Polyphenol-induced dissociation of various amyloid fibrils results in a methionine-independent formation of ROS ☆ Hila Shoval a,b , Lev Weiner c , Ehud Gazit b , Michal Levy b , Ilya Pinchuk a , Dov Lichtenberg a, ⁎ a Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel b Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel c Department of Organic Chemistry and Unit of Chemical Research Support, Weizmann Institute of Science, 76100 Rehovot, Israel abstract article info Article history: Received 17 February 2008 Received in revised form 21 July 2008 Accepted 4 August 2008 Available online 20 August 2008 Keywords: Amyloid disease Fibril dissociation Free radicals Polyphenols Electron spin resonance Self-Assembly Fibrillization of amyloid polypeptides is accompanied by formation of reactive oxygen species (ROS), which, in turn, is assumed to further promote amyloid-related pathologies. Different polyphenols, all of which are established antioxidants, cause dissociation of amyloid fibrils. This study addresses the latter, poorly understood process. Specifically, we have investigated the dissociation of Aβ 42 fibrils by six different polyphenols, using electron microscopy and spectrofluorometric analysis. Simultanously, we have monitored the production of ROS using electron spin resonance (ESR) and the commercially available peroxide assay kit. Using the same methods we found that curcumin, one of the most potent destabilizing agents of Aβ 42 , induced dissociation of fibrils of other amyloid polypeptides [Aβ 40 ,Aβ 42 Nle35, islet amyloid polypeptide and a fragment of α-synuclein]. When the solution contained traces of transition metal, all the dissociation reactions were accompanied by ROS formation, independent of the presence of a methionine residue. Kinetic studies show that the formation of ROS lags behind dissociation, indicating that if casual relationship exists between these two processes, then ROS formation may be considered a consequence and not a cause of dissociation. These findings open new avenues in amyloid research that will be required to gain further understanding of our results and of their implications. © 2008 Elsevier B.V. All rights reserved. 1. Introduction The formation of amyloid fibrils and/or early oligomers, via self- assembly of peptide and protein monomers [1], is assumed to be a crucial step in the pathogenesis of many amyloid diseases, including Alzheimer's disease (AD) [2], Parkinson's disease (PD) and type-2 diabetes (T2DM) [3]. Previous investigations have shown that fibrillization of several polypeptides [amyloid beta (Aβ) , α-synuclein and islet amyloid polypeptide (IAPP)] is accompanied by formation of free radicals [2]. In turn, reactive oxygen species (ROS), mainly free radicals, accelerate fibril formation [4], possibly via oxidation reac- tions [5], so that the free radicals formed during amyloid fibrillization enhance fibrillization [6]. The available data is insufficient to conclude whether amyloid formation is a direct cause or an outcome of ROS formation. Several recent studies proposed that aggregation of amyloid monomers and/ or oligomers into fibrils actually constitutes a protective mechanism [7] against free radicals by serving as either a metal-ion clearance system [8] and/or an antioxidant [9]. From this point of view, dissociation of the mature amyloid fibrils may enhance the oxidative damage rather than curing the disease [7]. This issue is of particular clinical importance because amyloid diseases are commonly diag- nosed at a stage when amyloid fibrils are already present. Therapeutic intervention is therefore possible only at a late stage. Hence, although several compounds that cause dissociation of amyloid fibrils appear to be safe and effective [10], the use of such compounds must be considered carefully, in view of the theoretically possible risks. Thus far, no systematic mechanistic study has been conducted on the outcome of dissociation of amyloid fibrils. In our previous study [6], we observed a strong correlation between the antioxidative potency of various antioxidants and their potency to cause dissocia- tion of amyloid fibrils. Assuming that amyloid fibrils are stabilized by ROS [11], it may be reasonable to expect that potent antioxidants destabilize the fibrils, as observed experimentally. Yet, the available data on the polyphenol-induced dissociation is insufficient to evaluate the interrelationship between dissociation of amyloid fibrils and ROS that may be formed during dissociation. In the first part of the present Biochimica et Biophysica Acta 1784 (2008) 1570–1577 Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer's disease; DMSO, dimethyl sulfoxide; ESR, electron spin resonance; EC, epicatechin; ECG, epicatechin gallate; EGCG, epigallocatechin gallate; GC, gallocatechin; GCG, gallocatechin gallate; IAPP, islet amyloid polypeptide; NAC, non-amyloid β component; PBN, Phenyl-tert-butylnitrone; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SOD, superoxide dismutase; T2DM, type-2 diabetes mellitus; TEM, transmission electron microscopy; ThT, thioflavin; TMI, transition metal ions ☆ This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Hila Shoval, Sackler Faculty of Medicine, Tel Aviv University, Israel. ⁎ Corresponding author. Tel.: +972 3 6407305; fax: +972 3 6409113. E-mail address: physidov@post.tau.ac.il (D. Lichtenberg). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.08.007 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap