. 8 Amiche M, Ladram A & Nicolas P (2008) A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae. Peptides, 29, 2074–2082. 9 Shai Y (2002) Mode of action of membrane active anti- microbial peptides. Biopolymers 66, 236–248. 10 Bechinger B & Lohner K (2006) Detergent-like actions of linear amphipathic cationic antimicrobial peptides. Biochim Biophys Acta 1758, 1529–1539. 11 Matsuzaki K (1999) Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys Acta 1462, 1–10. 12 Nilsson MR (2004) Techniques to study amyloid fibril formation in vitro. Methods 34, 151–160. 13 Jackson M & Mantsch HH (1995) The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol 30, 95–120. 14 Zandomeneghi G, Krebs MR, McCammon MG & Fa ¨ ndrich M (2004) FTIR reveals structural differences between native beta-sheet proteins and amyloid fibrils. Protein Sci 13, 3314–3321. 15 Krull LH, Wall JS, Zobel H & Dimler RJ (1965) Synthetic polypeptides containing side-chain amide groups: water-insoluble polymers. Biochemistry 4, 626–633. 16 Ferrone FA (2006) Nucleation: the connections between equilibrium and kinetic behavior. Methods Enzymol 412, 285–299. 17 Oosawa F & Asakura S (1975) Thermodynamics of the Polymerization of Protein. Academic Press, London. 18 Lopez de la Paz M & Serrano L (2004) Sequence deter- minants of amyloid fibril formation. Proc Natl Acad Sci USA 101, 87–92. 19 Kallberg Y, Gustafsson M, Persson B, Thyberg J & Johansson J (2001) Prediction of amyloid fibril-forming proteins. J Biol Chem 276, 12945–12950. 20 Aggeli A, Bell M, Carrick LM, Fishwick CW, Harding R, Mawer PJ, Radford SE, Strong AE & Boden N (2003) pH as a trigger of peptide beta-sheet self-assem- bly and reversible switching between nematic and iso- tropic phases. J Am Chem Soc 125, 9619–9628. 21 Spannhof L (1953) 54) Zur Genese, Morphologie und Physiologie der Hautdru ¨ sen bei Xenopus laevis. Wiss Z Humboldt Universita ¨ t Berlin 3, 295–305. 22 Lillywhite HB (2006) Water relations of tetrapod inte- gument. J Exp Biol 209, 202–226. 23 Krebs MR, Bromley EH & Donald AM (2005) The binding of thioflavin-T to amyloid fibrils: localisation and implications. J Struct Biol 149, 30–37. 24 Feder R, Dagan A & Mor A (2000) Structure– activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomeri- zation on selective cytotoxicity. J Biol Chem 275 , 4230–4238. 25 Sal-Man N, Oren Z & Shai Y (2002) Preassembly of membrane-active peptides is an important factor in their selectivity toward target cells. Biochemistry 41, 11921– 11930. 26 Kustanovich I, Shalev DE, Mikhlin M, Gaidukov L & Mor A (2002) Structural requirements for potent versus selective cytotoxicity for antimicrobial dermaseptin S4 derivatives. J Biol Chem 277, 16941–16951. 27 Korzeniewski C & Callewaert DM (1983) An enzyme- release assay for natural cytotoxicity. J Immunol Methods 64, 313–320. 28 Castle M, Nazarian A, Yi SS & Tempst P (1999) Lethal effects of apidaecin on Escherichia coli involve sequen- tial molecular interactions with diverse targets. J Biol Chem 274, 32555–32564. 29 Chiti F & Dobson CM (2006) Protein misfolding, func- tional amyloid, and human disease. Annu Rev Biochem 75, 333–366. 30 Skovronsky DM, Lee VM & Trojanowski JQ (2006) Neu- rodegenerative diseases: new concepts of pathogenesis and their therapeutic implications. Annu Rev Pathol 1, 151–170. 31 Sipe JD (1992) Amyloidosis. Annu Rev Biochem 61, 947–975. 32 Pepys MB (2006) Amyloidosis. Annu Rev Med 57, 223– 241. 33 Bucciantini M, Calloni G, Chiti F, Formigli L, Nosi D, Dobson CM & Stefani M (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxi- city. J Biol Chem 279, 31374–31382. 34 Gharibyan AL, Zamotin V, Yanamandra K, Moskaleva OS, Margulis BA, Kostanyan IA & Morozova-Roche LA (2007) Lysozyme amyloid oligomers and fibrils induce cellular death via different apoptotic ⁄ necrotic pathways. J Mol Biol 365, 1337–1349. 35 Petkova AT, Leapman RD, Guo Z, Yau WM, Mattson MP & Tycko R (2005) Self-propagating, molecular-level polymorphism in Alzheimer’s beta-amyloid fibrils. Science 307, 262–265. 