Báo cáo khoa học: The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain

The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain Michal Levy1, Nicolas Garmy2, Ehud Gazit1 and Jacques Fantini2

1 Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel 2 Universite´ Paul Ce´ zanne, Laboratoire de Biochimie et Physicochimie des Membranes Biologiques, Faculte´ des Sciences et Techniques

St-Je´ roˆ me, Marseille, France

Keywords amyloid; aromatic stacking; CH–p interaction; glycolipid; suramin

Correspondence J. Fantini, Universite´ Paul Ce´ zanne, Laboratoire de Biochimie et Physicochimie des Membranes Biologiques, Faculte´ des Sciences de St-Je´ roˆ me, 13397 Marseille Cedex 20, France Fax: +33 491 288 236 Tel: +33 491 28 27 54 E-mail: jacques.fantini@univ-cezanne.fr

(Received 15 September 2006, revised 25 October 2006, accepted 27 October 2006)

doi:10.1111/j.1742-4658.2006.05562.x

Several proteins that interact with cell surface glycolipids share a common fold with a solvent-exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH–p stacking interaction). Stacking interactions between aromatic residues (p–p stacking) also play a pivotal role in the assembly pro- cess, including many cases of amyloid ﬁbril formation. We found a structural similarity between a typical glycolipid-binding domain (the V3 loop of HIV-1 gp120) and the minimal amyloid-forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air–water interface, the NFGAILSS peptide speciﬁc- ally interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addi- tion, we observed that the full-length human islet amyloid polypeptide (1–37) did interact with a monolayer of lactosylceramide, and that the gly- colipid ﬁlm signiﬁcantly affected the aggregation process of the peptide. As glycolipid–V3 interactions are efﬁciently inhibited by suramin, a polyaromat- ic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amy- loid ﬁbril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken togther, our results indicate that the minimal amyloid-forming fragment of human islet amyloid polypeptide is a glycolipid-binding domain, and provide further experimental support for the role of aromatic p–p and CH–p stacking interactions in the molecular control of the amyloidogenesis process.

Abbreviations Dpmax, maximal variation of surface pressure; GalCer, galactosylceramide; HFIP, 1,1,1,3,3,3-hexaﬂuoro-2-propanol; hIAPP, human islet amyloid polypeptide; LacCer, lactosylceramide; pi, initial surface pressure; PrP, prion protein; TEM, transmission electron microscopy; ThT, thioﬂavin T.

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Stacking interactions, which consist of overlapping arrangements of parallel planar molecules at van der Waals distances of separation, play a major role in biology. On the one hand, base stacking is an enthalpy-driven process that signiﬁcantly contributes to the stability of the DNA double-helical conforma- tion in water [1]. On the other hand, the three-dimen- sional structure of globular proteins is often stabilized by entropy-driven p–p stacking interactions between aromatic residues located in their hydrophobic core [2]. Stacking interactions can also involve surface- exposed aromatic amino acid residues. Computational studies have recently shown that solvent-accessible aro- matic residues generally form part of a binding site for an external ligand [3]. This is probably due to the weak interactions between aromatic rings and water molecules, which can be advantageously replaced by more stable stacking interactions. In this respect, p–p

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[12]. The identiﬁcation of a short amyloid core peptide derived from human islet amyloid peptide (hIAPP) gave us an opportunity to establish a correlation between the potential to form amyloid ﬁbrils (a typical p–p stacking process) and glycolipid recognition (CH–p interaction). In the present article, we show that the minimal NFGAILSS amyloid core peptide derived from hIAPP is also the shortest glycolipid-binding domain so far characterized. We also show that suramin, a polyaro- matic compound known to bind to the glycolipid-bind- ing domain of HIV-1 gp120, has a bimodal effect on amyloid formation by hIAPP. Taken together, our data support the view that the surface-exposed aromatic resi- dues of amyloid peptides may be involved in both p–p and CH–p stacking interactions.

