Abstract
Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human β2-microglobulin (β2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.
Highlights
Oligomers have been the focus of amyloid research over decades because of their pivotal role in assembly and their potential cytotoxicity (Chiti and Dobson, 2017)
Previous results have shown that DN6 assembles rapidly into amyloid fibrils in vitro at pH 6.2, but not at pH 8.2 (Eichner et al, 2011), suggesting that lowering the pH increases the population of aggregation-prone species
To determine the kinetic mechanism by which DN6 aggregates into amyloid fibrils, experiments were performed in which DN6 fibril seeds (20 mM monomer equivalent concentration) were incubated with different concentrations of DN6 monomers (20 mM to 500 mM) and the rate of amyloid formation was monitored by the fluorescence of thioflavin T (ThT)
Summary
Oligomers have been the focus of amyloid research over decades because of their pivotal role in assembly and their potential cytotoxicity (Chiti and Dobson, 2017). Since hb2m is inert to aggregation at physiological pH and temperature in vitro, the oligomerization of the protein was stimulated by mutation and/or the addition of Cu2+ ions (Calabrese et al, 2008; Eakin et al, 2006; Mendoza et al, 2011; Mendoza et al, 2010), or by linkage of monomers via non-native disulfide bonds (Halabelian et al, 2015; Colombo et al, 2012) Some of these oligomers form under conditions in which WT hb2m may eventually form fibrils, the role of individual oligomeric species in the aggregation mechanism remains unclear. Enable the development of routes to combat disease by targeting the specific protein-protein interactions that define the early stages of assembly
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