36 Okada T, Wakabayashi M, Ikeda K & Matsuzaki K (2007) Formation of toxic fibrils of Alzheimer’s amyloid beta-protein-(1-40) by monosialoganglioside GM1, a neuronal membrane component. J Mol Biol 371, 481– 489. Self-assembly of anionic dermaseptin R. Go ¨ ßler-Scho ¨ fberger et al. 5858 FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... N 4a aggregates at neutral pH This supplementary material can be found in the online article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing... Supporting information The following supplementary material is available: Fig S1 CD spectra of soluble aDrs (100 lm) at various pH Fig S2 aDrs* has similar self-aggregation and cytotoxic properties to unlabelled aDrs Fig S3 Progression of the poisoning effect of aDrs aggregates on Sf9 insect cells Fig S4 No reactive oxygen species were induced by aDrs aggregates Fig S5 Amyloid-like characteristics of N 4a. .. biomembranes: insight into target vs host cell recognition Biochim Biophys Acta 1778, 983–996 40 Auvynet C, El Amri C, Lacombe C, Bruston F, Bourdais J, Nicolas P & Rosenstein Y(2008) Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9 FEBS J 275, 4134–4151 41 Simmaco M, Mangoni ML, Boman A, Barra D & Boman HG (1998) Experimental infections of Rana esculenta with... al ¨ ¨ 37 Fowler DM, Koulov AV, Balch WE & Kelly JW (2007) Functional amyloid – from bacteria to humans Trends Biochem Sci 32, 217–224 38 Zhao H, Mattila JP, Holopainen JM & Kinnunen PK (2001) Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin Biophys J 81, 2979–2991 39 Sood R, Domanov Y, Pietiainen M, Kontinen VP & Kinnunen PK (2008) Binding of LL-37 to. .. Lagueux M, Charlet M, Hegy G, Van Dorsselaer A & Hoffmann JA (1993) A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution J Biol Chem 268, 14893–14897 Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM & Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases Nature... with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora Scand J Immunol 48, 357–363 42 Boman HG (2000) Innate immunity and the normal microflora Immunol Rev 173, 5–16 43 Miele R, Bjorklund G, Barra D, Simmaco M & Engstrom Y (2001) Involvement of Rel factors in the expression of antimicrobial peptide genes in amphibia Eur J Biochem 268, 443–449 44 Poschner BC, Reed J, Langosch... bombinin H2 and H4 Toxicon, 52, 246–254 Byler DM & Susi H (1986) Examination of the secondary structure of proteins by deconvolved FTIR spectra Biopolymers 25, 469–487 Hultmark D, Steiner H, Rasmuson T & Boman HG (1980) Insect immunity Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia Eur J Biochem 106, 7–16 Bulet P, Dimarcq... & Hofmann MW (2007) An automated application for deconvolution of circular dichroism spectra of small peptides Anal Biochem 363, 306–308 45 Fodera V, Groenning M, Vetri V, Librizzi F, Spagnolo S, Cornett C, Olsen L, van de Weert M & Leone M (2008) Thioflavin T hydroxylation at basic pH and its effect on amyloid fibril detection J Phys Chem B 112, 15174–15181 46 Westermark GT, Engstrom U & Westermark P... The N-terminal segment of protein AA determines its fibrillogenic property Biochem Biophys Res Commun 182, 27–33 47 Moriarty DF & Raleigh DP (1999) Effects of sequential proline substitutions on amyloid formation by human amylin20-29 Biochemistry 38, 1811–1818 48 Zangger K, Gossler R, Khatai L, Lohner K & Jilek A( 2008) Structures of the glycine-rich diastereo- Self-assembly of anionic dermaseptin 49 50... re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 5849–5859 ª 2009 The Authors Journal compilation ª 2009 FEBS 5859 . An orphan dermaseptin from frog skin reversibly assembles to amyloid-like aggregates in a pH-dependent fashion Ruth Go ¨ ßler-Scho ¨ fberger 1 ,Gu ¨ nter. that aDrs can form amyloid-like aggregates at acidic pH. We tested the antibacterial or cytotoxic activity of these aggregates with the aim of obtaining insight