The Extracellular Chaperone Clusterin Prevents Primary and Secondary Nucleation of an Amyloidogenic Variant of ?2-Microglobulin
Australian journal of chemistry(2023)
Abstract
Amyloid fibril formation by the extracellular protein ss 2-microglobulin (ss 2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human ss 2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N ss 2m), which readily forms amyloid fibrils in vitro under physiological conditions. In this study, we examined the role of the extracellular molecular chaperone clusterin in modulating D76N ss 2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N ss 2m fibril formation, implying that electrostatic interactions are involved in the protein ' s aggregation. Thioflavin T (ThT) and 1-anilinonaphthalene-8-sulfonate fluorescence assays indicated that clusterin interacts via hydrophobic and electrostatic forces with the monomeric, prefibrillar and fibrillar species of D76N ss 2m. As a result, clusterin was incorporated into D76N ss 2m aggregates during the latter ' s fibril formation, as indicated by SDS-PAGE of depolymerised fibrils. SYPRO Orange and ThT fluorescence assays suggested that, compared to pure D76N ss 2m fibrils, those formed in the presence of clusterin are chemically more stable with a reduced ability to act as nucleation seeds. Detailed 15N NMR relaxation studies of mixtures of 15N-labelled ss 2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric ss 2m. Clusterin inhibits both primary and secondary nucleation of D76N ss 2m fibril formation. In doing so, clusterin binds to D76N ss 2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, ss 2m amyloid proliferation and deposition in tissues.
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Key words
amyloid fibril,amyloid fibril stability,clusterin,D76N ss 2-microglobulin,dialysis-related amyloidosis,molecular chaperone,primary nucleation,secondary nucleation.
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