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Rapid Generation of Amyloid from Native Proteins In vitro
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pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers

Kevin W Tipping1, Theodoros K Karamanos1, Toral Jakhria1

  • 1Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom.

Proceedings of the National Academy of Sciences of the United States of America
|April 23, 2015
PubMed

Insights

Amyloid disorders involve toxic protein aggregates. This study shows pH-dependent fibril disassembly releases membrane-disrupting oligomers at pH 6.4, but not at pH 7.4, revealing a key factor in amyloid disease.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Neuroscience

Background:

  • Amyloid disorders are linked to toxic protein aggregate formation.
  • The precise mechanisms of amyloid toxicity and the species mediating cellular dysfunction are not fully understood.

Purpose of the Study:

  • To investigate the role of pH in amyloid fibril disassembly and membrane disruption using beta2-microglobulin (β2m) as a model.
  • To identify the species responsible for membrane damage during amyloid aggregation.

Main Methods:

  • Thioflavin T (ThT) fluorescence assays
  • Nuclear Magnetic Resonance (NMR) spectroscopy
  • Electron Microscopy (EM)
  • Fluorescence Correlation Spectroscopy (FCS)
  • Synthetic membrane disruption assays

Main Results:

  • Fibril disassembly at pH 6.4 yielded nonnative, membrane-disrupting spherical oligomers.
  • Fibril dissociation at pH 7.4 produced nontoxic native monomers.
  • Chemical cross-linking and hsp70 interaction stabilized fibrils, reducing membrane disruption and cellular dysfunction.

Conclusions:

  • pH is a critical factor modulating the deleterious effects of preformed amyloid aggregates.
  • The shedding of membrane-active oligomers during fibril disassembly at acidic pH is a key mechanism of amyloid toxicity.
  • Endocytic trafficking through acidic cellular compartments may contribute to amyloid disease pathogenesis.
Keywords:
amyloiddisassemblyfibrilsmembrane disruptionoligomer

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