-Glutamylcysteine Ethyl Ester Protection of Proteins From A(1– 42)-Mediated Oxidative Stress in Neuronal Cell Culture: A Proteomics Approach Debra Boyd-Kimball, 1 Rukhsana Sultana, 1 H. Fai Poon, 1 Hafiz Mohmmad-Abdul, 1 Bert C. Lynn, 1,2 Jon B. Klein, 4 and D. Allan Butterfield 1,3 * 1 Department of Chemistry, Center for Membrane Sciences, University of Kentucky, Lexington, Kentucky 2 Core Proteomics Laboratory, University of Kentucky, Lexington, Kentucky 3 Sanders-Brown Center on Aging, University of Kentucky, Lexington, Kentucky 4 Kidney Disease Program and Proteomics Core Laboratory, University of Louisville School of Medicine and VAMC, Louisville, Kentucky Protein oxidation mediated by amyloid -peptide (1– 42) (A[1– 42]) has been proposed to play a central role in the pathogenesis of Alzheimer’s disease (AD), a neurode- generative disorder associated with aging and the loss of cognitive function. The specific mechanism by which A(1– 42), the primary component of the senile plaque and a pathologic hallmark of AD, contributes to the oxi- dative damage evident in AD brain is unknown. More- over, the specific proteins that are vulnerable to oxidative damage induced by A(1– 42) are unknown. Identification of such proteins could contribute to our understanding of not only the role of A(1– 42) in the pathogenesis of AD, but also provide insight into the mechanisms of neuro- degeneration at the protein level in AD. We report the proteomic identification of two proteins found to be ox- idized significantly in neuronal cultures treated with A(1– 42): 14-3-3 and glyceraldehyde-3-phosphate de- hydrogenase. We also report that pretreatment of neu- ronal cultures with -glutamylcysteine ethyl ester, a com- pound that supplies the limiting substrate for the synthesis of glutathione and results in the upregulation of glutathione in neuronal cultures, protects both proteins against A(1– 42)-mediated protein oxidation. © 2005 Wiley-Liss, Inc. Key words: amyloid -peptide; neurodegeneration; -glutamylcysteine ethyl ester; aging; proteomics; oxida- tive stress; glutathione Glutathione (GSH) is a tripeptide (-glutamyl- cysteine glycine) that is found in high millimolar concen- trations in the brain and functions as an important antiox- idant by reacting either directly or indirectly with reactive electrophiles, such as 4-hydroxynonenal (Cooper, 1997). GSH levels have been shown to decrease with aging, leaving neurons vulnerable to attack by reactive oxygen species (ROS) that led to subsequent protein oxidation and damage (Liu and Choi, 2000). Both in vitro and in vivo upregulation of GSH by -glutamylcysteine ethyl ester (GCEE) has been shown to protect brain cells against peroxynitrite-mediated oxidative damage (Drake et al., 2002, 2003a). Additionally, previous studies have shown that pretreatment of neuronal cell cultures with 1 mM GCEE protects neurons against A(1– 42)-induced pro- tein oxidation (Boyd-Kimball et al., 2005). Alzheimer’s disease (AD) is a neurodegenerative dis- order associated with cognitive decline, oxidative stress, and aging. One pathological hallmark of AD is the senile plaque, which is composed primarily of A(1– 42), a 39 – 43-amino acid peptide derived from the proteolytic pro- cessing of the transmembrane glycoprotein amyloid pre- cursor protein (APP). A(1– 42) has been shown to induce protein oxidation in vitro and in vivo (Yatin et al., 1999a; Drake et al., 2003b; Mohmmad-Abdul et al., 2004). There is increasing evidence that protein oxidation is involved in the pathogenesis of AD. Consequently, A(1– 42) has been implicated as a causative agent in AD as a mediator of oxidative stress (Markesbery, 1997; Butterfield et al., 2001, 2002a; Selkoe, 2001; Butterfield and Lauderback, 2002; Butterfield, 2002, 2003). Little is known about the targets of A(1– 42)- induced protein oxidation. Protein oxidation is indexed by protein carbonyls, a toxic intermediate (Butterfield and Contract grant sponsor: NIH; Contract grant number: AG-05119, AG- 10836. *Correspondence to: Professor D. Allan Butterfield, Department of Chem- istry, Center for Membrane Sciences, and Sanders-Brown Center on Aging, 121 Chemistry-Physics Building, University of Kentucky, Lexington, KY 40506-0055. E-mail: dabcns@uky.edu Received 15 July 2004; Revised 3 November 2004; Accepted 9 November 2004 Published online 25 January 2005 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.20393 Journal of Neuroscience Research 79:707–713 (2005) © 2005 Wiley-Liss, Inc.