Insoluble Off-Pathway Aggregates as Crowding Agents during Amyloid Fibril Formation

The study of drug candidates for the treatment of amyloidosis and neurodegenerative diseases frequently involves in vitro measurements of amyloid fibril formation. Macromolecular crowding and off-pathway aggregation (OPA) are, by different reasons, two important phenomena affecting the scalability of amyloid inhibitors and their successful application in vivo. On the one hand, the cellular milieu is crowded with macromolecules that drastically increase the effective (thermodynamic) concentration of the amyloidogenic protein. On the other hand, off-pathway aggregates, rather than amyloid fibrils, are increasingly appointed as causative agents of toxicity. The present contribution reveals that insoluble off-pathway aggregates of hen egg-white lysozyme (HEWL) are a peculiar type of crowding agents that, unlike classical macromolecular crowders, decrease the thermodynamic concentration of protein. Illustrating this effect, OPA is shown to resume after lowering the fraction of insoluble aggregates at a constant soluble HEWL concentration. Protein depletion and thioflavin-T fluorescence progress curves indicate that OPA rebirth is not accompanied by additional amyloid fibril formation. The crystallization-like model extended to account for OPA and time-dependent activity coefficients is able to fit multiple kinetic results using a single set of three parameters describing amyloid nucleation, autocatalytic growth, and off-pathway nucleation. The list of fitted results notably includes the cases of aggregation rebirth and all types of progress curves measured for different HEWL concentrations. The quantitative challenges posed by macromolecular crowding and OPA find here a unified response with broader implications for the development of on- and off-pathway inhibitors.