Amyloid fibrils. Many natural polypeptide chains are able to form amyloid fibrils in vivo or in vitro [4,14,18,23–33]. Current definitions of amyloid or amyloid fibrils can differ consid-erably depending on whether they are used in a biophysical or physiological context [34–37].
To circumvent confusion of terms, a structure based definition of amyloid fibrils and other polypeptide aggregates [35] will be used in the following. Regardless of the sequence or na-tive fold, amyloid fibrils can be defined as self-assembled, elongated and unbranched (fibrillar) polypeptide aggregates with cross-β conformation (see Figure 1.2) [28,32,38]. The cross-β architecture, as revealed by X-ray fiber diffraction data, is described as stacked β-strands that run perpendicular to the fibril axis and exhibit extensive hydrogen bonding along the length of
the fibril [39–42]. To this end, Aβ [43–45], amylin [46], fungal prion [47], and PrP peptide [48]
fibrils have been characterized by the application of solid-state NMR (ssNMR) [49] and all of them were found to be composed of in-register parallel β-sheets. Most notably, an in-register parallel β-sheet potentially maximizes favorable interactions between hydrophobic, as well as polar side chains by aligning the residues with themselves. Amyloid fibrils are polymorphic structures and a wide range from thin, straight fibrils to wider, striated, twisted ribbons have been observed [31,43,44,50,51].
Analyses of experimentally observed aggregation kinetics suggest different mechanistic explanations for possible pathways and rate-limiting steps of amyloid fibril formation [52–56].
Recent reports suggest that there may be similarities in the assembly mechanism of amyloidogenic peptides and proteins, as the overall growth process exhibits the characteristics of a nucleated growth [52,53,57,58]. In essence, the amyloidogenic self-association of peptides or proteins will initially populate high energy states, in which the sampled state of highest energy is termed the nucleus. Once the critical nucleus forms, either by addition of further assembly units or by stabilization through conformational change, the cooperative fibril formation process will be downhill in free-energy [29]. Nevertheless, it has not been possible to directly probe the nucleation event so far, thus leaving the details of the process not well understood.
Amyloidosis - a conformational disease. A number of human pathologies is associated with the deposition and accumulation of stable, ordered, filamentous aggregates of a specific protein or peptide in a variety of organs and tissues [59,60]. These include neurodegenerative diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD), type 2 diabetes, dialysis-related amyloidosis and familial systemic amyloidosis. Prion proteins that cause transmissible spongiform encephalopathies (TSEs) also form amyloid fibrils [61,62]. The self-propagating variations in the molecular structure of amyloid fibrils and amyloid-like aggregates are believed to be responsible for multiple strains of mammalian prions and yeast prion phenotypes [31].
Oligomers and nonfibrillar aggregates. Recently, evidence has accumulated suggesting that, instead of mature amyloid fibers, soluble oligomers are the more pathogenic species and primary causative agents of several types of amyloid diseases [4,63–70]. Oligomers were found to exhibit high levels of cytotoxicity in cell cultures and also have been found to localize in human tissue [64,71]. Furthermore, the presence of oligomers correlates better
Figure 1.2: Structure of amyloid fibrils. Electron micrograph of long, unbranched Aβ(1-40) fibrils with characteristic twist. Twist crossovers at regular distances are indicated by white arrow heads (A). Schematic representation of fibrils composed of 2, 3 and 4 protofilaments (B). Left-handed fibril chirality of Aβ(1-40) amyloid fibrils observed with transmission electron microscopy (TEM) after platinum side shadowing (C). On the left: Schematic representation of cross-βsheets architecture in a fibril. The black arrow indicates the orientation of the fibril main axis, backbone hydrogen bonds are represented by dashed lines. On the right: The typical fiber diffraction pattern with a meridional reflection at 4.7Å (black dashed box) and an equatorial reflection at 6-11Å (white dashed box), which correspond to the repetitive spacings of main chain and side chain atoms in the protofilament structure, respectively (D). Figure adapted from [18] and [35].
with the pathological changes than the insoluble fibrillar deposits do [72]. This indicates a possible origin for the relation between amyloid formation and cellular toxicity, namely the disruption of membrane integrity by oligomeric species. Several possible modes of membrane pertubations offer an explanation for cellular stress, e.g. through loss of chemical potential
and compartmentalization [13,70,73]. In addition, exposure or association with membranes was also found to be correlated with an increased rate of amyloid formation, leading to highly structured fibrillar states [74,75]. A variety of morphologies have been described for oligomers, which are usually observed during the incubation of amyloidogenic peptide solutions. These include prefibrillar and fibrillar oligomers, annular protofibrils, among others [56,63,65–68,71].
Molecular weights of the oligomeric precursor state are reported to span a range from a few ten to hundreds of kDa, corresponding from dimeric to multimeric aggregates, respectively.
However, it is difficult to obtain the biochemical properties as well as structural information of the oligomeric species in experiments. Most notably the investigations are hindered by the transient, polymorphic, and noncrystalline behavior of the oligomers [66,71,76]. Nevertheless, several studies report that: (a) amyloid oligomers contain β-sheet rich structures [77,78];
(b) oligomeric states are often heterogeneous and different sizes of the oligomers coexist in solution [79]; (c) antibodies recognize common structural features of oligomers formed from different amyloidogenic proteins [64]. Furthermore, given their qualitatively different morphologies from the characteristic appearance in TEM and AFM images, oligomeric precursor states and amyloid fibrils are surprisingly similar in molecular conformation and supramolecular structure [29,67,80]. In addition, crystal structures of peptide macrocyclics in an oligomeric (tetrameric) form were reported recently [81], providing additional structural constraints on this particular aggregation state. An interesting finding was that except for the observed sheet-to-sheet packing, they share most structural features of the fibrillar forms [82].