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Rapid Generation of Amyloid from Native Proteins In vitro
05:48

Rapid Generation of Amyloid from Native Proteins In vitro

Published on: December 5, 2013

Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta 2-microglobulin

Niels H H Heegaard1, Thomas J D Jørgensen, Noémi Rozlosnik

  • 1Department of Autoimmunology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark. nhe@ssi.dk

Biochemistry
|March 16, 2005
PubMed

Insights

A modified Beta(2)-microglobulin (beta(2)m) variant, DeltaK58-beta(2)m, shows significantly decreased stability and increased amyloid formation at body temperature. This finding sheds light on the mechanisms of dialysis-related amyloidosis.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Misfolding Diseases

Background:

  • Beta(2)-microglobulin (beta(2)m) is implicated in dialysis-related amyloidosis.
  • Mechanisms of beta(2)m fibrillogenesis in vivo remain largely unknown.
  • A specific beta(2)m variant, DeltaK58-beta(2)m, linked to cancer and inflammation, may be relevant in dialysis patients.

Purpose of the Study:

  • To investigate the conformational stability and amyloid formation propensity of the DeltaK58-beta(2)m variant.
  • To compare the behavior of DeltaK58-beta(2)m with wild-type (wt) beta(2)m under physiological conditions.

Main Methods:

  • Amide hydrogen/deuterium exchange monitored by mass spectrometry to assess unfolding rates.
  • Congo red binding assays to detect amyloid formation.
  • Thioflavin T fluorescence and intrinsic fluorescence measurements.
  • Size-exclusion chromatography and atomic force microscopy to characterize aggregates and fibrils.

Main Results:

  • DeltaK58-beta(2)m exhibits significantly decreased conformational stability with increased unfolding rates compared to wt-beta(2)m.
  • Unfolding of DeltaK58-beta(2)m is highly temperature-dependent, with a 170-fold faster half-time for unfolding at 37°C compared to 15°C.
  • DeltaK58-beta(2)m readily forms high molecular weight aggregates and extensive amyloid fibrils at 37°C, unlike wt-beta(2)m.

Conclusions:

  • The DeltaK58-beta(2)m variant is inherently unstable and prone to amyloid formation under near physiological conditions.
  • Limited proteolysis generating this variant can initiate stages of amyloid formation.
  • Findings provide insights into the pathogenesis of amyloidosis involving beta(2)m.

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