Results

stacking interactions between surface-exposed aromatic residues were suggested to play a major role in the aggregation process of amyloidogenic peptides [4]. The ﬁrst experimental demonstration of this hypothesis was provided by substitution of the Phe residue with Ala in the context of a minimal amyloid-forming octapeptide (NFGAILSS) of the islet amyloid polypeptide. This relatively minor substitution between two hydrophobic residues induced a dramatic decrease in its amyloido- genic potential to form ﬁbrils [5]. In contrast, replacing each of the other residue with alanine had no effect. Later studies that used high-resolution NMR and X-ray tools provided further direct experimental sup- port for the key role of aromatic interactions in the assembly of various amyloidogenic polypeptides [6–8]. The role of aromatic interactions in the hierarchical assembly of the amyloidal structures is also supported by molecular dynamics simulations performed by var- ious groups [9–11].

Structural similarity between hIAPP and the glycolipid-binding domain (HIV-1 gp120 V3 loop)

of both structures three-dimensional

for V3 hIAPP20)29). As

shown for

Surface-exposed aromatic residues have also been shown to be involved in the binding of proteins to the sugar headgroup of glycolipids [12]. In this case, the six-carbon sugar ring belonging to the polar head of the glycolipid receptor provides a complementary sur- face for the aromatic side chain. The stacking interac- tion is driven by the proximity of the aliphatic protons of the sugar ring, which carry a net positive partial charge, and the p-electron cloud of the aromatic ring. It can thus be considered as a limiting case of hydro- gen bonding, in which the acceptor group is the elec- tron cloud of the aromatic ring and the donor group is a C–H of the sugar ring. Thus, it is referred to as a CH–p interaction [13]. This particular type of stacking interaction appears to play an important role in the recognition of glycolipids by several cellular and pathogen proteins, including the HIV-1 surface envel- ope glycoprotein gp120, the prion protein, and the b-amyloid peptide of Alzheimer’s disease [14,15]. The last of these ﬁndings suggests that there may be struc- tural and functional homology between glycolipid- binding domains and amyloid peptides. Indeed, it is striking that the hallmark of these biochemical struc- tures is a surface-exposed aromatic residue.

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This may have profound biological signiﬁcance. By providing a complementary surface for amyloid and ⁄ or pathogenic proteins, cellular glycolipids may constitu- tively stabilize their ‘inoffensive’ conformation and inhi- bit their aggregation [12]. Replacing these protective heterologous CH–p interactions with homologous p–p interactions would then trigger the pathologic process [16]. A theoretical model accounting for the chaper- one ⁄ protective activity of membrane glycolipids for amyloid peptides and prions has been recently proposed the The hIAPP20)29 peptide and the V3 loop of HIV-1 gp120 have been studied in solution by NMR spectroscopy [17,18], and the atomic coordinates were downloaded from the Protein Databank (entries 1CE4 and 1KUW, for respectively, and hIAPP20)29, three distinct families of conformers could be identiﬁed (Fig. 1A,B). In all cases, the peptide adop- ted a distorted turn structure, leaving the aromatic side chain of the Phe23 residue available for a potential intermolecular interaction. Therefore, the three-dimen- sional structure of the glycolipid-binding domain of the HIV-1 gp120 V3 loop could be superposed on each type of hIAPP conformer. An example of such super- position is the hIAPP conformer C3 (Fig. 1C). According to this structural study, a com- mon motif consisting of a turn with a solvent-accessible aromatic residue (Phe20 for V3, Phe23 for hIAPP) was observed in the three-dimensional structure of these protein domains, which do not share any obvious sequence homology. As a matter of fact, the turn is induced by the presence of Gly and ⁄ or Pro residues in the motif. Molecular mechanics simulations were then conducted in order to generate a model of interaction between the glycolipid galactosylceramide (GalCer) and either V3 or hIAPP (Fig. 1D). One should note that, in both cases, the sugar ring of galactose in the polar head of the glycolipid provides a complementary surface for the aromatic side chain of Phe, allowing the establish- ment of a typical CH–p interaction. This suggested that hIAPP possesses a structural glycolipid-binding domain the hIAPP22)29 containing the Phe23 residue. As

