Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly

Abstract

Self-assembly of misfolded proteins into ordered fibrillar aggregates known as amyloid results in numerous human diseases. Despite an increasing number of proteins and peptide fragments being recognised as amyloidogenic, how these amyloid aggregates assemble remains unclear. In particular, the identity of the nucleating species, an ephemeral entity that defines the rate of fibril formation, remains a key outstanding question. Here, we propose a new strategy for analyzing the self-assembly of amyloid fibrils involving global analysis of a large number of reaction progress curves and the subsequent systematic testing and ranking of a large number of possible assembly mechanisms. Using this approach, we have characterized the mechanism of the nucleationdependent formation of ␤2-microglobulin (␤2m) amyloid fibrils. We show, by defining nucleation in the context of both structural and thermodynamic aspects, that a model involving a structural nucleus size approximately the size of a hexamer is consistent with the relatively small concentration dependence of the rate of fibril formation, contrary to expectations based on simpler theories of nucleated assembly. We also demonstrate that fibril fragmentation is the dominant secondary process that produces higher apparent cooperatively in fibril formation than predicted by nucleated assembly theories alone. The model developed is able to explain and predict the behavior of ␤2m fibril formation and provides a rationale for explaining generic properties observed in other amyloid systems, such as fibril growth acceleration and pathway shifts under agitation.AIC model comparison analysis ͉ amyloid fibril formation ͉ fibril brittleness ͉ global analysis S elf-assembly of misfolded forms of normally soluble and functional proteins or peptides into amyloid fibrils results in numerous human diseases (1). Understanding how amyloid self-assembly occurs, therefore, is of paramount importance for a molecular interpretation of amyloidosis and for the development of therapies against amyloid disease. Over the past decade, advances have been made toward a more complete description of amyoid fibril formation, including the determination of increasingly refined models of fibril structures (reviewed in ref. 1) and the identification of amyloid precursors and oligomeric states reviewed in ref. 2, one or more of which could be the culprits of cytotoxicity associated with several amyloid diseases (e.g., ref.3). However, the molecular events occurring during the self-assembly process itself remain obscure because of the heterogeneity and the complexity of the early association events.Amyloid fibril self-assembly reactions are generally accepted as a form of nucleated polymerization (4). These reactions are characterized by an initial lag phase where little or no change in fibril concentration can be detected. This is followed by an elongation phase where a large mass percentage of the starting protein material is converted into fibrils. A shared feature among amyloid formation and other nuclea...

Mechanism of amyloid plaque formation suggests an intracellular basis of Aβ pathogenicity

“…Although such biphasic growth profiles have been observed previously as bulk solution properties of whole samples or biological systems ( Fig. S4C) (21,24,25), the present analysis demonstrates this type of kinetic behavior also at the level of a single amyloid plaque.…”

“…3). This type of kinetic behavior is highly characteristic for amyloid fibrillation reactions and indicates its nucleationdependence (24). Although such biphasic growth profiles have been observed previously as bulk solution properties of whole samples or biological systems ( Fig.…”

Application and use of differential scanning calorimetry in studies of thermal fluctuation associated with amyloid fibril formation

“…Once the nucleus is formed, subsequent polymerization proceeds rapidly through the sequential incorporation of precursor molecules into the nucleated fibrils. Many experiments have demonstrated that fibrillation from various proteins involves a lag period that corresponds to the nucleation process before the formation of mature, welldefined amyloid fibrils (Lomakin et al 1996;Naiki et al 1997;Yagi et al 2005;Hamley 2007;Sasahara et al 2008;Xue et al 2008).…”

Nucleation of amyloid fibrils

“…[9][10][11][12][13][14][16][17][18]29,30,33,36,38,44,[52][53][54]68,70) or by the temperature at a fixed protein concentration (e.g., Refs. 9,10,14,19,25,26,70,74,76).…”