Proteomic Study of Amyloid Beta (25–35) Peptide Exposure to Neuronal Cells: Impact on APE1/Ref-1’s Protein–Protein Interaction Anil K. Mantha, 1,2 * Monisha Dhiman, 3 Giulio Taglialatela, 2 Regino J. Perez-Polo, 1,2 * and Sankar Mitra 1 1 Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas 2 Department of Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston, Texas 3 Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas The genotoxic, extracellular accumulation of amyloid b (Ab) protein and subsequent neuronal cell death are associated with Alzheimer’s disease (AD). APE1/Ref-1, the predominant apurinic/apyrimidinic (AP) endonucle- ase and essential in eukaryotic cells, plays a central role in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single-strand breaks (SSBs) in DNA. APE1/Ref-1 is also involved in the redox activation of several trans-acting factors (TFs) in various cell types, but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref-1’s role in neuronal cells vulnerable in AD and other neuro- degenerative disorders, as reflected in its nuclear accu- mulation in AD brains. An increase in APE1/Ref-1 has been shown to enhance neuronal survival after oxidative stress. To address whether APE1/Ref-1 level or its association with other proteins is responsible for this protective effect, we used 2-D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha-3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N-acetyl transfer- ase, sulfotransferase 1c), proteins involved in stress response (i.e., leucine-rich and death domain, anti- NGF30), and heterogeneous nuclear ribonucleoprotien- H (hnRNP-H) as being associated with APE1/Ref-1 in Ab(25–35)-treated rat pheochromocytoma PC12 and human neuroblastoma SH-SY5Y cell lines, two common neuronal precursor lines used in Ab neurotoxicity studies. Because the levels of some of these proteins are affected in the brains of AD patients, our study suggests a neuroprotective role for APE1/Ref-1 via its association with those proteins and modulating their cellular functions during Ab-mediated neuro- toxicity. V V C 2012 Wiley Periodicals, Inc. Key words: APE1/Ref-1; AP endonuclease 1; b; amyloid; neuroprotection; base excision repair; neurodegeneration; oxidative DNA damage The mammalian APE1/Ref-1 is an essential protein that has two known biological activities. The N-terminal segment, containing the nuclear localization signal (Jackson et al., 2005; Chattopadhyay et al., 2006), is essential for its transcriptional regulation activity, including redox activation (Xanthoudakis and Curran, 1992; Jayara- man et al., 1997; Evans et al., 2000; Bhakat et al., 2009), and protein–protein interactions (Chattopadhyay et al., 2008; Vascotto et al., 2009b; Sengupta et al., 2010), whereas the C-terminal domain contains amino acids required for the protein’s enzymatic activity on the en- dogenous and oxidatively generated AP sites and base excision repair intermediates in DNA (Masuda et al., 1998; Erzberger and Wilson, 1999; Evans et al., 2000; Mol et al., 2000; Mitra et al., 2002; Hegde et al., 2008). APE1/Ref-1’s neuronal expression level is gener- ally high (Ono et al., 1995; Wilson et al., 1996; Edwards et al., 1998a,b; Marcon et al., 2009) but is decreased in the hippocampus after hypoxic-ischemia (Walton et al., 1997), in the cortex after compression injury (Lewen et al., 2001), and in the spinal cord after ischemia (Sakurai et al., 2003). In contrast, upregulation during is- chemia (Edwards et al., 1998a) and after intracerebral administration of PACAP was observed in hippocampal neurons (Stetler et al., 2010). Overexpression of APE1/ Ref-1 in cultured neurons appeared to be neuroprotective Contract grant sponsor: USPHS; Contract grant numbers: RO1 CA53791, RO1 ES08457, P30 ES006676 (to S.M.). *Correspondence to: Anil K. Mantha, PhD or Regino J. Perez-Polo, PhD, Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555. E-mail: akmantha@utmb.edu, anilmantha@gmail.com, jperezpo@utmb.edu Received 11 August 2011; Revised 28 November 2011; Accepted 2 December 2011 Published online 20 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23018 Journal of Neuroscience Research 90:1230–1239 (2012) ' 2012 Wiley Periodicals, Inc.