Abstract
Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β2-microglobulin knock out mice, the D76N β2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.
Highlights
The mechanism of amyloid conversion of wild type β2m has been very extensively studied in the last two decades, much of this work was performed under non-physiological conditions making it difficult to relate the findings to the pathological processes which occur in vivo
We have tested the efficacy of previously characterized inhibitors of the fibrillary conversion of wild type β2m3,7 on the fibrillogenesis of the natural D76N β2m variant causing hereditary systemic amyloidosis and found that complete abrogation of amyloid conversion can be achieved only by a specific monoclonal nanobody raised against the full-length wild type β2m7,8
This result prompted us to compare the effect of Nb24 and doxycycline on the fibrillogenesis of D76N β2m using a combination of thioflavin T (ThT) assay and quantification of the soluble fraction
Summary
The mechanism of amyloid conversion of wild type β2m has been very extensively studied in the last two decades, much of this work was performed under non-physiological conditions making it difficult to relate the findings to the pathological processes which occur in vivo. Full-length wild type β2m cannot be converted into amyloid fibrils in physiological conditions and we cannot be completely confident that the effect in vitro may be reproduced in vivo. Extensive investigation of the mechanism of fibrillogenesis of this genetic variant has revealed that biomechanical forces, compatible with those present in vivo[6] can drive the amyloid conversion of this globular protein. We have tested the efficacy of previously characterized inhibitors of the fibrillary conversion of wild type β2m3,7 on the fibrillogenesis of the natural D76N β2m variant causing hereditary systemic amyloidosis and found that complete abrogation of amyloid conversion can be achieved only by a specific monoclonal nanobody raised against the full-length wild type β2m7,8
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