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Monomers, Oligomers and Fibrils: Structural Features Cristina Airoldi, Erika Sironi, Barbara La Ferla, Francisco Cardona and Francesco Nicotra* Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy Abstract: The misfolding, aggregation and accumulation of proteins in the brain, represents a common feature of diverse neurodegenerative diseases among which Alzheimer’s disease (AD). Important therapeutic strategies for this pathology aim at inhibiting the aggregation of misfolded amyloid  (A) peptides into different species, particularly intermediate oligomeric assemblies, which are believed to be the most neurotoxic species. Here we review the structural data present in the literature, with the purpose to supply useful information for the rational design of new potential molecules able to target A peptides and fibrils. In particular, structural information concerning the different A peptides assemblies, their supramolecular organization, their interaction with cations, biological membranes and known ligands are reported.

Keywords: -amyloid, A oligomers, A fibrils, A peptide structural features, Alzheimer’s disease. INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder that affects over 30 million individuals worldwide and it is the fourth common cause of death after heart disease, cancer and stroke in Western countries. A central pathological feature of AD is the accumulation of misfolded amyloid  (A) peptides in the form of oligomers and amyloid fibrils in the brain. Aggregated A species, particularly intermediate oligomeric assemblies, are believed to trigger a cascade of events that leads to the formation of neurofibrillary tangles, to the disruption of the neuronal cytoskeleton, to a widespread synaptic loss and to neurodegeneration. During the last two decades, several studies have been performed with the aim to investigate the structural features of amyloid peptides and their aggregates. As a matter of facts, therapeutic strategies based on the rational design of aggregation inhibitors require knowledge of the molecular structure of amyloid oligomers and fibrils. This review contains a resume of the structural data present in literature and was edited with the purpose to supply useful information for the design of new potential molecules able to target A peptides and thus acting as drugs against AD. A
MONOMER AND OLIGOMERS STRUCTURE Human A1-40 and A1-42 peptides present the following sequence: NH2-DAEFRHDSGY EVHHQKLVFF IIGLMVGGVV40 IA42-COOH

AEDVGSNKGA

The sequence of A1-40 is divided into two regions. Residues 1-28 make up a relatively hydrophilic domain with a high proportion of charged residues. In the amyloid precursor protein, this domain is extracellular. The carboxyl- terminal residues 28-40 represents a richly hydrophobic *Address correspondence to this author at the Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy; Tel: +39 0264483457; Fax: +39 0264483565; E-mail: [email protected]

1573-4072/11 $58.00+.00

domain associated with the cell membrane in the amyloid precursor protein. According to numerous different experimental evidences, the A1-40 and A1-42 peptides mainly appear as a random coil in aqueous solution, but contain some secondary structure elements: a poly-proline II helix (PII) in the N-terminus, and two -strands in the central part and in the C-terminus [1]. Monomers present a high tendency to aggregate and form A oligomers, which eventually produce A fibrils. In particular, the N-terminal domain exists as a soluble monomeric
-helical structure at pH 1-4 and pH greater than 7. Nevertheless, it rapidly precipitates and gives rise to an oligomeric -sheet structure at pH values between 4 and 7. The observation that -sheet formation by A was promoted at low pH is of particular interest as the pH in AD brain has been found to be slightly lower than in normal brain, and this acidosis may result in enhanced A deposition [2]. In the last few years, it has been shown that soluble oligomers might be pathologically more important than fibrillar-amyloid deposits [3]. A specific mechanism for the toxicity of oligomeric assemblies was suggested by solution studies of isolated A fragments. The great similarity between the A1–42 structure in apolar environments and that of a virus fusion peptide [4] suggests that membrane poration could be the key event for neurotoxicity. In fact, the
-helical peptide could induce formation of membrane channels, promoting the penetration of substances (such as metal ions) that can cause neuronal death [5]. The dynamics of monomeric Alzheimer A1–40 in aqueous solution was studied by Danielsson et al. using nuclear magnetic resonance (NMR) experiments [6]. The persistence length of the A1–40 monomer was found to decrease from eight to three residues when temperature was increased from 3 to 18°C. At 3°C the peptide shows structural propensities that correlate well with the suggested secondary structure regions of the peptide present in the fibrils, and with the
helical structure in membrane-mimicking systems. They proposed a structural model for the monomeric soluble peptide with six different regions of secondary structure propensities. The peptide has two regions with -strand propensity (residues 16–24 and 31–40), two regions with high PIIhelix propensity (residues 1–4 and 11–15) and two unstruc© 2011 Bentham Science Publishers

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tured regions with higher mobility (residues 5–10 and 25– 30) that connect the structural elements. In particular, their relaxation results are in agreement with the model in which the C-terminus forms an antiparallel -sheet with the central region. The central region is, mostly at low temperatures, a high fraction of PII, which is changed to a -strand propensity when raising the temperature. The core of the central region (residues 18–20) has a high -strand propensity at low temperatures. The mobile turn region (25–30) allows the Cterminus to approach the -strand, where side chains can be involved in hydrophobic interactions. The hydrophobic segment in the C-terminal domain of A is largely responsible for A peptide propensity to form -sheet, while the N-terminal domain has a major influence on the secondary structure of A conformation [7]. The helical propensity of the A is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation [8]. In addition, the substitution of glutamine for glutamic acid at position 22 (the “Dutch” peptide) decreased the propensity of the A N-terminal domain to adopt
-helical structure, with a concomitant increase in amyloid formation [9]. As indicated by numerous in vitro and in vivo studies, A1–40 and A1–42 peptides have different aggregation and deposition properties. A1–42 aggregation is much faster and its toxicity much higher. A1–40 and A1–42 thermal unfolding processes were investigated by parallel molecular dynamics (MD) simulations to explore the physical basis underlying the different dynamic behaviors of both A peptides [10]. In A1–40 peptide, due to their favorable spatial positions in R-helical conformations, residues 39-40 (VV) and 34-36 (LMV) form hydrophobic core in the C-terminus that certainly stabilizes the peptide. On the other hand, this hydrophobic core is destroyed in A1–42, because the additional residues 41-42 (IA) form hydrophobic interactions with residues 39-40 (VV). This causes the disruption of the hydrophobic interactions between residues 39-40 (VV) and residues 34-36 (LMV). As a consequence, residues 34-36 (LMV) tend to close with 31-32 (II) to form a new hydrophobic core (Fig. (1)). This finding provides some new clues for the understanding of the different aggregation and deposition propensities of both peptides. As previously introduced, NMR investigations on small A fragments [11] and A1-40 Met35ox [1b] suggest that in aqueous solution A peptides can be described as random coils, with only a small population of local nonrandom structures. Nevertheless, many studies suggest a critical role for in vivo conformational transitions between soluble
helical and  forms of the peptide. Unfortunately, direct observation of these conformational transitions is difficult, due to A peptides poor tendency to dissolve in water. For this reason, detailed structural studies are normally performed in mixtures of water and organic solvents, particularly fluorinated alcohols, such as trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), and micellar solutions [7, 9, 12]. A peptides do not aggregate when dissolved in these media and show a preferential
-helical conformation. Recently, it was demonstrated that, in addition to promoting
helices, mixtures of

Fig. (1). Stereo views of the hydrophobic interactions in the Cterminus of A1-40 (a) and A1-42 (b) at 1.5 ns of the MD simulations. The residues involved in the hydrophobic interactions are shown in Corey-Pauling-Koltun spheres.

water and TFE can induce -sheet structures. In particular, in the concentration range between 10 and 32.5% of TFE (v/v), A1–42 forms -sheet structures, whereas in the concentration range 50–100% TFE CD spectra are typical of
-helices. Tommaselli and co-workers [13] exploited this property to characterize A peptide conformational transitions from helical to  forms. By integrating NMR and CD experimental data with MD simulations, they demonstrated that the core sequence 25-35 plays a key-role in the
-to- conformational transition. The  conformation can be stable in solutions that contain 90–99% water. Moreover, they found that
-to- conformational transition is reversible, and that addition of appropriate amounts of HFIP turns the peptide conformation back from  to
; this reversion of  aggregate occurs slowly but unambiguously. Currently, attempts to identify peptide regions that are induced by surrounding medium drive conformational transitions, representing a possible approach to understand the molecular basis of Alzheimer’s disease (AD). In addition, the design of molecules able to interfere with the aggregation process can be rationalized starting from the structural characterization of a partially folded intermediate in the
-to- transition and vice versa. This has great potential for possible therapeutic applications. As previously introduced, A oligomers are soluble and are suggested to play an important role in the pathogenic cascade of AD by being toxic to neurons. The toxic mechanism of the oligomers is thought to be either a direct or indirect mechanism, mediated through oxidative stress or by inducing inflammatory processes [14].

