pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers

Abstract

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β 2 -microglobulin (β 2 m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.amyloid | fibrils | disassembly | oligomer | membrane disruption

Inducing protein aggregation by extensional flow

“…G-CSF C3 (37) was overexpressed in BL21[DE3]pLysS cells transformed with a pET23a_G-CSF C3 vector and purified as described in SI Appendix. Extensional flow experiments with G-CSF C3 were performed in filtered (0.22 μm) and degassed 25 mM sodium phosphate, 25 mM sodium acetate buffer, pH 7.0. β 2 m was purified as described (48) and extensional flow experiments performed in filtered (0.22 μm) and degassed 25 mM sodium phosphate buffer, pH 7.2. Antibodies were provided by MedImmune Ltd. Antibodies were prepared by dialyzing into 0.22 μm filtered and degassed 150 mM ammonium acetate buffer, pH 6.0, diluting before stressing experiments as appropriate.…”

Structural, morphological, and functional diversity of amyloid oligomers

“…Various small oligomers, up to hexamer, were observed, and, in fact, a hexamer has been proposed to serve as a critical nucleus for fibril formation under these conditions [86]. Depolymerization of b-2 microglobulin fibrils at pH 6.4 resulted in the formation of oligomers that appear less structured than the fibrils or monomers [87]. Association interface in the oligomers was shown to be mediated by the interactions between the D-strands of adjacent b-2m molecules based on structural and kinetic studies [88,89].…”

Neuron-Microglia Interactions in Motor Neuron Degeneration. The Inflammatory Hypothesis in Amyotrophic Lateral Sclerosis Revisited

“…In these conditions, glucose transport to CNS is not sufficient to cover demand, and lactate is formed within astrocytes and sent to neurons for its complete oxidation [87,88]. As a result, the tissue pH decreases, which alters protein folding and induces aggregate formation, as observed with Amyloid-β [89]. In ALS, this dynamic adaptation of the glial phenotype to the graded metabolic and structural modifications results in an overproduction of neurotoxic molecules and chronic neuroinflammation ( Figure 2) which drives, at least in part, the disease progression [77,[90][91][92] .…”