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B

A

D

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Fig. 1. Structural similarity between the glycolipid-binding domain (HIV-1 gp120 V3 loop) and hIAPP20)29. (A) Superposition of the backbone of the 40 lowest-energy conformers of the hIAPP peptide20)29 as determined by two-dimensional solution NMR spectroscopy according to Mascioni et al. [17]. (B) Structure of three hIAPP20)29 conformers, C1, C2 and C3, each representative of a distinct family of conformers. In all cases, the peptide adopts a turn structure with the aromatic side chain of Phe23 oriented towards the solvent. (C) Comparison of HIV-1 gp120 V3 loop (in orange) and hIAPP conformer C3 (in blue) structures and sequences. (D) Molecular modeling of V3 and hIAPP complexed with GalCer (molecular mechanics simulations). The Protein Data Bank entries used were 1CE4 (V3) and 1KUW (hIAPP fragment).

the minimal amyloid- (NFGAILSS) octapeptide is forming fragment of hIAPP, these modeling studies raised the intriguing possibility that this motif could also be a glycolipid-binding domain.

Speciﬁc interaction between hIAPP22)29 and glycolipids

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The ability of hIAPP to interact with glycolipids was assessed by incubating the synthetic NFGAILSS pep- tide with a monolayer of lactosylceramide (LacCer) spread at the air–water interface. In this experiment, the monomolecular ﬁlm of LacCer had a stable surface pressure of 18 mNÆm)1. This value was taken as the initial surface pressure (pi) of the monolayer. As shown in Fig. 2, addition of NFGAILSS in the aqueous phase underneath the LacCer monolayer induced a dramatic increase in the surface pressure. The maximal surface pressure increase (Dpmax) was 6.25 mNÆm)1. This interaction was speciﬁc, as the peptide did not alter the pressure of sphingomyelin or phosphatidyl- choline monolayers prepared under the same condi-

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)

m N m

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100 Time (min)

Fig. 3. Variations of maximal surface pressure increase (Dpmax) as a function of the initial surface pressure (pi) of LacCer monolayers incubated with NFGAILSS (s), NYGAILSS (d), or NLGAILSS (j).

Fig. 2. Physicochemical studies of the interaction between amyloid peptides and membrane lipids. (A) Monolayers of LacCer, sphingo- myelin or dipalmitoylphosphatidylcholine were prepared at an initial pressure of 18 mNÆm)1. After stabilization of the ﬁlm, the indicated peptide was added to the aqueous subphase at a ﬁnal concentra- tion of 8 lM. Surface pressure changes were continuously recorded with a microtensiometer. Wild-type hIAPP core peptide NFGAILSS under LacCer (d), sphingomyelin (n) or phosphatidylcholine (m) monolayers; NLGAILSS analog under a LacCer monolayer (j); amy- loid prion peptide P1 under a LacCer monolayer (s).

< 20 mNÆm)1. Thus the aromatic Phe23 residue of NFGAILSS appeared to be critical for LacCer recog- nition. To further evaluate the importance of the aro- matic side chain, we studied the interaction of a peptide analog with a Tyr residue in place of Phe23. As shown in Fig. 3, this peptide behaved exactly like the wild-type NFGAILSS, suggesting that it is the aro- matic nature of this residue rather than its exact struc- ture that is needed for glycolipid recognition. This is consistent with the molecular model presented in Fig. 1D, as sugar–aromatic CH–p interactions may occur with both Phe and Tyr side chains.

LacCer interacts with full-length hIAPP1)37 and inhibits its aggregation

to check whether

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In order the glycolipid-binding domain of hIAPP was accessible and functional in the full-length peptide, we probed a monolayer of LacCer with hIAPP1)37 (Fig. 4A). After a lag phase of 20 min, the surface pressure of the ﬁlm started to increase, and the maximal effect was observed after 2 h. This indica- ted that the full-length hIAPP recognized the mono- molecular ﬁlm of glycolipid. As the microtensiometer trough is compatible with real-time microscopic ober- vations, the aggregation process of hIAPP1)37 could be followed under the inverted microscope during surface pressure recordings. As shown in Fig. 4B, the presence of typical aggregates of hIAPP was obvious after 90 min of incubation of the peptide in aqueous solu- tion. In contrast, when the same amount of peptide injected in the aqueous phase underneath a was the aggregation process of monolayer of LacCer, tions. Conversely, the LacCer monolayer was recog- nized by a synthetic peptide derived from the human prion protein (PrP) (segment 185–208 of human PrP). This peptide, which shares a structural similarity with the V3 loop of HIV-1 gp120 and the Alzheimer b-amy- loid peptide [14], also forms amyloid aggregates [19]. Thus the ability of hIAPP22)29 to interact with mono- molecular ﬁlms of glycolipids is not unique among amyloid peptides. As molecular mechanics simulations suggested a critical role for aromatic residues in gly- colipid recognition, we tested the interaction of a pep- tide analog in which Phe23 was replaced with a Leu residue. This peptide did not interact with a monolayer of LacCer prepared at a pi of 18 mNÆm)1 (Fig. 2). These experiments were then performed with LacCer prepared at various values of pi (Fig. 3). For the wild- type NFGAILSS peptide, the interaction with LacCer was still detected at high pi values, and the critical pressure of insertion (extrapolated for Dpmax ¼ 0) was 31 mNÆm)1. For comparison, the mean surface pres- sure of the plasma membrane has been estimated to be 30 mNÆm)1 [20]. In contrast, the interaction of the pep- tide analog NLGAILSS with LacCer rapidly decreased as pi increased, with a critical pressure of insertion of

