The disruption of A-Beta peptide by transthyretin: A mechanism sensitive to Kunitz Protease Inhibitor

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the deposition of amyloid β-peptide (A-Beta) in the brain. The degree of A-Beta accumulation is dependent on its production but also on its removal. While the production of A-Beta is widely described, much less is known regarding peptide catabolism. Transthyretin (TTR) is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. Physiologically, TTR is responsible for the transport of thyroid hormone T4 and retinol, through binding to the retinol binding protein (RBP). TTR has been established, recently, as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We analyzed TTR/A-Beta interaction by SDS-PAGE, followed by western blot using an anti-A-Beta specific antibody. We then characterized the nature of this interaction by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. To assess the amyloidogenic potential of A-Beta fragments, we performed thioflavin binding assays and ultrastructural analysis by transmission electron microscopy (TEM). The effect of inhibitors was ascertained by SDS-PAGE, followed by western blot and by TEM. We found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27. A-Beta peptides (1-14) and (15-42) showed lower amyloidogenic potential than the full length counterpart. A-Beta cleavage by TTR was inhibited in the presence of an αAPP peptide containing the Kunitz Protease Inhibitor (KPI) domain but not in the presence of the secreted αAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.