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The structure of the oligomeric species discovered until now is not known in detail. Several aggregation states have been identified for amyloidogenic proteins before the formation of fibrils. As a matter of fact, the general term oligomer includes different kinds of assemblies such as dimers, trimers, protofibrils, ADDLs (A
-derived diffusible ligands) and annular or pore-like oligomers. Oligomers could also be classified into prefibrillar or fibrillar oligomers as they have different aggregation pathways. A
oligomers are most probably intermediates in amyloid fibrils formation. However, they are not necessarily required to form fibrils [15]. Generally, A
1-40 generates a mixture of monomers, dimers, trimers and tetramers, whereas A
1-42 forms pentamers/hexamers, as well as dodecamers, octadecamers, and other large assemblies. Of these two peptides, only A
1-42 has been observed to form ADDLs [16]. Recently, a specific A
oligomer has been described. It is a dodecamer able to bind specifically the dendritic portions of neurons and block the membrane potentiation [17], inducing a memory impairment in mice [18]. From the structural point of view, this dodecamer displays a micellar disposition of A
-peptides with the hydrophobic C-terminus hidden in the micelle center and at a critical micelle concentration of 17.6 μM [3c, 19]. The peptides in the oligomer appear to be unstructured mainly [20]. In 2005, Laurents et al. proposed new kinds of A
aggregated forms, the so-called
-balls, detected at pH lower than 4 [21]. According to their model,
-balls made up of A
1-40 peptides have a spherical micelle structure with the 12 Cterminal amino acid residues hidden inside an hydrophobic core and with the rest of the peptides exposed to solvent. These last 12 residues within the
-balls core are hydrogenbonded and form
-sheet structures, while the same residues in an extended conformation would be about 4.1 nm long, as detected by atomic force microscopy (8 nm). With the last 12 residues of A
1-40 modeled as a cylinder about 4.1 nm long with a radius of 0.25 nm, it can be estimated that about 350 A
1-40 C-terminal tails could fit into the micelle core. Other geometric forms, such as elliptical or cylindrical micelles or bilayers, also permit the solvent exposure of a polar head group and the burial of a nonpolar tail. Both cylindrical micelles and bilayers can grow indefinitely on their ends or edges, respectively, whereas the number of monomers in spherical or elliptical micelles is limited geometrically. The observation that the size distribution of
-balls is limited, more consistent with spherical or elliptical micelle geometries. The fact that
-balls have not been detected above pH 4 is consistent with the model prediction that
-balls will be unstable above this pH, where the deprotonation of the Cterminal carboxylic groups (pKa
3.8) will favor their solvent exposure and will break any hydrogen bond formed between the neutral carboxylic acids, therefore disrupting the hydrophobic
-ball core. Previously, Yong et al. [22] suggested the existence of a spherocylindrical A
micelle forming at pH 1. Similarly to the
-ball, this oligomer could contain hidden C-terminal carboxylic acid groups and have a definite size range. This spherocylindrical micelle contains fewer monomers (30–50), and its dimensions suggest that the nonpolar residues, both the central and C-terminal hydrophobic segments, are buried within the micelle core. The higher ionic strength present at pH 1 could more effectively

Airoldi et al.

screen the charge-charge repulsions allowing K28 from different monomers to be positioned closer together and thus account for its different geometry compared with
-balls. Other spherical A
peptide oligomers are present at endosomic or neutral pH prior to the formation of amyloid fibrils or protofibrils. Their C-terminal carboxylate groups are not buried but are charged and exposed, this is consistent with their smaller diameter. In contrast to the low pH A
oligomers, at neutral pH both positively and negatively charged groups are present and could be arranged to permit favorable electrostatic interactions in the oligomers [23]. The positioning of multiple positive charges on the exterior of the
-balls probably keeps them well separated in solution and prevents their evolution to fibrils. On the contrary, favorable electrostatic interactions in neutral pH spherical oligomers are likely to permit them to form progressively higher order aggregates. FIBRILS QUATERNARY STRUCTURE As masterfully described in a very recent paper [24], due to their noncrystalline, insoluble nature, determination of the full molecular structures of amyloid fibrils requires specific experimental approaches [25]. In particular, experimental information at the primary, secondary, tertiary, and quaternary structural levels are required. Primary structure is defined as the amino acid sequence, while secondary structure describes segments with standard backbone conformations. While monomeric peptides present prevalently random coil structures,
-strands are the predominant secondary structural elements in amyloid fibrils. The study of secondary structure in amyloid fibril therefore consists on the identification of
-strand and non-
-strand segments (i.e., turns, loops, or bends). This study was carried out experimentally exploiting different techniques: hydrogen/deuterium (H/D) exchange [26], proline-scanning mutagenesis [27], infrared and Raman spectroscopies [28], electron paramagnetic resonance (EPR) [29] and solid-state NMR [30]. EPR and solidstate NMR measurements [31] were exploited also for the characterization of fibrils tertiary structure. X-ray diffraction [32] and solid-state NMR [33] were used to characterize quaternary structure, defined as the positions and orientations of
-sheets relative to one another. Experimental determination of quaternary structure in amyloid fibrils has been quite difficult. The latest models for amyloid fibril structures support the idea that the core of an amyloid fibril contains two or more layers of
-sheets. The quaternary structure takes origin from a set of specific contacts involving amino acid side chains that project from adjacent
-sheets. In fibrils formed by relatively long peptides the adjacent
-sheets may be composed either by
-strands from the same peptide molecules or by
-strands from different molecules. This finding demonstrates that the side chain contacts from which quaternary structure takes origin can be either intramolecular or intermolecular. According to solid state NMR, hydrogen exchange and EPR data, in fibrils formed by the A
1-40 peptide residues 1-9 are structurally disordered, residues 10-22 and 30-40 form
-strands, and residues 23-29 form a bend or loop. The two
-strands form two separate in-registers, anti-parallel
sheets, which can make contact with one another because of

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the intervening bend segment. Shivaprasad et al. [34] obtained the more recent information about A
1-40 quaternary contacts. They performed disulfide cross-linking experiments on A
1-40 double cysteine mutants, and the data obtained suggest a quaternary structure in which side chains of L17 and L34 are in proximity. As L17C/L34C, L17C/M35C, and L17C/V36C mutants were all able to form amyloid fibrils after oxidation in their monomeric states, they concluded that other quaternary structures for A
1-40 fibrils are also possible. At the same time, it has been demonstrated that A
1-40 modified with a lactam cross-link between residues D23 and K28 gives rise to amyloid fibrils significantly more rapidly than the wild-type peptide [35]. These experiments are in agreement with the observation by solid-state NMR of the presence of a salt bridge interaction between side chains of D23 and K28 in fibrils formed with gentle agitation, while this kind of salt bridge is absent in fibrils formed under purely quiescent conditions. This is an evidence of the polymorphism at molecular-level in A
1-40 fibrils. In 2006, Petkova and co-workers [24] obtained evidence that each layer of molecules consists of two
-sheet layers. Mass-per-length (MPL) measurements by scanning transmission electron microscopy showed that the basic structural unit in these fibrils contains two layers of A
1-40 molecules in a cross-
motif. Specifically, the experimentally observed structural unit with minimum MPL, called “protofilament”, is a four-layered
-sheet structure with both “internal” and “external” quaternary contacts (Fig. (2)). In particular, internal quaternary contacts exist between side chains of L17 and F19 and side chains of I32, L34, and V36, while external quaternary contacts involve the side chain of I31 and the peptide backbone at G37, and external quaternary contacts between the side chain of M35 and the peptide backbone at G33. These data support the C2z quaternary structure depicted in (Fig. (2)).

