Ubiquitin binds the amyloid b peptide and interferes with its clearance pathways† F. Bellia, a V. Lanza, a S. Garc ´ ıa-Vi ˜ nuales, a I. M. M. Ahmed, a A. Pietropaolo, b C. Iacobucci, c G. Malgieri, d G. D'Abrosca, d R. Fattorusso, d V. G. Nicoletti, e D. Sbardella, f G. R. Tundo, f M. Coletta, f L. Pirone, g E. Pedone, g D. Calcagno, h G. Grasso * h and D. Milardi * a Several lines of evidence point to a compromised proteostasis associated with a reduction of the Ubiquitin Proteasome System (UPS) activity in patients affected by Alzheimer's Disease (AD) and suggest that the amyloid b peptide (Ab) is an important player in the game. Inspired also by many reports, underlining the presence of ubiquitin (Ub) in the amyloid plaques of AD brains, here we set out to test whether Ub may bind the Ab peptide and have any effect on its clearance pathways. By using an integrated array of MALDI-TOF/UPLC-HRMS, fluorescence, NMR, SPR, Microscale Thermophoresis (MST) and molecular dynamics studies, we consistently demonstrated that Ab40 binds Ub with a 1 : 1 stoichiometry and K d in the high micromolar range. In particular, we show that the N-terminal domain of the Ab peptide (through residues D1, E3 and R5) interacts with the C-terminal tail of Ub (involving residues K63 and E64), inducing the central region of Ab ( 14 HQKLVFFAEDVGSNK 28 ) 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 of the entire pool of cytosolic Ub binding proteins. Ub-bound Ab has a lower tendency to aggregate into amyloid-like fibrils and is more slowly degraded by the Insulin Degrading Enzyme (IDE). Finally, we observe that the water soluble fragment Ab1–16 significantly inhibits Ub chain growth reactions. These results evidence how the non-covalent interaction between Ab peptides and Ub may have relevant effects on the regulation of the upstream events of the UPS and pave the way to future in vivo studies addressing the role played by Ab peptide in the malfunction of proteome maintenance occurring in AD. Introduction Alzheimer's disease (AD), the most common form of dementia worldwide, is an age-related, fatal neurodegenerative disorder. A hallmark of AD is the presence of extracellular proteinaceous deposits (senile plaques) in the brain of affected people. The prevalent component of senile plaques is b-amyloid (Ab). 1 Although a rm relationship between the occurrence of different Ab aggregates in the AD brain and the severity of the disease has not been established yet, Ab misfolding and self- assembly are widely believed to be crucial pathogenic events in AD (amyloid hypothesis). 2–4 Unfortunately, all clinical trials of amyloid-targeting drugs have failed so far 5,6 suggesting that the amyloid hypothesis needs to be reconsidered. Intriguingly, several reports put in evidence that transgenic mice models of AD show an intracellular Ab immunoreactivity 7 which occurs before cognitive loss and massive amyloid plaques deposition and may be more closely associated with the disease progress. 8 Therefore, studies focusing on the interplay between Ab amyloid growth and protein clearance in the cell represent a promising arena for the design of more effective AD therapies. 9 The removal of misfolded and potentially toxic cytosolic proteins is mostly regulated by the ubiquitin proteasome system (UPS). 10 The rst component of the UPS is ubiquitin (Ub), a small protein composed of 76 amino acids, with a Consiglio Nazionale delle Ricerche, Istituto di Biostrutture e Bioimmagini, Via P. Gaifami 18, 95126 Catania, Italy. E-mail: danilo.milardi@cnr.it b Dipartimento di Scienze della Salute, Universit` a degli Studi Magna Graecia di Catanzaro, Viale Europa, 88100, Catanzaro, Italy c Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, 06120 Halle/Saale, Germany d Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, Via Vivaldi 43, 81100 Caserta, Italy e Dipartimento di Scienze Biomediche e Biotecnologiche (BIOMETEC), sez. Biochimica medica, Universit` a di Catania, Via Santa Soa 97, 95124 Catania, Italy f Dipartimento di Scienze Cliniche e Medicina Traslazionale, Universit` a di Roma Tor Vergata, Via Montpellier 1, 00133, Roma, Italy g Consiglio Nazionale delle Ricerche, Istituto di Biostrutture e Bioimmagini, Via Mezzocannone, 16, Naples I-80134, Italy h Dipartimento di Scienze Chimiche, Universit` a di Catania, V.le Andrea Doria 6, 95125 Catania, Italy. E-mail: grassog@unict.it † Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc03394c Cite this: DOI: 10.1039/c8sc03394c All publication charges for this article have been paid for by the Royal Society of Chemistry Received 31st July 2018 Accepted 9th January 2019 DOI: 10.1039/c8sc03394c rsc.li/chemical-science This journal is © The Royal Society of Chemistry 2019 Chem. Sci. Chemical Science EDGE ARTICLE Open Access Article. Published on 10 January 2019. Downloaded on 1/19/2019 12:52:32 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal