Fiber-dependent amyloid formation as catalysis of an existing reaction pathway

Abstract

A central component of a number of degenerative diseases is the deposition of protein as amyloid fibers. Self-assembly of amyloid occurs by a nucleation-dependent mechanism that gives rise to a characteristic sigmoidal reaction profile. The abruptness of this transition is a variable characteristic of different proteins with implications to both chemical mechanism and the aggressiveness of disease. Because nucleation is defined as the rate-limiting step, we have sought to determine the nature of this step for a model system derived from islet amyloid polypeptide. We show that nucleation occurs by two pathways: a fiber-independent (primary) pathway and a fiber-dependent (secondary) pathway. We first show that the balance between primary and secondary contributions can be manipulated by an external interface. Specifically, in the presence of this interface, the primary mechanism dominates, whereas in its absence, the secondary mechanism dominates. Intriguingly, we determine that both the reaction order and the enthalpy of activation of the two nucleation processes are identical. We interrogate this coincidence by global analysis using a simplified model generally applicable to protein polymerization. A physically reasonable set of parameters can be found to satisfy the coincidence. We conclude that primary and secondary nucleation need not represent different processes for amyloid formation. Rather, they are alternative manifestations of the same, surfacecatalyzed nucleation event.amylin ͉ fibers ͉ islet amyloid polypeptide ͉ nucleation T he noncovalent, fibrous self-assembly of proteins, including hemoglobin S, actin, microtubules, and amyloid fibers, occurs by a nucleation-dependent mechanism (1-5). If polymer product formation is monitored as a function of time in a reaction where all of the protein is initially monodisperse, there is a lag phase in which little product is formed, followed by a period of rapid growth (2). Such observations are consistent with a rate-limiting step in which change occurs to a high-energy intermediate known as the nucleus. In many systems such as actin, collagen, and microtubules, the nucleus is modeled as an oligomeric species that is in a highly unfavorable equilibrium with monomer (1, 3-6). The nucleus may also be a high-energy conformation of monomer, such as that reported for polyglutamine (7). Regardless, the rate-limiting step involves a nucleus because it is defined as the species with the highest free energy and therefore lowest population (1, 4).This model of nucleation alone cannot account for the high apparent cooperativity of conversion observed in many systems. Historically, hemoglobin S was the first biological polymerization reaction to demonstrate a transition time that is much shorter than its preceding lag phase (8). This same phenomenon is commonly reported for amyloid systems including islet amyloid polypeptide (IAPP) from type II diabetes (9), A␤ from Alzheimer's (10), PrP from the mammalian prion (11), and Sup35 from the yeast prion (12, 13), as well as mode...

Cutting Edge Structural Protein from the Jaws of Nereis virens

“…[48][49][50] Factors such as pH, ionic strength, and metal coordination have been shown to influence fiber formation and morphology. 24,27,[51][52][53][54][55] Based on our data we can reasonably predict that similar features are involved in Nvjp-1 fibrillization.…”

Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism

“…The general method underlying the kinetic analysis builds on earlier work (10, 14-17, 18, 19, 21, 22) and considers all of the possible sources of new aggregates, which consist of two or more monomers, from the species present in the system, as shown in Table 1, from both primary (10,19,(23)(24)(25) and secondary (11,12,14,(26)(27)(28) pathways. Primary pathways, such as homogeneous nucleation (10,19), generate new aggregates at a rate dependent on the concentration of monomers alone and independent of the concentration of existing fibrils.…”

“…Secondary pathways are the complementary class of mechanisms that generate new aggregates at a rate dependent on the concentration of existing fibrils. The latter class can be subdivided into monomer-independent processes, such as fragmentation (11,12,18,26), with a rate depending only upon the concentration of existing fibrils, and monomer-dependent processes, such as secondary nucleation (14,22,27,28), where the surfaces of existing fibrils catalyze the nucleation of new aggregates from the monomeric state, with a rate dependent on both the concentration of monomers and that of existing fibrils. Together, these three classes of mechanism, shown in Table 1, form the basis of a general description of protein aggregation (15,17), because they account for the generation of new aggregates from mechanisms that involve monomers alone, existing aggregates alone, or both monomers and existing aggregates.…”

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

“…By contrast, if the secondary process involves heterogeneous nucleation on fibril surface (26), then the seeding efficiency should be independent of the number of fibril ends, because the total surface area of thin linear aggregates will be essentially independent of agitation. To determine whether this is the case, fibrils were formed in the absence of agitation but in the presence of the same amount seeds by weight that had been fragmented to different extents by vigorous agitation (see Materials and Methods).…”