Fig. (2). Representation of candidate quaternary structure for A
140 fibrils with C2z symmetry [24].

According to the model they proposed, side chains of L17, F19, I32, L34, and V36 create hydrophobic cluster that

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apparently stabilizes the fold of a single molecular layer by internal quaternary contacts. Glycine residues 33, 37, and 38 create grooves into which the side chains of I31 and M35 fit at the interface between molecular layers, generating the external quaternary contacts. Oppositely, charged D23 and K28 side chains interact in an intermolecular fashion in the interior of a single molecular layer with STAG-(±2) stagger. Besides, this model contains channels that are lined by side chains of F19, A21, D23, K28, A30, and I32 and that may contain “water wires”. In addition, they demonstrated that the internal quaternary contacts in agitated and quiescent fibrils are different. In fact, A
1-40 fibrils grown under different conditions can have different dimensions, morphologies, MPL values, and NMR spectra, and therefore qualitatively different molecular structures. COMPARISON BETWEEN OLIGOMERS AND FIBRILS STRUCTURE Spherical A
intermediates were studied by S. Chimon et al. in a recent work, in which they have verified if substantial structural transitions precede fibril formation in the amyloid misfolding of the full length A
. They studied the structural transition of A
1-40 in a 100 μM solution, in phosphate saline buffer at 4°C, and they observed that monomeric A
140 peptides in random coil conformation self-assemble into aggregates without amyloid-like
-sheet structure, then they generate toxic spherical intermediates predominated by well ordered
-sheet structures. These spherical intermediates have higher neurotoxicity than monomers or matured fibrils. These intermediates, analyzed by solid-state NMR, showed that they have well ordered structures: there is a structural disorder, for the first ten residues at the N-terminal region, while in the C-terminus the structural disorder is probably minimal. Comparing the structures of these intermediates with that of the fibrils, the dihedral angles and the suggested
-sheet regions were nearly identical between fibrils and intermediates. The measurements of the interstrand distance suggest that both have in-register parallel
-sheet structure. In conclusion their hypothesis is that A
1-40 misfolds into an intermediate state that has well-defined fibrils-like parallel
-sheet structures (secondary structure) but with only a metastable supramolecular packing (tertiary structure) before final fibrillization. The higher toxicity displayed by the intermediates may be attributed to the unique supramolecular structures that they assume in addition to the conformational changes of A
1-40 [36]. It was already known from previous studies carried out on A
oligomers that there is a dominance of
-sheet structure, but there was no distinction between parallel and antiparallel structures. By using ATR-FTIR spectroscopy, it has been demonstrated that parallel and anti-parallel
-sheet structures can be distinguished based on the analysis of the Amide I (1700-1600 cm-1) region. In antiparallel
-sheet structures the amide I region displays two typical components, while for parallel
-sheet structures the amide I region displays only the major component around 1630 cm-1. FTIR spectra of A
1-42 in fibril forming conditions showed typical parallel
-sheet feature, characterized by a maximum of absorbance at 1630 cm-1 in the amide I region, while spectra of A
1-42 oligomers were significantly different, indicating

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that these entities adopt a different structure: the amide I region is characterized by the presence of the two characteristic components of antiparallel
-sheet structure, at 1630 and 1695 cm-1. A quantitative analysis of the amide I region showed that random coil and/or helical structures represented 20-26 and 5-10% for oligomers and fibrils respectively, whereas
-sheet structures were the most abundant (48-57% and 75% respectively). So, even if fibrils and oligomers are rich in
-sheets, they do not adopt the same conformation. The comparison of the overall shape of the A
1-42 spectra in the amide I and II range with the entire database by means of a cluster analysis based on Euclidian distance measurement, clearly indicated that A
fibrils spectra do not cluster with other protein spectra and that A
oligomers spectra are clustered with five antiparallel
-sheet proteins (avidin, concanavalin A, lentil lectin, xylanase). IR spectra of A
oligomers are very similar with bacterial outer membrane porins which are folded as
-barrels, with antiparallel
sheet organization (Fig. (3)). This
-barrel conformation is ideally suited to insert into a cellular membrane, spanning a lipid bilayer. Such an organization can potentially lead to permeabilization of cells, contributing to the toxicity associated with these species [15].

Fig. (3). A schematic representation of A
oligomers in a putative pore-forming conformation.

Liping Yu et al. identified soluble forms of A
1-42 called preglobulomers and globulomers. Preglobulomers have a molecular weight of 16 kDa (about 4 peptides/soluble aggregate), while A
1-42 globulomers have a molecular weight of 64 kDa (corresponding to 12-16 peptides/soluble aggregates). By NMR analysis the preglobulomers show characteristic chemical shifts of the backbone atoms, a pattern of protected amides, and NOE data which are all consistent with the presence of two
-strands from V18 to D23 and from K28 to V40. Residues V18-D23 form one strand of an intrachain antiparallel
-sheet connected by a
-hairpin to the other intrachain strand K28-G33, while L34-V40 forms an interchain in-register parallel
-sheet. So the model proposed is that of a dimer (Fig. (4)). Globulomers, instead, were not suitable for structural studies, however amide exchange experiments gave a pattern consistent with the hypothesis that both pre-globulomers and

Airoldi et al.

globulomers adopt a similar secondary structure and that the A
1-42 globulomer is an oligomer of pre-globulomer units. The amide exchange data of the preglobulomer and globulomers were very different from the fibril forms. In fibril form the amides 11, 12, 15-22, 24, 25 and 28-30 are fully protected, while in the globulomer form only the last 11 residues of the peptide are fully protected. A
fibrils are composed of two in-register interstrand parallel
-sheets connected by a bend between residues 25 and 30. Both the fibrils and the preglobulomers structures exhibit an interstrand parallel
-sheet for the C-terminal residues (34-42). However, in contrast to the fibril structures, the preglobulomers have an intrastrand antiparallel
-sheet connected by a
hairpin between D23 and K28. Residues 10-16, which are parts of the first
-sheet in fibrils, are disordered in the preglobulomer. Probably, a bend versus a turn between residues 22 and 30 is the trait that leads to fibrils versus soluble globulomers (Fig. (5)) [37]. A
OLIGOMER AND FIBRIL POLYMORPHISMS The heterogeneity of amyloid fibrils reflects different types of polymorphism: (1) variations in the protofilament number, (2) variations in the protofilament arrangement and (3) different polypeptide conformations. Structurally polymorphic amyloid fibrils are not found only in in vitro preparations, since several tissue-extracted amyloid fibrils also show significant structural polymorphism. Amyloid fibril polymorphism implies that fibril formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, starting from the same protein sequence, only one ordered conformation and only one set of inter-residue interactions. Amyloid formation differs from this scenario as the same polypeptide sequence can assume multiple conformationally stable states that are defined by very different inter-residue contacts. These conclusions are in agreement with concepts that describe amyloid fibrils as a generic conformational state or a ‘polymer-state’ of the polypeptide chain. Their nature as organic polymers enables polypeptide chains to form structural states for which sequence specificity is less important than for native protein folding reactions, thus leading to amyloid fibril polymorphism [38]. The precursor-to-product relationship of these species remains to be understood [39], and this polymorphic range raises the questions of the major pathways through which they originate, the conditions favoring each ones, and the presence of certain intermediate states that could be targeted. Even if A
oligomers are off fibril formation pathways, they share some structural similarity with protofibrils [40]. The cross-
sheet structure constructs the core of amyloid protofilaments, which represent the filamentous substructures of mature fibrils. Although the basic structural arrangement of the cross-
structure is conserved for different fibrils, they can pack into the three-dimensional quaternary structure in different ways. Such variable protofilament arrangements can give rise to several distinct amyloid fibril morphologies [38]. NMR data of globulomer intermediates, “preglobulomers”, suggested parallel in-register C-terminal
-sheet structures, with different N-terminal conformations. The major difference between fibril-forming oligomers and