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80 100 120 140 160 180 Time (min)

the interactions

A

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+ LacCer

Interestingly,

B

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(B) Without LacCer.

Fig. 4. Interaction of full-length hIAPP1)37 with LacCer. (A) Kinetics of interaction of full-length hIAPP1)37 (8 lM) with a LacCer mono- layer. Phase contrast micrographs of hIAPP1)37 aggregates as observed after 90 min of incubation of the peptide in the microten- siometer trough. (C) Underneath a LacCer monolayer. Magniﬁcation: · 20.

hIAPP was signiﬁcantly affected. Taken together, these data indicated that the full-length peptide did interact with LacCer, and that the glycolipid could interfere with the aggregation process of hIAPP.

chloride)

Suramin, a polyaromatic compound that binds to glycolipid-binding domains, may interact with hIAPP22)29

Fig. 5. Chemical structure of suramin. Figure created using CHEMDRAW (Cambridge Soft, Gainesville, FL, USA).

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compounds on glycolipid–protein interactions [16]. An interesting example of such aromatic compounds is suramin (Fig. 5), which binds to the V3 loop of HIV- 1 gp120 and interferes with a wide range of HIV-1– glycolipid interactions [21]. Molecular modeling stud- ies using a molecular mechanics approach allowed us to predict the regions of suramin that could poten- tially interact with the V3 loop, as well as the phys- involved in the ical nature of formation of a V3–suramin complex. As shown in Fig. 6A,B, there are three main regions of interaction between V3 and suramin: (a) an electrostatic interac- tion between Arg18 and one sulfate group of suramin (zone 1); (b) a hydrophobic interaction between the methyl group linked to ring B of suramin and the side chain of Ala19 (zone 2); and (c) a T-shaped p–p stacking interaction involving Phe20 and suramin ring A (zone 3). An hIAPP–suramin complex was also modeled (Fig. 6C,D). in the model obtained, the region of suramin interacting with hIA- PP was the same as that involved in V3 binding. Indeed, three zones of interaction could also be pre- dicted in this case: (a) a CH–p interaction between the methylene group of the Asn21 side chain and ring B of suramin (zone 4); and (b) two T-shaped p–p stacking interactions involving Phe23 and suramin rings A and B (zones 5 and 6). Altogether, these data suggested that suramin could interact with hIAPP and thus affect the amyloidogenic properties of this peptide. Indeed, hIAPP ﬁbril formation was efﬁciently inhibited by phenol red, a small polyphenol molecule [22]. These data prompted us to evaluate the ability of phenol red to interfere with the formation of a complex between suramin and a glycolipid-binding domain. This was analyzed by using a solid-phase ra- dioassay in which the V3 peptide was adsorbed onto a poly(vinyl surface and probed with [3H]suramin [23]. To ensure that suramin had free access to the V3-binding motif (GPGRAF), we used a multimeric synthetic V3 loop peptide with eight GPGRAF motifs radially branched on an inert matrix [24]. As shown in Fig. 7, phenol red induced The key role of aromatic residues in CH–p stacking interactions provides a molecular interpretation for inhibitory activity of water-soluble aromatic the

