Abstract
Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. β2-Microglobulin (β2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving long-term hemodialysis. Although amyloid fibrils of β2m in patients are observed at neutral pH, formation of β2m amyloids in vitro has been difficult to discern at neutral pH because of the amyloid-resistant native structure. Here, to further understand the mechanism underlying in vivo amyloid formation, we investigated the relationship between protein folding/unfolding and misfolding leading to amyloid formation. Using thioflavin T assays, CD spectroscopy, and transmission EM analyses, we found that β2m efficiently forms amyloid fibrils even at neutral pH by heating with agitation at high-salt conditions. We constructed temperature- and NaCl concentration-dependent conformational phase diagrams in the presence or absence of agitation, revealing how amyloid formation under neutral pH conditions is related to thermal unfolding and breakdown of supersaturation. Of note, after supersaturation breakdown and following the law of mass action, the β2m monomer equilibrium shifted to the unfolded state, destabilizing the native state and thereby enabling amyloid formation even under physiological conditions with a low amount of unfolded precursor. The amyloid fibrils depolymerized at both lower and higher temperatures, resembling cold- or heat-induced denaturation of globular proteins. Our results suggest an important role for heating in the onset of dialysis-related amyloidosis and related amyloidoses.
Highlights
Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. 2-Microglobulin (2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving longterm hemodialysis
To confirm that the conformational change under stirring led to amyloid formation, we carried out thioflavin T (ThT) assays (Fig. 1, E and F, and Fig. S1)
In the absence of stirring, neither ThT fluorescence nor light scattering (LS) changed up to 90 °C. Both ThT fluorescence and LS increased markedly beginning at ϳ66 °C, which was near the midpoint temperature (Tm) for heat unfolding of 2m under these conditions (Fig. 1, E and F, and Fig. S1)
Summary
Amyloidosis-associated amyloid fibrils are formed by denatured proteins when supersaturation of denatured proteins is broken. 2-Microglobulin (2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving longterm hemodialysis. 2-Microglobulin (2m) forms amyloid fibrils and causes dialysis-related amyloidosis in patients receiving longterm hemodialysis. Fibrillar aggregates of denatured proteins or peptides stabilized by intermolecular cross- structures, are associated with many amyloidoses, including neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases, and dialysis-related amyloidosis (DRA)2 [1,2,3,4,5]. The kinetics of amyloid formation are common to amyloidogenic proteins and are typically separated into nucleation, elongation, and equilibrium phases. Seed-dependent elongation is a common property of amyloid formation, in which the nucleation phase is shortened or escaped Another type of protein misfolding leads to amorphous aggregates [6]. Amyloid formation under neutral pH conditions, under which patients develop diseases, does not occur because of the amyloid-resistant native structure [18, 19]. Based on previous studies performed with N-terminal truncated or mutant 2m [20] or those in the presence of additives, such as collagen, heparin [21], SDS [14], or
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