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Fig. (4). Summary of NOE constraints obtained from the N-Met-A
1-42 samples. Dashed lines indicate observed NOEs. Circles indicate the backbone amides that exhibit slow exchange in the NH/ND exchange experiments [37].

Fig. (5). Representation of the two stable forms of amyloid
-peptides; the soluble globulomer form and that of insoluble fibril. The parallel
-sheet which involves the hydrophobic C terminus of the peptide and common to both forms is highlighted in blue.

ADDL-like oligomers (globulomers) could be the exposure of M35 patches. Although the M35 patches are necessarily exposed in fibril-forming oligomers to allow their maturation into fibrils, the M35 patches in the globulomer are covered by other residues in the orthogonally packed A
peptides. Using a single layer A
fibril oligomer
-sheet model, molecular dynamic experiments show that the C-terminal
sheet in the fibril oligomer is mostly curved, preventing it from quickly forming a fibril and leading to its breaking into shorter pieces. After the formation of the C-terminal
-sheet, there are two ways for it to interact: the first is by using the M35 patch surface, which leads to fibril formation, and the second is using the surface without the M35 patch, which would favor globulomer formation. The simulations also indicate that
-sheets packed orthogonally could be the most stable species for A
dodecamers. These results raise the hypothesis of the existence of certain “critical intermediates” that can lead to both seeds and other soluble ADDL-like oligomers. Because the formation of a C-terminal
-sheet is

critical in both pathways, it may constitute a “critical intermediate”. Together, these findings lead to one of the most difficult questions related to aggregate polymorphism: are the aggregates likely to have different preferred architectures depending on such physical factors? [40]. It was demonstrated for samples of A
(1-40) fibrils in the presence of different salts that salts tend to stabilize fibrils with a smaller width, nevertheless fibril heterogeneity was observed in fibril samples with and without salts [38]. Experimental data, computational investigation on four classes of A
dodecamers fibril, fibril oligomer, prefibril/preglobulomer cluster, and globulomer models showed that all conformations and selfassembly states pre-exist according to the energy landscape theory, and any external or internal condition can only lead to shifts in their relative populations. The broad A
oligomer variability represents a problem in AD drug design and in planning other therapeutic approaches. Targeting toxicity in an AD late stage may miss

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the reservoir of polymorphic forms. If common links among polymorphic states can be established, then targeting the bottleneck would be more effective [40]. N-TERMINAL TRUNCATED AND PYROGLUTAMATE MODIFIED A
FORMS A
extracted from AD brains present extensive truncations, yielding peptides of differing N- and C-terminal composition. The brains of AD patients are highly enriched in Nterminally truncated A
, generated by the loss of D-A preceding the glutamate at position 3, and pyroglutamate modified forms of these truncated A
peptides [41]. Pyroglutamate-A
(pE-A
) has a higher propensity to oligomerisation and aggregation than full-length A
, triggering the accumulation of neurotoxic A
oligomers and amyloid deposits. In addition, pE-A
has increased resistance to clearance by peptidases [41b, 41c] causing the persistence of these peptides in biological fluids and tissues [41a]. pE-A
formation requires as a substrate amino-terminally truncated A
beginning at glutamate (3 or 11), followed by the cyclization of exposed glutamate to pyroglutamate. This process causes a loss of three charges for A
3-pE, and six charges for A
11pE, increasing the hydrophobicity and promoting the rapid adoption of
-sheet structures, and then toxic aggregate formation. Moreover, the cyclization exerts an effect on the calculated isoelectric points (pI) of the amyloid peptides, and makes their pI more basic [41c]. However, pE-A
is known to increasingly adopt
-sheet structures in both aqueous and hydrophobic environments, suggesting that the increased propensity to oligomerisation and aggregation of these peptides is not due to an increased hydrophobicity alone [41a]. Many experiments confirmed that N-terminally truncated A
and pE-A
are more toxic than wt A
. The investigation of fibril formation from monomeric A
1-40, A
3-40, and pE-A
3-40 (50 μM) by a ThT fluorescence assay at pH 7.0 and 8.0 values, showed that at pH 7.0 fibrils are very rapidly generated from pE-A
3-40, while A
1-40, which shows better solubility at pH 7.0, aggregates with a prominent lag phase. Similarly, such fibril formation has been also observed with A
3-40. The truncation results in a shortened lag phase but slower fibril elongation as concluded by the lower slope in the exponential phase. Thus, the truncation seems to accelerate the seed formation process, which is further enhanced by the cyclization of N-terminal glutamic acid. Further investigation of this process with dynamic light scattering (DLS) confirmed that at a 5 μM peptide concentration there is a fast occurrence of larger aggregates for the pE-A
samples at time point 0 [41c]. A
3pE-40 has specifically been found to be more cytotoxic to hippocampal and cortical neurons than A
1–40, A
1–42 and A
3pE-42 [41a]. A model mouse expressing only N-truncated pyroglutamate A
peptides was generated by Wirths and co-workers [41b]. TBA2 transgenic mice revealed obviously macroscopic abnormalities, including growth retardation, cerebellar atrophy, Purkinje cell loss and premature death; their expression level of N-truncated Abeta was anyway lower than that of a six month old APP/PS1KI mice expressing usual AD pathology [41b]. The rapid aggregation of pE-A
3-40 compared to A
140 is also accompanied by faster structural changes. Both

Airoldi et al.