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A

B

D

C

Fig. 6. Molecular modeling of suramin complexed with V3 (A,B) or hIAPP fragment (C, D). Numbers 1–6 indicate the main zones of inter- actions.

dose-dependent inhibition of the binding of [3H]sur- amin to the glycolipid-binding domain of the HIV-1 gp120 V3 loop. Taken together, these data strongly support the view that glycolipid-binding domains are recognized by both suramin and phenol red through heteroaromatic p–p stacking interactions.

signiﬁcant stimulation the of Effect of suramin on hIAPP ﬁbril formation

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Given the prominent role of p–p stacking interactions in amyloid formation [4,5] and the suppressive effect of various aromatic compounds, including phenol red [22,25], it was logical to investigate whether suramin could inhibit hIAPP amyloid formation. The sponta- neous aggregation of hIAPP1)37 alone was followed with a thioﬂavin T (ThT) assay. Under these condi- tions, amyloid formation was evidenced by a ﬂuores- lag phase of cence increase occurring after a approximately 20 h (Fig. 8A). Addition of suramin to hIAPP1)37 resulted in opposite effects on ﬁbril for- mation, according to the concentration of suramin in the assay (Fig. 8). Strong inhibition was observed for suramin concentrations lower than 5 lm (Fig. 8B). However, at concentrations higher than 5 lm, suram- in induced a marked increase of ﬂuorescence, indica- aggregation ting process. Morphologic transmission electron microsco- py (TEM) studies conﬁrmed the opposite effects of suramin on amyloid formation: stimulation in pres- ence of 50 lm suramin (Fig. 8C,D), and inhibition in the presence of 2 lm and 3 lm suramin (Fig. 8E–G). The bimodal effect of suramin on amyloid formation was particularly evident when the percentage of ﬂuorescence was plotted as a function of suramin concentration (Fig. 9). Indeed, it is clearly apparent suramin induced dose-dependent that in Fig. 9

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[3H]suramin binding to Fig. 7. Phenol immobilized multivalent V3 loop peptide. The eight-branched multi- meric V3 GPGRAF motif was coated onto a poly(vinyl chloride) 96-well plate and then incubated with the indicated concentrations of phenol red. The wells were then incubated with [3H]suramin. The radioactivity associated with the wells at the end of the incuba- tion was counted, and the data were expressed as the mean ± SD of three independent determinations.

inhibition of hIAPP1)37 ﬁbril formation in the 2– 4 lm range, and then induced dose-dependent activa- tion of amyloid formation at concentrations higher than 5 lm.

Discussion

the interaction. Taken together, these data strongly suggest that the minimal amyloid NFGAILSS peptide interacts speciﬁcally with LacCer. Thus, it is clearly a glycolipid-binding peptide. As a CH–p interaction appeared to be involved in the recognition of LacCer, it is not surprising that the replacement of Phe by a Tyr residue did not affect the binding of the amyloid peptide to LacCer. Indeed, it has been shown previ- ously that Tyr has a higher propensity to interact with sugar rings than does Phe [27]. However, the NYG- AILSS peptide has a reduced ability to form amyloid ﬁbrils in comparison to the wild-type NFGAILSS motif [28]. Thus, similar aromatic-containing peptide fragments derived from amyloid proteins may bind to the same glycolipid but differ with respect to their amyloidogenic properties. Understanding the bioche- mical speciﬁcities of the p–p and CH–p stacking inter- actions will be a major issue in future research on conformational diseases. However, one of the major outcomes of the present study is the structural similar- ity between the hIAPP core amyloid peptide and the domain of HIV-1 gp120 (i.e. the crown of the V3 loop) involved in glycolipid recognition (Fig. 1). In both cases, a Phe residue oriented towards the solvent pro- vides a complementary planar surface that is optimally presented to establish a CH–p stacking interaction with the ring of a sugar belonging to the polar head of the glycolipid. As aromatic residues are usually found in the functional core of many amyloid peptides [5], our data suggest that glycolipid recognition could be an intrinsic property of amyloid proteins. We are aware that further studies will be necessary to conﬁrm this potential correlation. Nevertheless, the recent ﬁnd- ing that the Alzheimer b-amyloid peptide interacted with GalCer in a monolayer assay [14] is in line with this theory.

