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Rapid Generation of Amyloid from Native Proteins In vitro
Published on: December 5, 2013
Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops
D M Hatters1, C E MacPhee, L J Lawrence
1Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.
Biochemistry
|July 13, 2000
Summary
Human apolipoprotein C-II (apoC-II) forms amyloid-like aggregates in vitro. This study characterizes apoC-II self-assembly, revealing a novel looped-ribbon morphology and increased beta-sheet structure, offering insights into apolipoprotein aggregation.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biophysics
Background:
- Human apolipoprotein C-II (apoC-II) is crucial for lipid metabolism.
- Apolipoproteins are known to form aggregates, but the mechanisms are not fully understood.
- Amyloid formation is associated with various diseases.
Purpose of the Study:
- To investigate the self-assembly properties of human apolipoprotein C-II (apoC-II) in solution.
- To characterize the structural features of apoC-II aggregates.
- To explore the potential for apolipoproteins to form amyloid structures in vitro.
Main Methods:
- Recombinant apoC-II expression and purification from E. coli.
- Gel filtration chromatography and analytical ultracentrifugation to assess aggregation.
- Circular dichroism (CD) spectroscopy to analyze secondary structure changes.
- Thioflavin T fluorescence and Congo Red binding assays to detect amyloid characteristics.
- Electron microscopy to visualize aggregate morphology.
Main Results:
- Purified apoC-II initially exists as soluble, low molecular weight species with predominantly unordered structure.
- Upon incubation, apoC-II self-associates into high molecular weight aggregates, confirmed by gel filtration, ultracentrifugation, and light scattering.
- CD spectroscopy revealed an increase in beta-sheet content during aggregation.
- Electron microscopy identified a novel looped-ribbon morphology (12 nm width) for apoC-II aggregates.
- Aggregates exhibited amyloid-like properties, including Congo Red birefringence and Thioflavin T fluorescence.
Conclusions:
- Human apoC-II self-associates in vitro to form amyloid-like fibrils.
- The aggregation process involves a transition from unordered structure to increased beta-sheet content.
- The observed looped-ribbon morphology is a novel finding in apolipoprotein aggregation.
- This in vitro system provides a model to study the propensity of apolipoproteins to form amyloid structures, potentially relevant to in vivo conditions.
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