peptides are initially in random coil structure, conformational changes of the pE-A
-containing samples then starts after day 3, while the first structural changes of A
1-40 are observed on day 5, and after twelve days a lower negative CD signal at 217 nm clearly supports higher
-sheet content in pE-A
3-40 compared with A
1-40. Differences in the fibrils morphology were evaluated by transmission electron microscopy. Amyloid peptides without N-terminal pE showed the typical morphology of mature fibrils. In contrast, however, the pyroglutamate aggregates form a dense meshwork of short fibrils that self-associate laterally to form irregular bundles [41c]. The origins of amino-truncated A
substrates for pyroglutamate formation are not well understood. The loss of DA preceeding the glutamate at position 3 is necessary for pEA
3 formation; however, it is unclear whether this truncated precursor is directly liberated from APP by endoproteolysis, or the full length. A
is post-translationally processed by aminopeptidases. Following the generation of aminotruncated A
, exposed glutamate is converted to pyroglutamate by the enzyme glutaminyl cyclase (QC). QC-catalysed conversion of glutamate to pyroglutamate is favoured under acidic conditions, such as those present in secretory compartments where APP and QC partially co-localise. There is intriguing evidence suggesting that A
truncation may also result from a non-enzymatic process, such as attack from reactive oxygen species (ROS). The interaction of A
fragments with redox-active Cu can cause oxidation of the peptide backbone, resulting in peptide bond cleavage [41a]. METHIONINE 35 OXIDATION Oxidized methionine in position 35 of A
peptides (M35ox) has been detected in samples extracted from postmortem AD plaques [42], in which they represent 10-50% of total brain A
. This methionine could play an important role in AD-associated oxidative stress and may act as an electron donor for the reduction of A
bound Cu2+ to Cu1+ [43]. Few studies have been performed to explore the consequences of the presence of M35ox on peptide chemical characteristics and biological actions. There are, however, some intriguing findings and some contradicting reports. The substitution of synthetic A
1-42 with methionine sulfoxide M35ox results in enhanced A
1-42 mediated cellular toxicity. Nowadays it remains unclear if these differences are a consequence of attenuated or accelerated fibril formation. A
1-40 M35ox is dramatically less prone to aggregation and fibril formation as compared with A
1-40, as shown in circular dichroism studies [42]. Most significantly, Met35ox prevents formation of the A
protofibril, which is believed to be a common toxic intermediated in the amyloidosis of many proteins [43]. This is also confirmed by the inhibition of conformational switching of A
1-40 M35ox from random coil to
-sheet, observed with NMR using millimolar peptide concentrations [42]. To investigate the relationship between the oxidation state of M35 and A
-aggregation, Zagorski et al. [43] utilized NMR spectroscopy to obtain site-specific structural information. The studies were performed in aqueous solution, at pH=7.2 and 5°C, on the peptides A
1-40 M35red, A
1-40 M35ox, A
1-42 M35red, A
1-42 M35ox, kept in almost monomeric form by previous elimination of pre-aggregated

A
Monomers, Oligomers and Fibrils

seeds. The data collected supports the absence of any welldefined secondary or tertiary structures, as the four peptides are all largely random extended chains. On the basis of NOE and chemical shift data, the A
1-40 M35red and the A
1-42 M35red peptides have residual
-strand structure. However, two hydrophobic regions (L17-A21 and I31-V36) have turn or bend-like conformation at two largely hydrophilic regions (D7-E11 and F20-S26). Aging studies performed at 37°C demonstrated that the M35ox reduces gradual upfield chemical shift movements on the 2H NMR signals for the H6, H13 and H14 side chains. On the basis of these data, the authors propose that the conversion of M35red to M35ox inhibits amyloid formation by preventing early, site specific hydrophobic and electrostatic associations. So, different association steps may be involved in amyloid formation for the A
1-40 and A
1-42 peptides, respectively. In particular these peptides adopt predominantly random and
-strand conformations and M35 oxidation reduces the propension for
-strand structures at both the C-terminal and central hydrophobic segments. In general, the driving force for
-aggregation and fibrillation is hydrophobic, with electrostatic interactions playing an important but secondary role. In the first steps of A
assembly formation, both hydrophobic and bend-like structures are necessary to produce amyloid fibrils. Thus, the A
1-40M35ox peptide, which displays the least tendency for turn or bend-like structures, and essentially no
-strand structure, is the least prone to aggregation and does not form amyloid fibrils. On the other extreme, the A
1-42-M35red peptide has turn or bend-like tendencies and has the greatest number of residues favoring
-strand structure, and consequently undergoes very rapid
-aggregation [43]. MD simulations were performed by Triguero et al. with the GROMACS software package on full-length wt-A
1-40 and wt-A
1-42 monomers and their oxidized forms, to explore the conformational dynamics of these peptides in aqueous solution. The aim of these simulations was the exploration of the conformational changes that occur within the secondary structure of the regions 17-21, 23-28 and 29-35 of the A
monomers, which have been proposed to play a critical role in aggregation mechanisms. In the monomeric form of wt-A
1-40, the regions 17-21 and 23-28 exist in a well-defined helical conformation. The first fifteen residues (D1-E15) adopt an overall
-hairpin structure with the
-bend in the S8-Y10 region and tails extending over D1-D7 and E11-Q15 residues. During the simulation, the
-bend conformation in the S8-Y10 region changes into a turn and a
-sheet. The next 20 residues (K16-M35) constitute the most interesting part of the peptide, which contains all the critical regions. In fact, this segment (K16-M35) exhibits overall
-hairpin-like structure with the
-bend between K16-A20 and tails stretched in the E11-Q15 and E21-M35 regions. Quite early in the simulation the helical structure of the 17-21 region completely transformed into a bend conformation. This bend or turn conformation is stabilized by the presence of hydrogen bonds between Q15-L17 and L17-F20 residues. The V24-N27 turn segment, stabilized by the D23-K28 salt bridge, is involved in the formation of collapsed coil and
-helical structures in solution. The loop formed by this segment is known to exist in the A
fibrils. The overall structure of monomer possesses three well defined bend regions separated by coils segments, while wt

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209

A
1-40 exists in an overall
-hairpin-like conformation [16]. The first bend structure of A
1-40 M35ox monomer is present in the H6-Y10 region; the second bend lies in the V12F20 region and the third bend is in the C-terminus (S26-L34) segment. The N23-K28 salt bridge, which promotes the hairpin-like structure of the wt A
1-40 monomer, has not been observed for A
1-40 M35ox. The absence of this salt bridge with subsequent destabilization of the turn region could be the main reason for the absence of
-hairpin structure in A
1-40 M35ox monomer. Another difference between these two monomers is the lack of a close interaction between the N- and C-terminal regions of A
1-40 M35ox. This interaction gives rise to the loop-like conformation observed in wt A
140. Overall, A
1-40 M35ox monomer possesses 16% more bend conformations and 13% less
-sheets in comparison to wt A
1-40. These results indicate that the oxidation of M35 in the monomeric form induces significant structural changes in the secondary structure, which may be associated with the attenuation of the aggregation of A
1-40 M35ox. In A
1-42 M35ox the 17-21 region exists in a helical conformation, whereas in A
1-40 M35ox, this region is dominated by coil and bend conformations. In the former, the turn 24-27 segment adopts the hairpin-like conformation stabilized by the D23-K28 salt bridge. The second hydrophobic region in A
1-42 M35ox mostly forms the turn, whereas in A
1-40 M35ox exists in a stable bend conformation. These structural differences could contribute to the difference in the aggegation rates of these two peptides [16]. INTERACTION WITH CATIONS Amyloid deposits contain high concentrations of redox active metals, such as copper, zinc, and iron [44]. Cu2+and Zn2+ are known to significantly affect the rate of aggregation and morphology of A assemblies in vitro [45]. In addition, the in vitro binding of the A
peptide with the metal ions (Cu2+, Zn2+, and Fe3+) seems to be able to promote [46] or hinder amyloidosis [47]. It is thought that these binding events in vivo could trigger or prevent amyloidosis in the AD brain [28a], as supported by encouraging results from clinical trials of drugs with metal chelating properties. In addition, there is evidence that APP is involved in copper homeostasis [48]. APP has a selective metal-binding site and is able to reduce bound Cu2+ to Cu+. It is also involved in the regulation of copper levels, and its expression is affected by copper concentration. Also other metal ions, such as Zn2+ and Fe2+, are known to interact with APP. Recently, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of such free zinc pulses in promoting A
aggregation has never been established. Significant progress has been made in the last years about the Cu2+ and Zn2+-binding site, but the exact coordination of these metals is still unknown. Cu2+ and Zn2+ A complexes are very dynamic. The co-ordination environment for Cu2+- A changes with pH and, at neutral pH, at least two binding modes coexist and are in fast equilibrium. NMR spectroscopy experiments suggest that Zn2+ and Cu2+ bound to A undergo metal-ion exchange between the peptides in the ms or sub-ms time range [49]. A assemblies formed in the presence of metals are not considered as amyloid, because of the absence of tinctorial properties and fibrillar morphology, and they can be com-

