Abstract
It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 nm, an oligomer height of about 2– 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aβ peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aβ40 peptide. Less ordered fibrils are observed with the synthetic Aβ42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aβ40, fibrils of Aβ42 are more protected, showing less ordered organization compared to that of Aβ40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.
INTRODUCTION
An important model for studying the process of amyloid formation is the amyloid-β (Aβ) peptide [1]. It has attracted the attention of many researcher groups, being a marker of such a severe disorder as Alzheimer’s disease. At present, synthetic Aβ peptide batches supplied by different manufacturers, as well as preparations of recombinant Aβ peptide, are used in research experiments [2, 3]. Despite the large number of publications devoted to studying Aβ fibril formation, there is no consistent model describing the molecular mechanism of Aβ amyloid formation [4].
During recent decades, significant attempts have been made to identify, isolate, and describe oligomeric particles formed in solution prior to appearance of the fibrils. The interest in studying the oligomer formations of Aβ was driven, in particular, by detection of such particles in the brains of patients with Alzheimer’s disease [5], and also in lysates and solutions with cells expressing the precursor protein of Aβ [6, 7]. The experimental data show that structured protofibrils can form either by sticking to each other or through rearrangement of small relatively unstructured oligomers formed at the very beginning of aggregation [8]. The role of oligomers in propagating the growth of amyloid fibrils has been pointed out in many studies, especially the role of nonamers and dodecamers, which are more toxic than monomers and tetramers, and can associate with the loss of the cognitive ability in rat models of Alzheimer’s disease [9–12]. This interest in studying the oligomers has two explanations: firstly, it appears that the formation of such particles is a vital stage in amyloidogenesis; secondly, a growing body of evidence suggests that oligomers are the greatest menace in terms of pathogenesis. Recently, it has been demonstrated that Aβ42 oligomers have a direct toxic effect on the blood-cerebrospinal fluid barrier, that negatively affects brain homeostasis [13].
The aggregation of Aβ peptide is preceded by the development of a number of metastable non-fibril structures, detected by using the atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques [14–17]. Some of the formations look like small spherical beads, 2-5-nm in diameter, and others look like small beads on a string, with individual beads having also the diameter of 2– 5 nm. The third type form some ring structures, which have likely been created as a result of closure of the structures similar to the beads on a string.
There is no basic understanding of how both structural and kinetic parameters of the macromolecule interactions can be translated into the information about the mechanism of fibril formations. The understanding molecular mechanism of Aβ amyloid formation will allow developments of the high resolution biological models of protein conformational changes and searching pharmacological chaperones to correct misfolded forms of proteins. In this paper we propose a new model of structural organization of an amyloid fibril.
MATERIALS AND METHODS
Synthetic samples
Synthetic samples of human Aβ40 (purity 95%, lot No 054M4822V) and Aβ42 (purity 95%, lot No SLBJ2877V) peptides used in our experiments were purchased from Sigma-Aldrich.
Recombinant Aβ40 and Aβ42
Due to high hydrophobicity of Aβ peptide we use a construction coding a fusion protein to obtain the sample. The sequence of Aβ peptide was fused with the sequences of thioredoxin protein, His6 and cleaving site for factor X. The fusion protein yielded by affine chromatography was cleaved with factor Xa (GE Healthcare, UK) that released Aβ peptide. After the proteolysis the sample was separated by affine chromatography while free Aβ peptide was in breakthrough fraction and thioredoxin bound with the column due to His6. Then the breakthrough fraction was additionally purified by size exclusion chromatography. The purified Aβ40 and Aβ42 peptides were stored at – 70°C.
Electron microscopy
To facilitate dissolving, all the samples were initially dissolved in DMSO (5% final concentration), then samples were concentrated up to 0.1– 0.2 mg / ml and the buffer (50 mM Tris-HCl, pH 7.5) was added. Samples for electron microscopy (EM) studies were prepared by the method of negative contrast. A copper grid (400 mesh) coated with a formvar film (0.2%) was mounted on a sample drop (10μl). After 5– 10 min absorption, the grid with the preparation was negatively stained for 1.5– 2.0 min with 1% (weight/volume) aqueous solution of uranyl acetate. The excess of the staining agent was removed with filter paper. The preparations were analyzed using a JEM-1200EX transmission electron microscope at the accelerating voltage of 80 kV. Images were recorded on the Kodak electron image film (SO-163) at nominal magnification of 40,000.
Mass spectrometry analysis
Mature fibrils were obtained in the same way as for electron microscopy except but the time of incubation which was 24 h. Amyloid fibrils were isolated from monomer forms and oligomers by pelleting. After that they were treated with a mixture of proteases (trypsin, chymotrypsin, and proteinase K). The fragments split off by the proteases were washed, and the remaining fragments included in fibril structures were pelleted once again and isolated. Optimal conditions and times of protein treatment with the proteases were chosen so that the monomer form would be completely degraded while the fibril moiety would maintain long fragments. Then the isolated residual fibril structures were dried using a vacuum concentrator, after which they were dissolved in a small formic acid volume and thereafter in 10 mM ammonium acetate buffer.
