In his 1972 Nobel-Prize acceptance speech, Anfinsen stated that "the native conformation [of proteins] is determined by the totality of inter-atomic interactions and hence by the amino acid sequence, in a given environment" which does not always favor the normally functional and folded state of proteins. Consistent with Anfinsen's theory, the conformational-change hypothesis postulates that one of 17 normally soluble and functional human proteins could undergo structural alterations under partially denaturing conditions leading to self-assembly and amyloid fibril formation [235
]. Besides disease-associated amyloid-forming proteins, and proteins that naturally form non-pathological, functional amyloid-like fibrils (reviewed in [236
]), disease-unrelated proteins [237
] and artificially designed peptides [238
] have been found to form amyloid under particular non-native conditions. To the best of our knowledge, the ability of disease-unrelated peptides and proteins to form amyloid fibrils was first reported by Guijarro et al.
] and Litvinovich et al.
]. The src-homology 3 (SH3) domain of bovine phosphatidyl inositol 3-kinase (PI3K), an 85-residue, β-structured protein, was shown to aggregate slowly and form amyloid fibrils under acidic pH [241
]. Thenceforth, the disease-unrelated SH3 domain has served as an excellent model for systematic studies examining structural properties of amyloid fibrils and molecular mechanisms of amyloid formation [243
]. The PI3K-SH3 was shown to adopt a compact denatured state under acidic conditions before formation of amyloid fibrils [247
]. Limited proteolysis studies showed that PI3K-SH3 at low pH had a partially folded conformation [247
] and progressively displayed enhanced susceptibility to proteolysis, suggesting that the protein became more unfolded in the early stages of aggregation [247
]. In contrast, the amyloid fibrils that formed over longer periods of time were resistant to proteolysis [247
]. It was suggested that the protein aggregates formed initially were relatively dynamic species and this flexibility allowed dynamic species and this flexibility allowed for the particular interactions leading to formation of the highly ordered fibrils [247
After Litvinovich et al.
demonstrated formation of amyloid-like fibrils by self-association of murine fibronectin type III module [242
], others reported that similar conversions in a number of disease-unrelated proteins could be induced in vitro
by a deliberate, rational choice of excipient conditions [248
], Examples (reviewed in [237
]) include human apolipoprotein CII, ADA2H, amphoterin, stefin B and endostatin, murine VI domain, equine acylphosphatase (AcP) and apomyoglobin, monellin (Dioscoreophyllum camminsii
), and yeast phosphoglycerate kinase. Fezoui et al.
reported de novo
design of a monomeric α-helix-turn-α-helix peptide (αtα) which converted to β-sheet-rich amyloid-type, protease-resistant, 6–10-nm fibrils at 37°C in a neutral aqueous buffer [238
]. Formation of fibrils from full-length proteins requires solution conditions that partially or completely disrupt the native structure of the protein but not completely disturb hydrogen bonds [248
]. It was observed that proteins with as few as four residues, and amino-acid homopolymers that are unable to fold into stable globular structures, form fibrils readily [237
]. Therefore, it has been suggested that the ability to form amyloid fibrils could be a generic property of polypeptide chains [237
In one study of pre-fibrillar assemblies of disease-unrelated proteins, tapping-mode AFM was used to follow the process of HypF-N aggregation which was induced by incubating the protein in the presence of trifluoroethanol [252
], HypF-N was shown to aggregate hierarchically through a number of distinct steps with morphologically different intermediates [252
]. Initially, globular assemblies appeared, which subsequently self-assembled into beaded chains, similar to those found for amyloidogenic proteins [253
]. Subsequently, these organized into crescents, large annular and ribbon-like structures (), and eventually assembled into mature fibrils of different sizes [252
]. The globule height was measured to be 2.8–3.0 nm [252
]. Although HypF-N and AcP are similarly prone to conversion from a predominantly α-helical conformation to one rich in β-sheet, HypF-N aggregation rate was found to be dramatically higher (~1,000-fold) than AcP, possibly due to the higher hydrophobicity and lower net charge of HypF-N compared to AcP [256
Hierarchical aggregation process of HypF-N
In contrast to fibrils of disease-causing amyloidogenic proteins, those formed by disease-unrelated proteins did not cause cytotoxicity in cell-culture experiments. For example, fibrils formed by the aforementioned αtα peptide displayed no neurotoxicity, even though they were morphologically indistinguishable from Aβ and IAPP fibrils, which were toxic [238
]. It was therefore unexpected that the pre-fibrillar assemblies of PI3K-SH3 and HypF-N were shown to be highly toxic to PC12 cells and murine fibroblasts in vitro
]. The extent of cellular injury caused by the cytotoxic oligomers was comparable to that of Aβ42 oligomers, whereas the corresponding fibrils of both PI3K-SH3 and HypF-N were benign.
Early pre-fibrillar HypF-N assemblies were shown to permeabilize artificial phospholipid membranes more efficiently than mature fibrils, indicating that this disease-unrelated protein displayed the same toxic properties as pre-fibrillar assemblies of pathological peptides and proteins [252
]. Further investigation of the cellular effects of HypF-N oligomers revealed that they entered the cytoplasm and caused an acute rise in ROS levels and [Ca2+
, leading to cell death [258
]. In a study where murine fibroblasts and endothelial cells were treated with pre-fibrillar HypF-N assemblies, the two cell types underwent two different death mechanisms—fibroblasts exposed for 24 h to 10 µM HypF-N oligomers underwent necrosis, whereas endothelial cells treated similarly sustained apoptosis [259
]. A similar study comparing cytotoxic effects of pre-fibrillar and fibrillar HypF-N assemblies using a panel of normal and pathological cell-lines showed that cells were variably affected by the same amount of pre-fibrillar aggregates, whereas mature fibrils showed little or no toxicity [260
]. This difference in the extent of compromise of cell viability was significantly related to the cell-membrane cholesterol content and to different cellular Ca2+
-buffering and antioxidant capacities of the various cell types [260
]. Recently, it has been shown that microinjection into rat brain nucleus basalis magnocellularis of PI3K-SH3 or HypF-N assemblies, but not the corresponding mature fibrils, compromised neuronal viability dose-dependently [8
]. Taken together, these data clearly demonstrate that the pre-fibrillar assemblies of disease-unrelated proteins are highly toxic whereas the corresponding mature fibrils are not [8
]. The toxic effect of the oligomers may arise when these assemblies assume a "misfolded" conformation which may expose hydrophobic residues that are natively entombed within the core structure. Such aggregation-prone regions may interact with membranes and other cellular components modifying their structural/functional homeostasis.
Dobson and co-workers have proposed that evolutionary mechanisms may have been in force to ensure propagation of proteins that resist aggregation for efficient function [261
]. However, genetic, environmental and metabolic factors that decrease the solubility or increase the concentration of susceptible proteins in vivo
may act against those forces and induce protein misfolding disorders including neurodegenerative diseases [261
The fact that aggregates of some disease-unrelated proteins could function similarly to those formed by amyloidogenic, disease-related peptides and proteins, has profound implications for understanding the mechanistic fundamentals of abnormal protein deposition in amyloidoses. These observations facilitate investigation and discovery of the general mechanistic features underlying protein misfolding and aggregation [237
] and help defining likely targets for drug design.