Unraveling Amyloid Toxicity Pathway in NIH3T3 Cells by a Combined Proteomic and 1 H-NMR Metabonomic Approach ANNALISA VILASI, 1 SILVIA VILASI, 2 ROCCO ROMANO, 3 FAUSTO ACERNESE, 3 FABRIZIO BARONE, 3 MARIA LUISA BALESTRIERI, 4 ROSA MARITATO, 4 GAETANO IRACE, 4,5 AND IVANA SIRANGELO 4,5 * 1 Laboratory of Mass Spectrometry and Proteomics, Institute of Protein Biochemistry-CNR, Naples, Italy 2 Institute of Biophysics-CNR, Palermo, Italy 3 Department of Pharmaceutical and Biomedical Sciences, Salerno University, Fisciano, Sa, Italy 4 Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy 5 Istituto Nazionale Biostrutture e Biosistemi, Rome, Italy A range of debilitating human diseases is known to be associated with the formation of stable highly organized protein aggregates known as amyloid fibrils. The early prefibrillar aggregates behave as cytotoxic agents and their toxicity appears to result from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca 2þ that lead to apoptotic or necrotic cell death. However, specific signaling pathways that underlie amyloid pathogenicity remain still unclear. This work aimed to clarify cell impairment induced by amyloid aggregated. To this end, we used a combined proteomic and one-dimensional 1 H-NMR approach on NIH-3T3 cells exposed to prefibrillar aggregates from the amyloidogenic apomyoglobin mutant W7FW14F. The results indicated that cell exposure to prefibrillar aggregates induces changes of the expression level of proteins and metabolites involved in stress response. The majority of the proteins and metabolites detected are reported to be related to oxidative stress, perturbation of calcium homeostasis, apoptotic and survival pathways, and membrane damage. In conclusion, the combined proteomic and 1 H-NMR metabonomic approach, described in this study, contributes to unveil novel proteins and metabolites that could take part to the general framework of the toxicity induced by amyloid aggregates. These findings offer new insights in therapeutic and diagnostic opportunities. J. Cell. Physiol. 228: 1359–1367, 2013. ß 2012 Wiley Periodicals, Inc. A large group of severe degenerative conditions are caused by the aggregation of proteins in the form of deposits known as amyloid fibrils (Carrell and Lomas, 1997; Sunde and Blake, 1998; Dobson, 2001, 2003). These disorders include cerebral conditions such as Alzheimer’s disease, Parkinson’s disease, and Creutzfeldt–Jakob disease, and also a series of systemic amyloidoses in which amyloid deposition occurs in a wider variety of organs within the body (Stefani and Dobson, 2003; Stefani, 2004). In each of these pathological conditions, a specific peptide or protein, that is normally soluble, is deposited into insoluble fibrils which accumulate in one or more types of tissue (Lansbury, 1999; Merlini and Bellotti, 2003; Stefani and Dobson, 2003). The amino acid sequence and the native structure of the proteins associated with amyloid diseases have been found to be highly variable, but structural studies have revealed that amyloid fibrils from different sources share a common ultrastructure (Sunde and Blake, 1997; Stefani and Dobson, 2003; Sorrentino et al., 2012). Electron and atomic force microscopy have shown that amyloid fibrils are typically straight and unbranched and are formed from an assembly of protofilaments 2–5 nm wide. X-ray diffraction analysis has indicated a characteristic structure, that is, the b-cross motif, in which the polypeptide chains form b-strands oriented perpendicular to the long axis of the fibril, and b-sheets propagating in the fibril direction (Sunde and Blake, 1997; Stefani and Dobson, 2003). The ability to form amyloid fibrils is not a peculiar property of the relatively few amino acid sequences associated with specific diseases, but it is a generic phenomenon of polypeptide chain. In fact, a considerable number of proteins, not involved in any amyloid disease, including those adopting full a-helical structures under native conditions, have been shown to form amyloid fibrils in vitro (Chiti et al., 1999; Dobson, 2001, 2003; Bucciantini et al., 2002). Myoglobin belongs to this class of proteins being able to aggregate and form amyloid fibrils under appropriate experimental conditions (Fandrich et al., 2001; Vilasi et al., 2011). In particular, the replacement of both indole residues located at positions 7 and 14 in the N-terminal region of the protein with phenylalanine residues, that is, W7F/W14F, renders the protein highly susceptible to aggregation and formation of amyloid fibrils under experimental conditions not drastically different from the natural setting (pH 7.0 and room temperature; Sirangelo et al., 2002, 2004; Infusini et al., 2012). The authors declare no conflict of interest. Annalisa Vilasi and Silvia Vilasi contributed equally to this work. Additional supporting information may be found in the online version of this article. Contract grant sponsor: Regione Campania; Contract grant number: DGR 2270. *Correspondence to: Ivana Sirangelo, Via L. De Crecchio 7, 80138 Naples, Italy. E-mail: ivana.sirangelo@unina2.it Manuscript Received: 18 September 2012 Manuscript Accepted: 16 November 2012 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 28 November 2012. DOI: 10.1002/jcp.24294 ORIGINAL RESEARCH ARTICLE 1359 Journal of Journal of Cellular Physiology Cellular Physiology ß 2012 WILEY PERIODICALS, INC.