Ubiquitin binds the amyloid b peptide and interferes with its clearance pathways

Several lines of evidence point to a compromised proteostasis associated with a reduction of the UbiquitinProteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that theamyloid b peptide (Ab) is an important player in the game. Inspired also by many reports, underlining thepresence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub maybind the Ab peptide and have any effect on its clearance pathways. By using an integrated array ofMALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and moleculardynamics studies, we consistently demonstrated that Ab40 binds Ub with a 1 : 1 stoichiometry and Kd inthe high micromolar range. In particular, we show that the N-terminal domain of the Ab peptide (throughresidues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducingthe central region of Ab (14HQKLVFFAEDVGSNK28) to adopt a mixed a-helix/b-turn structure. ELISA assays,carried out in neuroblastoma cell lysates, suggest that Ab competitively binds Ub also in the presence ofthe entire pool of cytosolic Ub binding proteins. Ub-bound Ab has a lower tendency to aggregate intoamyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, weobserve that the water soluble fragment Ab1-16 significantly inhibits Ub chain growth reactions. Theseresults evidence how the non-covalent interaction between Ab peptides and Ub may have relevant effectson the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressingthe role played by Ab peptide in the malfunction of proteome maintenance occurring in AD.