210 Current Bioactive Compounds 2011, Vol. 7, No 3

pletely dissociated with the use of chelating agents or by a slight increase in pH. Intriguingly, it seems that metal binding induces a fold which effectively protects against further propagation into fibrillar form and that the removal of the bound metals causes dissociation of the peptide aggregates rather than further assembly into amyloid. Turbidity measurements showed that the aggregation rates of both A
1-40 and A
1-42 were significantly increased in the presence of either Cu2+ or Zn2+, in agreement with previous investigations. Aggregates formed in the presence of a molar excess of divalent metal ions did not have the classic morphology of amyloid fibrils. Nevertheless, it was impossible to morphologically distinguish aggregates formed in the presence of Cu2+ from those formed in the presence of Zn2+. Taken together, these results suggest that A
aggregates formed in the presence of Cu2+ or Zn2+ are assembled from A
s with altered secondary structure and are less compact than fibrillar complexes. Thioflavin T analysis shows that Zn2+-A
complex may tolerate assembly into a more fibrillar-like fold, whereas Cu2+ more efficiently prevents formation of a fibrillar assembly. Furthermore, all metal bound A
aggregates could be efficiently converted to monomers through the addition of a chelating agent, suggesting that their mode of assembly is different from that of the fibrillar forms [45]. The binding affinity of the metal ions to A
is pHdependent. Cu2+ shows a higher affinity under mild acidic conditions, while Zn2+ affinity is less pH-dependent over the pH range 6.5-7.5 [50]. When pH is lower than 6.0, Zn2+ binding does not take place; at these pH values His residues are mainly protonated, suggesting a prominent role for these residues in metal binding. As a consequence, under physiological conditions, A
shows an higher affinity for Zn2+, while under mild acidic conditions, such as those that follow an inflammatory process, A
binds preferentially Cu2+ [51]. This suggests that, under normal conditions, Zn2+ is able to protect against Cu2+–induced A
toxicity, which is a consequence of physiologic acidosis [52]. In the light of these considerations, detailed knowledge of the metal-A
co-ordination environment could aid in the development of compounds with more effective and specific metal chelating properties as eventual treatments in AD. In 2006 Danielsson and co-workers [53] described a detailed molecular model of Zn2+ binding on the A
full-length by the combined use of heteronuclear NMR, fluorescence and CD spectroscopy. They identified a selective Zn2+-binding site composed of H6, H13, H14 and the N-terminal residue D1. The binding to this region seems to be specific and affects mainly the ligands and the neighboring residues. There is a turn at E3 that bend the N-terminal towards H6 and a second turn at G9, that put H13 and H14 near Zn2+ (Fig. (6)). A second binding site for Zn2+ can be hypothesized, constituted of D23, V24, S26 and K28, with an induced turn at G25. By using time-resolved structural and spectroscopic techniques, Noy and co-workers [54] showed that interactions between Zn2+ and A
1-40 occurs in less than a millisecond and initiates A
1-40 conformational changes and aggregation in a few milliseconds. On a longer time-scale, transmission electron microscopy (TEM) and analytical

Airoldi et al.

N-terminus

H N

N

HN

N N NH

C-terminus

Fig. (6). Schematic representation of the structural model of A
binding zinc or copper. The structure was constructed using a combination of signal intensity changes, relaxation data and induced amide proton stability.

ultracentrifugation (AUC) indicated that these interactions prevent the formation of the typical amyloid fibrils, and induce the accumulation of large unorganized aggregates of smaller non-fibrillar forms of A
. In particular, the rapid binding of zinc ions to multiple A
1-40 sites may interfere with realignment by practically locking the relative positions of the
-sheet peptide cores, thus preventing realignment and inhibiting fibril growth (Fig. (7)). Consequently, the lifetimes of pathogenically related non-fibrillar intermediates are prolonged. Authors propose a general pathogenic pathway, in which Zn2+ represents a class of molecular pathogens that effectively stabilizes transient, toxic pre-aggregate amyloid forms by interfering with the process of A
selfassembly which leads to insoluble, non-pathological fibrillar forms. Hou and Zargorski [55] characterized the binding of Cu2+ to A
1-40 by heteronuclear NMR. Their data, obtained on the native A
1-40, support a model in which the metal binding does not induce formation of typical amyloid aggregates or folding into a well-defined conformation. In this model, the histidine side chains first anchor Cu2+ binding to the A
monomer (fast exchange rate), followed by deprotonation and/or severe line broadening of the backbone amide NH for E3-V18 (intermediate exchange rate). By contrast, Cu2+ binding to soluble A
aggregates leads to rapid aggregation and nonfibrillar amorphous structures. Without metal, the A
can undergo the normal time-dependent aggregation, eventually producing more ordered, late stage-parallel
-sheet structures. These anomalous binding events may account for the unique proposed “dual role”, where sequestration of metal ions by the monomer is neuroprotective, while that by
-aggregates generates oxygen radicals and causes neuronal death. Using quenched H/D exchange in combination with NMR spectroscopy [45] it was demonstrated that the presence of either Cu2+ or Zn2+ during aggregate/fibril growth had a significant influence on the solvent protection pattern

A
Monomers, Oligomers and Fibrils

Current Bioactive Compounds 2011, Vol. 7, No. 3

211

Also the C-terminal region of the metal complexed A
1-40 aggregates show pronounced differences from the fibrillar form as it also happens for the turn region of the peptide. Differences in solvent protection are observed between the Cu2+ induced and the Zn2+ induced aggregates, reflecting the differences in metal binding properties of A
. Although the properties of fibrils and metal bound aggregates of A
peptides differ significantly, the H/D protection patterns of metal bound A
aggregates still suggests a partial conservation of the
-strand-turn-
-strand fold, similar to the fibrillar form. However, assembly in the presence of metals is unlikely to involve the same intermolecular interactions as within fibrils, as removal of the metals causes dissociation into monomers instead of the continued assembly into amyloid fibrils expected from a more fibril-like fold [45]. A
SDS-STABLE CROSS-LINKED FORMS A positive diagnosis of AD requires both the clinical confirmation of dementia and post-mortem detection of amyloid plaques and neurofibrillary tangles in the neocortex of the brain [56]. Nevertheless, according to different authors, plaques containing fibrillar, insoluble forms of A
do not correlate with severity of dementia, which is mediated by non-fibrillar soluble forms of A
peptides [57]. In facts, a strong correlation exists between the levels of water-soluble A
and the severity of cognitive impairment [58].

Fig. (7). Proposed mechanism of the role of Zn2+ in A
aggregation process. The rapid interchelation of Zn2+ ions to this complicated network of multiple intra- and intermolecular Zn2+-binding sites prevents realignment and formation of fibrillar aggregates.

for both A
1-40 and A
1-42. If compared to the protection ratios in the absence of metal, the overall protection ratio in the presence of metal was highly reduced. In the metalcomplexed aggregates, the D1-K16 region does not have solvent protection. Metal binding occurs within the first 14 residues, inducing a low degree of secondary structure. The remaining parts of both A
1-40 and A
1-42 still display two well-protected bell-shaped regions with an intervening minimum centred on Q27. This is a clear demonstration that metal-complexed A
aggregates reach a
-strand-turn-
strand structural arrangement similar to that of mature fibrils.

