Crucial role of nonspecific interactions in amyloid nucleation

Protein oligomers have been implicated as toxic agents in a wide range of amyloid-related diseases. However, it has remained unsolved whether the oligomers are a necessary step in the formation of amyloid fibrils or just a dangerous byproduct. Analogously, it has not been resolved if the amyloid nucleation process is a classical one-step nucleation process or a two-step process involving prenucleation clusters. We use coarse-grained computer simulations to study the effect of nonspecific attractions between peptides on the primary nucleation process underlying amyloid fibrillization. We find that, for peptides that do not attract, the classical one-step nucleation mechanism is possible but only at nonphysiologically high peptide concentrations. At low peptide concentrations, which mimic the physiologically relevant regime, attractive interpeptide interactions are essential for fibril formation. Nucleation then inevitably takes place through a two-step mechanism involving prefibrillar oligomers. We show that oligomers not only help peptides meet each other but also, create an environment that facilitates the conversion of monomers into the β-sheet–rich form characteristic of fibrils. Nucleation typically does not proceed through the most prevalent oligomers but through an oligomer size that is only observed in rare fluctuations, which is why such aggregates might be hard to capture experimentally. Finally, we find that the nucleation of amyloid fibrils cannot be described by classical nucleation theory: in the two-step mechanism, the critical nucleus size increases with increases in both concentration and interpeptide interactions, which is in direct contrast with predictions from classical nucleation theory.During the process of amyloid formation, normally soluble proteins assemble into fibrils that are enriched in β-sheet content and have diameters of a few nanometers and lengths up to several micrometers. This phenomenon has been implicated in a variety of pathogenic processes, including Alzheimer’s and Parkinson’s diseases, type 2 diabetes, and systemic amyloidoses (1–3). The association with human diseases has largely motivated a long-standing effort to probe the assembly process, and numerous studies have aimed at elucidating the mechanism of amyloid aggregation (4). The basic nature of the aggregation reaction has emerged as a nucleation and growth process (5, 6), where the aggregates are created through a not well-understood primary nucleation event and can grow by recruiting additional peptides or proteins to their ends (7, 8). In this paper, we focus on the nature of this primary step in amyloid nucleation and the fundamental initial events that underlie amyloid formation.Amyloidogenic peptides and proteins, when in their nonpathological cellular form, can range in the structures from mainly α-helical to β-sheet and even random coil, whereas the amyloid forms of proteins possess a generic cross–β-structure (9–14). The formation of amyloid is, hence, accompanied by marked changes in the conformations of the peptides and proteins that undergo this process. A pertinent question is whether this conformational change takes place simultaneously with the nucleation process or whether nucleation takes place first and is then followed by conformational change. These two possible scenarios of nucleation have been extensively discussed in the experimental and theoretical literature (5, 8, 15–19). We will refer in this work to the two scenarios simply as one-step nucleation (1SN), in which the β-sheet–enriched nucleus forms directly from the solution, and two-step nucleation (2SN), where soluble monomers first assemble into disordered oligomers, which subsequently convert into a β-sheet nucleus. Disordered oligomers, ranging in size between dimers and micrometer-sized particles, have been observed in some experiments (20–28). These findings highlight a central question regarding the role of disordered oligomers in fibril formation: are such clusters a necessary step in the process of fibril formation or just a byproduct?From a biological and biomedical perspective, it is important to understand the conditions under which oligomeric clusters form, because such species exhibit high cytotoxicity (1, 29–31). Indeed, there is strong evidence that the disordered oligomers rather than fully grown fibrils are the main pathogenic species in protein aggregation diseases (31–33). As such, defining the role of the prefibrillar oligomers during amyloid formation will be crucial to develop intervention strategies that target these species (1, 30, 34, 35).Mutations in the polypeptide sequence and extrinsic changes in the experimental conditions are known to alter the concentrations of aggregated species, their size, and their cytotoxicity (25, 36–39). For instance, mutations that increase hydrophobicity of the Alzheimer’s β-peptide (Aβ1–42) have a pronounced effect on its aggregation behavior and the size distribution of the resulting oligomers (23–26, 40), promoting toxicity and expediting the fibrillization process. In the same spirit, two extra hydrophobic residues in Aβ1–42 are believed to contribute to the more pronounced oligomerization and faster fibrillization compared with its alloform Aβ1–40 (24, 25, 40). Temperature, pH, and concentration of certain metals also affect oligomerization and pathways of fibrillization (41–44).The common feature of the above experiments is that they modify the internal free energy difference between the soluble and the β-sheet–forming state, also called the β-sheet propensity, which has been extensively studied in the literature (45–48). However, they also modify interactions between peptides that aggregate, a crucial contribution that has not yet been systematically addressed.In this paper, we study the effect of nonspecific interactions between peptides on the amyloid nucleation process. Such nonspecific interactions do not depend on the atomistic details of the amino acids involved, allowing us to address question about amyloid aggregation and nucleation using a coarse-grained model. In particular, generic hydrophobic stretches in the sequence of Aβ have been shown to be sufficient to promote aggregation (49, 50). Mutations of nonpolar residues to other nonpolar residues had little or no effect on aggregation, whereas mutations that reduce charge and/or increase hydrophobicity enhanced it (50, 51). Furthermore, atomic force microscopy measurements have shown that the strength of overall interactions between amyloidogenic proteins correlates with their tendency to aggregate (52, 53).We have performed extensive computer simulations that allowed us to observe both the 1SN and the 2SN mechanisms. These simulations reveal that 1SN and 2SN can be viewed as two limits of the same process, something that several previous studies have suspected (16, 18). Importantly, we observe that only 2SN is possible at low peptide concentrations, comparable with the levels that are found in vivo. Another key observation is that fibril nucleation typically does not proceed through the most prevalent oligomeric species but rather, through an oligomer with a size that is only observed as a result of rare fluctuations. As a consequence, such oligomers will be hard to capture experimentally, although their presence is required for nucleation to take place. Our simulations show that the free energy barrier for fibril nucleation through the two-step mechanism decreases with increasing strength of the interpeptide interactions. Furthermore, the critical nucleus size in the two-step mechanism is found to grow with the increase in the peptide concentrations as well as with stronger interpeptide interactions, which is in direct contrast with the classical nucleation. These results imply that weakening the nonspecific interactions between peptide monomers in solution and thereby, simultaneously increasing both the free energy barrier for oligomer formation and the free energy barrier for peptide conversion at a given oligomer size may be a crucial step in preventing amyloid aggregation.