The obtained set of peptides was analyzed by the method of tandem mass spectrometry. To this end, the mixture of peptides obtained after hydrolysis was separated by HPLC, the peptides washed off from the column were analyzed by a high-resolution mass spectrometer (Orbitrap Elite mass spectrometer, Thermo Scientific, Germany). Then the peptides were identified using the Peaks Studio 7.5 program. This program makes it possible not only to identify peptides but also to estimate their relative concentration in the probe.
Modeling Aβ42 amyloid structure based on the size of folding nuclei of fibrils
As a basis for the design of the structure of the Aβ42 peptide in fibril we chose the available structure of wild-type peptide Aβ40 (PDB entry 2M4J [18]). This structure was obtained from samples seeded with material taken from the brain of an Alzheimer’s sufferer [18]. Unlike the structure of Aβ42 (PDB entry 2BEG) [19], where the N- and C-termini are close to each other, in the 2M4J structure, the N- and C-termini of the Aβ40 peptide are remote, and furthermore, the C-terminus is located deep inside the fibrils. It has been shown that some inter- and intra-contacts for Aβ40 and Aβ42 fibrils were the same, as demonstrated by solid-state NMR data where these structures were found to be an in-register parallel β-sheet pattern [20–22]. The side chain packing registry within the β-turn-β motif of Aβ42 fibrils was similar to the one observed previously in Aβ40 fibrils. Also, the β-strand-turn-β-strand backbone is similar for the fibrils formed from both peptides. This data is consistent with the previous studies showing that the mixture of Aβ40 and Aβ42 peptides is homogeneous in amyloid fibrils, suggesting that Aβ40 and Aβ42 fibrils are likely to have the same structural architecture. Moreover, recent solid state NMR studies of Aβ42 fibrils [23] have demonstrated the packing of single monomers inside the fibril to be almost identical to that of Aβ40 reported in [18]. Therefore two amino acid residues Ile and Ala were added at the C-terminus of each chain in the pdb file 2M4J. Using the program WHATIF/YASARA [24] the dodecamer model structure of the Aβ42 peptide wasobtained.
RESULTS AND DISCUSSION
Morphology of recombinant and synthetic Aβ fibrils
A mixture of fibrils of different morphology is always present in solution, and several different types of fibrils can be seen under the same conditions. This brings about a difficulty in image analyses, requiring sorting of fibrils according to their morphology, and performing separate analyses for every type of insulin, Aβ peptide, β2-microglobulin, glucagon, amylin, calcitonin, etc. It is not completely excluded that different fibril morphologies result from similar but not identical pathways of fibril formation. The differences may appear at initial steps of fibril formation, including the step of nucleation. For almost all proteins and peptides, the size of a protofibril nucleus is still unknown. Nucleation is the restricting step in the amyloid fibril formation, as the former can start only when the concentration of amyloid proteins exceeds the critical one. After the nucleation, the fibrils grow rapidly. For Aβ42 the size of the primary nucleus of the amyloid protofibril is larger (three monomers) than that of Aβ40 (two monomers), although the mechanism of amyloid fibril formation must be similar (Fig. 1) [25]. The sizes of secondary nuclei are also different: two monomers for Aβ42 and one monomer for Aβ40.
According to the data of the EM analysis, at 50 mM Tris-HCl (pH 7.5) the Aβ40 and Aβ42 peptides form fibrils up to several μm long, and the diameter of single fibrils of about 8-9 nm (see Figs. 2, 3).
Aβ40 fibrils are characterized by a tendency to lateral association of individual fibrils with the formation of structures in the form of tapes of various widths [26, 27]. Association of two thin fibrils from the recombinant Aβ42 peptide can be seen on Fig. 2. Aβ42 fibrils are characterized by a greater polymorphism of the diameter of mature fibrils compared to Aβ40 fibrils. For the recombinant Aβ42 fibril diameters vary between 8– 35 nm, and for the synthetic fibrils it ranges from 8– 20 nm. At the same time, the Aβ42 fibrils are much rougher compared to the Aβ40 fibrils and do possess a tendency to branching. Figure 3 shows the images of the fragments of thin fibrils (8-9 nm) formed by the recombinant and synthetic Aβ40 and Aβ42 peptides. The fibrils look as if they are being composed of two filaments. However, at higher magnification it can be seen that both types of fibrils are formed by circular shaped oligomers with diameter similar to the one seen in the thin fibrils (Fig. 5).
The ring-like oligomers in fibrils are packed either in a directly ring-to-ring, or ring-on-ring with a slight shift. Resulting from this packing are the thinnest fibrils. If such fibrils are viewed under low magnification, they seem to consist of two filaments. But this is a staining phenomenon: the staining agent penetrates the holes in the oligomers and forms a joint band when viewed under low magnification (Fig. 4d). Such ring oligomers are seen in microphotographs from the very start of fibril formation, and their number decreases as the incubation time increases. This shows that fibrils grow due to inclusion of ring oligomers into fibrils. Oligomers enter fibrils not only in the oligomer-to-oligomer way, but they also interact with fibrils chaotically, resulting in fibril thickening (Fig. 4c). Time-lapse AFM studies allowed observing how the sides of early fibrils pick up oligomers through lateral interactions that make fibrils thicker[8, 28].