the initial pressure of

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In the present study, we show that the minimal NFG- AILSS octapeptide core of hIAPP is a glycolipid-bind- ing domain. The interaction of the synthetic peptide with LacCer, a prototype glycolipid, has been studied at the air–water interface by using Langmuir ﬁlm bal- ance technology. This method has been used previ- ously to formally demonstrate the interaction of the V3 domain of HIV-1 gp120 with various glycolipids [26]. The increase in surface pressure following the introduction of the peptide in the aqueous phase underneath the LacCer monolayer is indicative of the the interaction was interaction. The speciﬁcity of assessed by: (a) the lack of surface pressure increase when the hIAPP peptide was incubated with a mono- layer of sphingomyelin, i.e. a sphingolipid with phos- phorylcholine as polar head instead of carbohydrate, or with a monolayer of the glycerophospholipid phos- phatidylcholine; (b) the gradual decrease of measured Dpmax as the LacCer ﬁlm increased; and (c) the decreased interaction of the syn- thetic peptide when Phe was replaced with Leu, indica- ting that the aromatic residue played a major role in In the second part of this study, we focused on the potential link between polyaromatic compounds, amy- loid peptides and glycolipid-binding domains. In a ﬁrst set of experiments, we showed that phenol red, which is known to inhibit amyloid ﬁbril formation [22], recognized a glycolipid-binding domain (HIV-1 gp120 V3 motif) in an indirect solid-phase assay (Fig. 7). We used tritiated suramin as a ligand for immobilized syn- thetic V3 motifs [23], and we demonstrated that phenol red induced dose-dependent inhibition of the binding of suramin to V3. This allowed us to establish that both suramin and phenol red recognized the glyco- lipid-binding region of the V3 loop. As phenol red has previously been shown to interact with hIAPP and to inhibit its amyloidogenesis [22], these data demonstrate that hIAPP and the glycolipid-binding domain were recognized by the same polyaromatic compound. It

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Amyloid peptides and glycolipids

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Fig. 8. Aggregation of the hIAPP peptide1)37 in the presence of various concentrations of suramin. Human IAPP was dissolved in HFIP and diluted to a ﬁnal concentration of 5 lM with a gradual increase in concentration of suramin. (A, B) ThT ﬂuorescence kinetic values of hIAPP1)37 in the absence or in the presence of the indicated concentration of suramin. (C–G) Ultrastructural morphology determined using TEM of 5 lM hIAPP incubated alone for 24 h (C) or with 50 lM suramin for 24 h (D). TEM of 5 lM hIAPP was also performed after 48 h when it was incubated alone (E) or in the presence of 3 lM suramin (F) and 2 lM suramin (G). Suramin solutions of different concentrations were used as references in the assay. Samples were negatively stained with 2% uranyl acetate. Scale bar, 200 nm.

same problem with suramin. Nevertheless, the results obtained with the radioassay (Fig. 7) solid-phase strongly support the notion that phenol red and suramin could interact with various glycolipid-binding peptides including hIAPP. Our data are also consistent with two recent studies showing that suramin can interfere with the amyloidogenesis process of PrP and b2-micro- globulin [29,30].

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Molecular modeling studies using molecular mechan- ics simulations suggested a potential mechanism of should be emphasized that the use of a microtensiometer in glycolipid-binding studies did not allow the direct evaluation of polyaromatic compounds as potential inhibitors of peptide–glycolipid interactions. Indeed, we observed that phenol red alone dramatically increased the pressure of a LacCer monolayer (data not shown). Thus, although phenol red actually binds to hIAPP, it was not possible to detect any inhibitory effect of this compound on the hIAPP–LacCer reaction by means of surface pressure measurements. We encountered the

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Fig. 9. Biphasic effects of suramin on the aggregation of hIAPP1)37. The relative endpoint ﬂuorescence values after incubation of hIAPP1)37 for 5 days in the presence of the indicated suramin con- centrations (the 100% value was measured in the absence of sur- amin) are shown.