These small A
species extracted from the AD-affected post mortem brain specimens migrate on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) as SDS-, urea-, and formic acid-resistant oligomers [59, 60]. An efficient protocol for isolation of low-n amyloid
-protein oligomers from cultured cells was recently reported [61]. McLean and co-workers [62] demonstrated that the buffersoluble fraction of human cerebral cortex contains monomeric 4 kDa A
, and low-n sodium dodecyl sulphate (SDS)stable A
oligomers (8 and 12 kDa). As described by Shankar et al. [63], soluble A
species isolated directly from human AD brains induce several AD-like phenotypes in normal adult rodents by decreasing dendritic spine density, inhibiting long term potentiation and facilitating long term depression in hippocampus. From the mechanical point of view, soluble A
dimers from AD cortex induce their effects by perturbing glutamatergic synaptic transmission. A
containing SDS-stable dimers and trimers at subnanomolar concentration is able to induce cofilin-actin rods in neurites of hippocampal neurons. Rods represent a likely mechanism to explain the synaptic loss associated with early stages of AD and thus represent a novel target for therapeutic intervention because they block transport and cause distal atrophy of the neurites in which they form without death of the neuron [64]. Very recently Selkoe’s group isolated A
dimers from the cerebral cortex of typical AD cases and demonstrated that they first induce tau phosphorylation at specific epitopes characteristic of AD in primary hippocampal neurons, and then produce cytoskeletal collapse and neuritic degeneration [65]. They also examined a synthetic human A
40 peptide in which S26 is mutated to C (A
40 S26C), enabling the formation of stable, disulfide bonded dimers under oxidizing conditions, and this pure dimer was sufficient to induce an

212 Current Bioactive Compounds 2011, Vol. 7, No 3

Airoldi et al.

increasing alteration of the microtubule cytoskeleton and neuritic architecture in a dose dependent manner. Soluble dimers from the human brain appear to have a conformation that is highly potent in inducing these neuronal changes, at concentrations in the subnanomolar range. Two distinct antibodies to the free D1 of A are more potent in preventing the effects of soluble dimers from the AD cortex on the tau cytoskeleton than is a C-terminal specific antibody. This result suggests that endogenous dimers and other oligomers with an exposed N terminus have a conformation that is particularly able to induce neurofibrillary degeneration and synapse loss. Synthetic human A can be converted into this more highly active form by oxidative conditions, suggesting that it is tyrosine oxidation products that are the most active species. In fact, they normally migrate as monomers on SDSPAGE, but they are also able to form higher-molecular mass species upon incubation [66], being the process accelerated by adding Cu2+ and by exposure to oxidative systems [67]. Incubation of A with Cu oxidation system damages tyrosine and histidine residues [68], that co-ordinate Cu in A species. Electrons that cause a reduction in the oxidation state of Cu can originate from the peptide itself, in particular from M35, or from biological reducing agents such cholesterol, ascorbate and dopamine [69]. Barnham et al. [70] used density function theory (DFT) to propose a model according to which covalently linked soluble oligomers can originate from the formation of a reactive tyrosyl radical on Tyr10 and the subsequent tyrosine dimerization, that generates a dityrosine cross-linkage (Fig. (8)) [71]. R CH2

R CH2

R CH2

R CH2

R CH2

O-

OH

OH

H 2O 2 + OH

O-

Fig. (8). Dityrosine formation under oxidative conditions [72].

The pivotal role of the tyrosine residue was further demonstrated by substituting Y10 with A in A sequence (AY10A) [70]. AY10A maintained the ability to form oligomers but, unlike the wt peptide containing dityrosine linked oligomers, it was not toxic to cortical cultures. A
INTERACTION BRANE

WITH

BIOLOGICAL MEM-

A is produced by the cleavage of APP inside the membrane where the peptide is initially located. After cleavage, the peptide leaves the membrane and ends up in monomeric, oligomeric and than aggregated amyloid forms. The oligomeric forms of the peptide alter the membrane integrity of the cell. In vitro, A peptide has been shown to form selective cation channels; as a matter of fact, A changes the Ca2+ homeostasis, increasing intracellular Ca2+ levels [5]. An AFM study suggests that the channels have a well-defined structure and consist of 4-6 peptides (Fig. (9)).

Fig. (9). Hypothetical A pore architectures.

The peptide may create pores by assembling into a barrel formed by -sheets. Alternatively the peptide-membrane interaction induces the formation of
-helices and the helical peptides then assemble into pores. A induces leakage of sodium, potassium and calcium into negatively charged lipid vesicles [73]. The peptide does not insert itself into neutral membranes, while in negatively charged membranes A both inserts into the membrane and induce leakage [74]. In the presence of Cu2+ and Zn2+ the peptide exhibits a structural transition from a prevalently  structure to a high
-helical content as a consequence of the interaction with negatively charged vesicle. This suggests that, in the presence of Cu2+ and Zn2+ the oligomeric aggregate in the membrane is formed by a small number of transmembrane helices, and this may build up the channel, disrupting the membrane integrity. As demonstrated by Curtain et al. [75] the shorter fragment A1-28 and the reversed sequence A1-40 neither insert into the vesicles nor cause any leakage. The hydrophobic region in the shorter fragment A1-28 is too short to penetrate the membrane. The hydrophobic region in the membrane corresponds to 20-23 amino acids in
-helical conformation. The structure of the membrane bound A peptides has been studied in various membranes mimicking media, such as SDS micelles or TFE/water mixtures. The data collected show that two regions, the Cterminal region including residues 29-36 and a central region including residues 16-24, adopt an
-helical conformation. These regions could correspond to the transmembrane segment of the membrane bound soluble A. INTERACTION WITH LIGANDS Despite a great number of different ligands for A peptides that have been proposed, the structural features of their interaction with A are mainly unknown, with sporadic exceptions. Four significant cases are reported. The first example concerns the interaction between A and cyclodestrins, torus-shaped rings built up by different numbers of glucose residues. Qin et al. [76] reported that cyclodextrin interacts with the A-peptide and the interaction inhibits the formation of soluble oligomers with an IC50 of 5mM [77]. The stoichiometry of the A -peptide and

A
Monomers, Oligomers and Fibrils

-cyclodextrin complex is 1:1 [78]. The main interaction involves the aromatic rings of phenylalanines and the hydrophobic cavity of cyclodextrin. The interaction between Apeptide and -cyclodextrin was studied by NMR in 2004 [79]. In their work Danielsson and co-workers demonstrated that the A-peptide has four putative aromatic binding sites represented: F4, Y10, F19 and F20. When the phenylalanines at positions 19 and 20 in the fragment A12-28 are replaced with glycines, and so no other aromatic side chain is present, no interaction occurs. Phenylalanines are in fact necessary for -cyclodextrin-peptide interaction. The central fragment A12-28, containing phenylalanines, interacts with a Kd = 3.8 mM. The most important residue for interaction is phenylalanine 19, as suggested by induced chemical shift changes. In the full-length peptide, a two-site binding involving F19 and Y10 with a total apparent dissociation constant of Kd = 4 mM has been hypothesized. Assuming that the sites are independent, the individual Kd for the two sites are Kd F = 4.7 mM and Kd Y = 6.6 mM, respectively. The fragment A1-9 does not bind -cyclodextrin, and this is in agreement with the idea that the N-terminal aromatic amino acid F4 does not bind -cyclodextrin with any measurable affinity. Other types of cyclodextrins showed no binding. This may be because the hydrophobic pocket of -cyclodextrin has optimal dimensions for the interaction with the aromatic side chains. As a matter of fact,
- and -cyclodextrins have too small or too large cavities. The second example regards the study of the interaction between gangliosides (glycosphingolipids containing sialic acid) and A1-40 peptide. This interaction was characterized in 2004 by Mandal et al. [80] in a membrane mimic environment, dissolving the gangliosides asialo-GM1 and GT1b in a SDS (sodium dodecyl sulphate) solution. As previously demonstrated in literature [81], A peptides associate with gangliosides in Alzheimer’s brains and form specific complexes with gangliosides in vitro. Gangliosides present two main structural components: an hydrophilic oligosaccharide chain, to which one or more sialic acid groups (Nacetylneuraminic acid) are attached, and an hydrophobic ceramide unit, anchoring the gangliosides to the plasma membrane. These compounds are abundant components of neuronal membranes and are involved in important neurobiological events such as synaptic transmission, synaptogenesis and neurodifferentiation. Gangliosides binding induces A peptide oligomerization, as demonstrated by several experimental evidences [82]. GM1 and GT1b gangliosides have four sugar moieties in common; however, GT1b has three additional sialic acids compared to asialo-GM1 that does not contain sialic acid units. The binding of asialo-GM1 and GT1b ganglioside to A1-40 peptide were characterized by NMR spectroscopy. At variance with GT1b, that is unable to bind A, asialo-GM1 addition induces appreciable chemical shift variations in A1-40 amide protons. In particular, asialo-GM1 binding site is defined by region I (E3–V24) and region II (G29–V40) of the A peptide sequence. In the light of these evidences it is possible to understand why GM1 does not bind the shorter A1-28 peptide [82a] . According to the proposed model, in fact, the sugar moiety of GM1 is sandwiched between the two regions of the A peptide. In the case of A1-28, the absence of the main bind-

