The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin
Manjeet Kumar A H , Cristina Cantarutti B , David C. Thorn A I , Vittorio Bellotti C D , Gennaro Esposito E F , Mark R. Wilson G , Heath Ecroyd G and John A. Carver
A *
A Research School of Chemistry, The Australian National University, Acton, ACT 2601, Australia.
B Department of Medicine, University of Udine, 33100 Udine, Italy.
C Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London, London NW3 2PF, UK.
D Department of Molecular Medicine, Institute of Biochemistry, University of Pavia, 27100 Pavia, Italy.
E Science & Math Division, New York University Abu Dhabi, Abu Dhabi, UAE.
F Istituto Nazionale Biomolecole e Biostrutture, 00136 Rome, Italy.
G Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia.
H Present address: Department of Biophysics and Chemistry, The University of Michigan, Ann Arbor, MI 48109-1055, USA.
I Present address: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Australian Journal of Chemistry - https://doi.org/10.1071/CH23082
Submitted: 30 April 2023 Accepted: 19 June 2023 Published online: 11 July 2023
Abstract
Amyloid fibril formation by the extracellular protein β2-microglobulin (β2m) and its subsequent accumulation in periarticular tissues have been linked to dialysis-related amyloidosis. A natural variant of human β2m responsible for aggressive systemic amyloidosis contains an aspartate to asparagine mutation at residue 76 (i.e. D76N β2m), 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 β2m fibril formation in vitro under physiological conditions. The presence of extrinsic charged amino acids modulated D76N β2m 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 β2m. As a result, clusterin was incorporated into D76N β2m 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 β2m 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 β2m with clusterin confirmed that the chaperone interacts transiently and non-specifically with monomeric β2m. Clusterin inhibits both primary and secondary nucleation of D76N β2m fibril formation. In doing so, clusterin binds to D76N β2m fibrils and stabilises them to prevent possible fragmentation. In vivo, the multifaceted chaperone action of clusterin may delay, if not prevent, β2m amyloid proliferation and deposition in tissues.1
Keywords: amyloid fibril, amyloid fibril stability, clusterin, D76N β2-microglobulin, dialysis-related amyloidosis, molecular chaperone, primary nucleation, secondary nucleation.
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