NFGAILSS amyloid core peptide is also the shortest monomeric peptide able to interact with a glycolipid in our monolayer binding assay. Most importantly, this minimal peptide has no charged residues (either acid or basic), so its ability to interact with glycolipids could be essentially ascribed to its unique aromatic Phe residue. This means that the stacking CH–p inter- action is by itself sufﬁcient to initiate and stabilize a glycolipid–peptide interaction, provided that the aro- matic residue is accessible enough to the sugar head- group of the glycolipid. This conclusion is in line with our modeling study presented in Fig. 1, where the CH–p stacking interaction represents the main force for stabilization of the glycolipid–peptide complex. We recently obtained similar data with a synthetic glycolipid-binding peptide derived from a bacterial adhesin [31].

that

type 1 diabetes

that glycolipids

type 2 diabetes,

inoffensive functional and ⁄ or

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through recognition between suramin and hIAPP, heteroaromatic p–p stacking (Fig. 6) interactions [22,25]. Thus, it was logical to investigate the potential effects of suramin on amyloid formation by hIAPP. The data obtained in this study were somewhat surpri- sing. A bimodal effect of suramin on amyloid forma- tion was observed, depending on the concentration of suramin used in the assay. At concentrations £ 3 lm, suramin induced dose-dependent inhibition of amyloid ﬁbril formation. However, at concentrations > 3 lm, suramin appeared to enhance ﬁbril formation by hIAPP (Figs 8 and 9). Phenol red did not show such a biphasic effect, as it gradually interfered with the pro- cess of amyloid formation over the concentration range 0.5–40 lm [22]. The chemical structures of phenol red and suramin may shed some light on this strikingly dis- tinct behavior. Both molecules are polyaromatic, but suramin is a symmetric compound (Fig. 5). In addi- tion, it is a polysulfonated compound, whereas phenol – group. Our data suggest that at red has just one –SO3 high concentrations, suramin could cross-link several hIAPP molecules, probably through nonspeciﬁc elec- – trostatic interactions involving its numerous –SO3 groups. This could lead to the establishment of a com- plex network including both suramin and hIAPP ﬁbrils. Consistent with this hypothesis, hIAPP1)37 is a basic peptide (theoretical pI of 8.9) with two basic resi- dues (Lys1 and Arg11) and neither Asp nor Glu resi- dues. In any case, our data showed that under speciﬁc conditions, multivalent aromatic compounds did not inhibit but instead promoted amyloid formation. This should be kept in mind before considering using poly- aromatic molecules as therapeutic p–p breaker agents. synthetic analogs soluble [22] or Our data may have several biological implications. the minimal First, we have demonstrated that Second, the ﬁnding of a glycolipid-binding domain in hIAPP may be relevant to the etiology of type 2 diabetes. Indeed, hIAPP is synthesized in the b-cells of the pancreas and cosecreted with insulin [32]. It is the major protein of the islet amyloid deposits fre- quently seen in the pancreas of patients affected by type 2 diabetes [33]. Recently, it has been shown that the glycolipid sulfatide mediates the conversion of insulin hexamers to the biologically active monomers sulfatide has an [34]. These data indicate important chaperone activity for the insulin hor- mone. Consistently, sulfatide treatment could prevent in a mouse the development of model [35]. On the basis of our data, one can tenta- tively propose expressed by the b-cells of the pancreas (either sulfatide or other gly- could also interact with hIAPP colipid species) through CH–p interactions, thereby impairing the establishment of the p–p stacking interactions leading to amyloid formation. The preliminary observation that LacCer seemed to interfere with the aggregation process of hIAPP (Fig. 4) is consistent with this view. As amyloid deposits are assumed to play a key role in the pathogenesis of such CH–p interactions between hIAPP and glycolipids may prevent (or delay) the development of this dis- ease. Altogether, these data support the concept that glycolipids of the b-cells of the pancreas act as chap- erones that are able to lock insulin and hIAPP in conformations their through a common molecular mechanism involving CH–p interactions. A therapeutic option for type 1 and type 2 diabetes would be to restore these pro- tective interactions with nontoxic polyaromatic com- pounds of glycolipids [23].

M. Levy et al.

Amyloid peptides and glycolipids

Experimental procedures

recorded until equilibrium was reached. The data were analyzed with the filmware 2.5 program (Kibron Inc.). The accuracy of the system under our experimental condi- tions was ± 0.25 mNÆm)1 for surface pressure.

