Dissociation of a BRICHOS trimer into monomers leads to increased inhibitory effect on Aß42 fibril formation.

ABSTRACT

The BRICHOS domain is associated with human amyloid disease, and it efficiently prevents amyloid fibril formation of the amyloid ß-peptide (Aß) in vitro and in vivo. Recombinant human prosurfactant protein C (proSP-C) BRICHOS domain forms a homotrimer as observed by x-ray crystallography, analytical ultracentrifugation, native polyacrylamide gel electrophoresis, analytical size exclusion chromatography and electrospray mass spectrometry. It was hypothesized that the trimer is an inactive storage form, as a putative substrate-binding site identified in the monomer, is buried in the subunit interface of the trimer. We show here increased dissociation of the BRICHOS trimer into monomers, by addition of detergents or of bis-ANS, known to bind to the putative substrate-binding site, or by introducing a Ser to Arg mutation expected to interfere with trimer formation. This leads to increased capacity to delay Aß(42) fibril formation. Cross-linking of the BRICHOS trimer with glutaraldehyde, in contrast, renders it unable to affect Aß(42) fibril formation. Moreover, proSP-C BRICHOS expressed in HEK293 cells is mainly monomeric, as detected by proximity ligation assay. Finally, proteolytic cleavage of BRICHOS in a loop region that is cleaved during proSP-C biosynthesis results in increased capacity to delay Aß(42) fibril formation. These results indicate that modulation of the accessibility of the substrate-binding site is a means to regulate BRICHOS activity.