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ing region G29–V40 would prevent GM1 binding. Gangliosides follow the order (GT1b > GD1a > GD1b > GM1) to induce
-sheet formation, as demonstrated by Matzukaky [83]. These NMR data are in agreement with previous evidences; when the size of the gangliosides increases (GT1b > GD1a > GD1b > GM1) with the addition of sialic acids, it becomes difficult to accommodate the bulky gangliosides between regions I and II of A and -sheet formation proceeds. The third example reports Chen et al. [84] characterization of the interaction between A14-23 and A1-40 fibrils and the short synthetic peptides iA5 (LPFFD) and iA5inv (DPFFL) by NMR spectroscopy. In a previous work, made by Soto and coworkers [85], the -sheet breaker peptide iA5 (LPFFD) was designed from the central hydrophobic region in the N-terminal domain of A, made by amino acids 17-20 (LVFF), in order to specifically bind to the A region implicated in -sheet formation. The second peptide inhibitor shares a common core sequence (PFF), possessing exchanged N-terminal (L for D) and C-terminal (D for L) residues. The superposition of the NMR structures of the two peptides iA5 and iA5inv bound to A14-23 fibrils show different backbone structures in the bound state, but remarkably similar orientations of side-chains with identical properties. Superposition of the two structures of iA5 and iA5inv shows the side-chains of L1 and D5 of iA5 placed at the positions of L5 and D1 in iA5inv. This indicates an identical spatial binding pattern for the different peptides at probably the same binding site of A14-23. At the same time, iA5inv binds to fibrils formed from A14-23 and A140 adopting different structures that display different sidechain orientations for FFL. This suggests a different geometry with respect to binding to A14-23 and A1-40 fibrils. The different structures are consequence of the differences in the A14-23 and A1-40 fibril structures. An antiparallel organization of the -strands along the fibril axis was described for the short amyloid peptide A14-23. The central residues L17VFFA form an hydrophobic core at a single molecular layer, flanked by alternating patterns of positive and negative charges, due to the presence of the charged Cterminal (K16) and N-terminal (E22 and D23) residues. These alternate charges are responsible for interaction of these “building blocks” along and orthogonal to the fibril axis. The parallel arrangement of -strands along the fibril axis for the full-length amyloid peptide A1-40 shows a completely different interaction pattern for possible ligand binding: A1-40 fibrils consist of two -strands that are separated by a 180° bend, that form two in-register parallel -sheets that interact mainly through intramolecular hydrophobic side chain contacts. At the surface of a single molecular layer (cross- unit), the positively (K16) and negatively charged (D22) residues of the first -strand are arranged with respect to each other in a row along the fibril axis. The two charged rows are separated by hydrophobic region (V18, F20). Hydrophobic side-chains of the second -strand form hydrophobic face at the opposite side of the cross- unit. iA5 displays more interactions to A14-23 than iA5inv, reflecting the different affinity of the two peptides. Two general orientations of iA5inv along the hydrophilic phase of the A1-40 fibril surface are allowed. The orientation in which the two negative charges of the ligand are oriented towards

214 Current Bioactive Compounds 2011, Vol. 7, No 3

the row of positively charged lysine residues (K16) is energetically preferred compared to the opposite ligand orientation, in which iA
5inv is rotated around the fibrils axis and the N-terminal positive charge is interacting with the negative charges (E22) on the fibril surface. The negative charges of D1 and of the C terminus of the ligand iA
5inv are interacting with K16(I+2) and K16(I+3) of the fibril, respectively. Furthermore, the aromatic residues F3 and F4 of the ligand are in van der Waals contact with a train of aromatic residues, F20(I), F20(I+1), and F20(I+2), located in three successive
-strands. The hydrophobic residue L5 is hidden between two subsequent hydrophobic residues V18(I+2) and surrounded by aromatic residues. Summarizing, Chen et al. studies demonstrated that iA
5 and iA
5inv peptides bind to the hydrophobic core of A
. Residue K16 and F20 of the A
amyloid fibril serve as an anchor for iA
5 and iA
5inv. Different peptide inhibitors are associated with amyloid fibrils at similar binding sites. Authors hypothesize that competition for hydrophobic and electrostatic interactions, and, at the same time, the incapability of forming hydrogen-bonded
-sheet structures, yield a weakening of hydrogen bonds among subsequent
-sheets of the amyloid fibril, and therefore induce dissolution of
amyloid fibrils. In the last example, our recent findings concerning tetracycline interaction with oligomeric A
1-40 and A
1-42 are reported [86]. Tetracycline possesses an anti-amyloidogenic activity on a variety of amyloidogenic proteins both in in vitro and in vivo models. To understand the mechanism of action of tetracycline on A
1-40 and A
1-42 oligomers we carried out a series of experiments using different biophysical techniques, in particular NMR spectroscopy, FTIR spectroscopy, DLS and AFM. NMR and FTIR data have showed tetracycline ability to interact with A
peptides in a non-conventional manner, characterized by the absence of a specific epitope in the drug and the absence of a well defined binding site on the A
oligomers. DLS and AFM also demonstrated that supramolecular complexes are immediately formed when A
peptides are co-dissolved with tetracycline, preventing the formation of aggregates. Incubation of both A
peptides with tetracycline led to the formation of colloidal particles that specifically sequester oligomers, improving their solubility and preventing in this way the progression of the amyloid cascade. We have hypothesized that the internal structure of aggregates formed by tetracycline with A
oligomers is disordered and non-homogeneous, governed by hydrophobic and charge multiparticle interactions. CONCLUSIONS Several studies concerning the structure of A
peptides and their assemblies have been performed. The main structural features of A
monomers, oligomers and fibrils have been obtained by employing several biochemical and biophysical techniques such as hydrogen/deuterium (H/D) exchange, proline-scanning mutagenesis, infrared and Raman spectroscopies, electron paramagnetic resonance (EPR), solid-state NMR and X-ray diffraction. In this review we collected these interesting scientific results. In particular,

Airoldi et al.

structural information regarding the different A
peptides assemblies, their supramolecular organization, their interaction with cations, biological membranes and known ligands are reported. These data are not conclusive, as some A
assemblies are not yet suitable for structural studies, but allow us to shed light not only on A
peptide structure, but also on the dynamic of their folding and aggregation processes. Both these kind of information are fundamental for the development of therapeutics and diagnostics for Alzheimer’s disease. ACKNOWLEDGEMENT Funding support has been received from the European Community Seventh Framework Programme (FP7/2007e2013) under grant agreement no 212043. REFERENCES [1]

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Accepted: June 23, 2011