Synthetic peptides and preparation of stock solutions

(HFIP)

A 50 lL aliquot of the eight-branched V3 peptide (100 ng) was incubated in poly(vinyl chloride) 96-well plates over- night at 4 (cid:2)C, as described previously [23]. The wells were washed three times with 200 lL of NaCl ⁄ Pi, and subse- quently treated with NaCl ⁄ Pi containing 1% gelatin for 90 min at 37 (cid:2)C to reduce nonspeciﬁc binding. The plates were rinsed in NaCl ⁄ Pi and incubated with various concen- trations of phenol red for 1 h at 37 (cid:2)C. After washing, the [3H]suramin plates were incubated with 100 lL of (1 lCiÆmL)1). After 1 h at 37 (cid:2)C, the plates were washed ﬁve times with 200 lL of NaCl ⁄ Pi, each well was individu- alized, and the radioactivity was determined in a b-scintilla- tion counter (Packard United Technologies, Downers Grove, IL, USA).

(GPGRAF)8-(K)4-(K)2-K-bA [20] was

[3H]Suramin-binding assay

The synthetic peptides derived from hIAPP (NFGAILSS and analogs NYGAILSS and NLGAILSS) were purchased from Peptron Inc. (Taejeon, Korea). Human IAPP1)37 was purchased from Calbiochem (San Diego, CA, USA). The synthetic peptide P1 (KQHTVTTTTKGENFTETDVKM- MER), derived from human PrP, [14] was purchased from Euro Sequence Gene Service (Evry, France). The peptides were puriﬁed by HPLC (purity > 95%) and characterized by ESI MS. Stock solutions of NFGAILSS and peptide analogs were prepared by dissolving the lyophilized form of the peptides in dimethylsulfoxide at a concentration of 100 mm. The stock solution of hIAPP was prepared by dis- solving the lyophilized form of the peptide in 1,1,1,3,3,3- hexaﬂuoro-2-propanol concentration of 500 lm. To avoid any preaggregation, all stock solutions were sonicated for 2 min before each experiment, as previ- ously reported [24]. The multivalent eight-branched V3 pep- generously tide provided by J-M Sabatier (IFR Jean Roche, Marseille, France). LacCer, sphingomyelin and dipalmytoyl phosphat- idylcholine of the highest available purity were purchased from Sigma (St. Louis, MO, USA).

ThT ﬂuorescence assay

After sonication, dissolved hIAPP was diluted with 10 mm sodium acetate buffer (pH 6.5) or in suramin solutions, also in 10 mm sodium acetate buffer (pH 6.5), to a ﬁnal concen- tration of 5 lm, with a ﬁnal HFIP concentration of 1% (v ⁄ v). After dilution, the protein was centrifuged for 20 min at 20 817 g and 4 (cid:2)C (Eppendorf centrifuge 5417R, rotor F-45-30-11). After centrifugation, the supernatant fraction was taken for incubation at 25 (cid:2)C for the inhibition experi- ment. ThT was added to the incubated samples, giving an hIAPP ﬁnal concentration of 0.5 lm and a ThT ﬁnal con- centration of 0.4 lm. Fluorescence was measured using an Yvon Horiba Fluoromax3 ﬂuorimeter (Edison, NJ, USA) (excitation at 450 nm, 2.5 nm slit; emission at 480 nm, 5 nm slit).

Molecular structures were visualized using the swiss-pdb viewer program [36]. Protein Data Bank identiﬁcation num- bers were 1CE4 (HIV-1 gp120 V3 loop peptide) and 1KUW (hIAPP fragment). Molecular mechanics simula- tions (with the MM+ ﬁeld force) of V3 and hIAPP pep- tides complexed with GalCer and suramin were performed with hyperchem 7 (Cambridge Soft), and the models were visualized with the swiss-pdb viewer as previously reported [37].

Molecular modeling

Ten-microliter samples were placed on 400-mesh copper grids covered by carbon-stabilized formvar ﬁlm (SPI Sup- plies, West Chester, PA, USA). After 2 min, excess ﬂuid was removed, and the grids were negatively stained with 2% uranyl acetate in water for a further 2 min. Samples were viewed in a JEOL 1200EX electron microscope (Jeol, Croissy-sur-Seine, France) operating at 80 kV.

TEM Surface pressure measurements

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