Abstract
Formation of large protein fibrils with a characteristic cross β-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path.
Highlights
Deposits of insoluble protein fibrils with cross b-sheet structure are the molecular hallmark of an increasing number of human disorders, including Alzheimer’s disease, Parkinson’s diseases and type II diabetes [1,2,3,4,5]
We determined the nucleation and growth kinetics of lysozyme amyloid fibrils grown at different concentrations of sodium chloride by combining dynamic light scattering (DLS) with correlated atomic force microscopy (AFM) [28]
The above data indicate that aggregation of amyloidogenic lysozyme falls into two broad categories: up to modest salt concentrations (,350 mM) lysozyme assembles into amyloid fibers while, at elevated salt concentration, disordered protein precipitation sets in
Summary
Deposits of insoluble protein fibrils with cross b-sheet structure are the molecular hallmark of an increasing number of human disorders, including Alzheimer’s disease, Parkinson’s diseases and type II diabetes [1,2,3,4,5]. Oligomeric intermediates, transiently formed during fibril assembly, are consistently implicated as main culprits responsible for cellular toxicity of both neuropathetic and systemic forms of amyloidoses [6,7,8]. Amyloid polymerization can proceed along multiple assembly pathways, not all of which give rise to oligomeric intermediates [9,10,11,12]. Given the significance of oligomeric intermediates to amyloid toxicity, it is important to elucidate the protein and solution attributes regulating fibril assembly pathways. Exposing these mechanisms will improve our basic understanding of amyloid fibril self assembly but could help devise new treatment strategies by directing amyloid formation towards non-toxic assembly pathways
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