Polymorphism of Aβ fibrils, an important characteristic of Aβ peptides, is achieved by varying association of oligomers. Aβ42 peptide is more prone to the association of oligomers in various manners, seen in a rougher surface of fibrils and a larger spread of diameters of Aβ42 fibrils. Nevertheless, it is possible to measure an approximate diameter of the ring, which coincides with that of the fibril (8-9 nm) and determine an approximate diameter of the hole in the oligomer (3-4 nm), accounting for the negative staining resolution (±2 nm). Moreover, it is possible to estimate an approximate height of the ring, which corresponds to the fibril diameter in the inflection point (2-3 nm) (Fig. 5). Thus, using the EM method, we have demonstrated that fibrils of these peptides are formed by association of ring-like structures (Fig. 5). Negatively stained TEM images of fibrils extracted from AD brain tissues are very similar to our EM images [18]. Similar structures are observed elsewhere [29].
It should be noted that both peptides with such mechanism of fibril formation according to the X-ray analysis possess the diffraction patterns with reflections which are typical for a cross-β structure[30].
Determination of amyloidogenic regions in Aβ40 and Aβ42 peptides
Regions of Aβ40 and Aβ42 peptides, forming intermolecular interactions in amyloids, were determined using a combination of limited proteolysis and high-resolution mass spectrometry (MS). It is known that the amyloid core of a fibril is inaccessible to proteases [31]. Thus, it is possible to determine regions of a monomer involved in this core by cleaving non-incorporated regions and identifying the non-cleaved region of molecules. In this work, mature fibrils were treated with a mix of proteases (trypsin, chymotrypsin, and proteinase K) to cut off the regions of Aβ peptide not incorporated into the amyloid core. After the proteolysis, amyloid cores were isolated by centrifugation and treated with formic acid to release peptides that formed these cores. Amyloidogenic peptides were separated using the liquid chromatography/tandem MS technique, and identified by PEAKS Studio 7 program (see Methods for details). The accumulated fragments of amyloid structures of Aβ40 and Aβ42 peptides are shown in Fig. 6 (see also Supplementary Tables 1 and 2, Supplementary Material). The obtained data showed that protease-resistant regions were the ones from 26 to 40 amino acid residues in Aβ40 peptide molecule, whereas a more complicated distribution of protected regions of the chain is observed for Aβ42. In some molecules, the region from 26 to 42 amino acid residues is protected, but in other molecules only several N-terminal amino acid residues are excised, and the remaining part is inaccessible to proteases. These data agree with the EM data showing that the diameter of the recombinant Aβ42 fibrils varies much greater (8– 35 nm) as compared to that of Aβ40 (8-9 nm).
The main structure element of these peptides is a ring-like oligomer with the diameter of about 8-9 nm, the internal cavity of about 2-3 nm, and the height of ring about 3-4 nm. Fibrils are generated through the interaction of such oligomers (dodecamers, Fig. 7), which gives rise to fibrils of several microns in length. The packing of ring oligomers in the fibrils is varying due to their varying orderliness, resulting in a polymorphism. Most ordered association of ring oligomers is observed for the recombinant Aβ40 peptide. Moreover oligomers in fibrils may interact with each other with large overlaps, causing an increase of the fibril width in the sites of its twisting (see Figs. 4 and 7). The most disordered interaction of ring oligomers is observed in the synthetic Aβ42 peptide (Figs. 3 and 5). The polymorphism of this preparation is revealed only in varying widths of fibrils. These observations permit us to conclude that the formation of fibrils of short peptides proceeds via ring-like oligomer structures.
Two explanations for amyloid polymorphism have been proposed [32]. (1) Different morphology is the result of different models of lateral association of protofilaments without significant changes in the molecular structure [33]. (2) Different morphology is the result of significant variability in the molecular structure on the protofilament level. In this paper, based on the data of studying the formation of amyloid fibrils in synthetic and recombinant Aβ40 and Aβ42, we demonstrated that the first alternative, rather than the second as stated above, is likely to be correct. It should be noted that the mechanisms of the association of oligomers into fibrils might be different, but the structure of oligomers is determined by the primary structure of the protein. Taking into account such molecular mechanism of Aβ40 and Aβ42 amyloid formation it is easier to explain such process as different polymorphism (different laboratories are studying different polymorphs), affinity of specific antibodies to protofibrils and oligomers [11], discrepancies between H/D exchange and solid state NMR [34], small fluorescence intensity of thioflavin T, fragmentation, spontaneous fibril breaking and rejoining [35], dodecamer is the terminal species observed in the Ion Mobility-MS experiments [36], and the presence of water in the holes of ring-like oligomers [37, 38]. Understanding the molecular mechanism of amyloid formation should help designing new drugs to block this process.
