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Rapid Generation of Amyloid from Native Proteins In vitro
Published on: December 5, 2013
Pathogenic D76N Variant of β2-Microglobulin: Synergy of Diverse Effects in Both the Native and Amyloid States
Éva Bulyáki1, Judit Kun1, Tamás Molnár2
1ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, 1117 Budapest, Hungary.
Abstract:
β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.
Insights
The D76N mutation in beta-2 microglobulin (β2m) significantly increases amyloid formation. This pathogenic variant exhibits destabilized structure, enhanced fibril stability, and greater affinity for extracellular components, contributing to systemic amyloidosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Beta-2 microglobulin (β2m) is the light chain of the MHC-I complex and is implicated in dialysis-related amyloidosis (DRA).
- A hereditary systemic amyloidosis linked to a naturally occurring D76N β2m variant has been identified, exhibiting structural similarity to wild-type (WT) β2m but with reduced stability and increased amyloidogenicity.
Purpose of the Study:
- To investigate the mechanisms by which the D76N mutation in β2m promotes amyloid formation.
- To explore the impact of specific point mutations on the stability and amyloidogenic potential of β2m.
- To analyze the intermolecular interactions of WT and mutant β2m with potential in vivo binding partners.
Main Methods:
- Utilized a range of biophysical techniques to assess conformational stability, partial unfolding, and amyloidogenic propensity of β2m variants.
- Investigated the stability of amyloid fibrils formed by WT and mutant β2m under various conditions.
- Studied the binding interactions of WT and mutant β2m with relevant extracellular components.
Main Results:
- The D76N β2m variant displays exceptional amyloidogenicity compared to WT β2m.
- This increased amyloidogenicity results from a combination of destabilized native structure, heightened sensitivity to anionic molecules (lipids, polyphosphate), more efficient fibril nucleation, and increased fibril stability.
- The D76N variant shows an elevated affinity for extracellular components, including extracellular matrix proteins.
Conclusions:
- The D76N mutation confers a significantly higher risk of systemic amyloidosis due to multiple synergistic effects on β2m's structure and interactions.
- Understanding these mechanisms is crucial for developing therapeutic strategies against β2m-related amyloid diseases.
- The study highlights the critical role of specific mutations in protein misfolding and aggregation pathways.
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