S-Glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function. We have shown that S-glutathionylation of protein disulfide isomerase (PDI) disrupts protein folding and leads to the activation of the unfolded protein response (UPR). PDI is a molecular chaperone for estrogen receptor alpha . Our present data show in breast cancer cells that S-glutathionylation of PDI interferes with its chaperone activity and abolishes its capacity to form a complex with . Such drug treatment also reverses estradiol-induced upregulation of c-Myc, cyclinD1, and , gene products involved in cell proliferation. Expression of an S-glutathionylation refractory PDI mutant diminishes the toxic effects of PABA/NO. Thus, redox regulation of PDI causes its S-glutathionylation, thereby mediating cell death through activation of the UPR and abrogation of stability and signaling. 1. Introduction Glutathione S-transferase pi (GSTP) is a biomarker protein in drug-resistant solid epithelial tumors, including ovarian, breast, liver, pancreatic, lung, and lymphoma [1]. In some cases, GSTP can be the most abundant protein in the tumor and, consequently, has the potential to serve as an important drug target [2–4]. One therapeutic approach has been to develop prodrugs that are substrates for GSTP and become cytotoxic when liberated in cancer cells, yet exhibit diminished activation/toxicity in normal tissue. PABA/NO (O2-[2,4-dinitro-5-[4-(N-methylamino) benzoyloxy]phenyl] 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate) [5] is a GSTP-activated prodrug that releases high levels of nitric oxide (NO) at physiological pH. This reaction results in the formation of a Meisenheimer-complex intermediate and subsequently the leaving group of the reaction generates two molecules of NO. Elevated NO levels lead to cytotoxic effects by forming RNS/ROS intermediates that can alter protein function directly through posttranslational modifications on redox sensitive cysteine residues (S-nitrosylation, P-SNO or S-glutathionylation, P-SSG) [3, 6]. Prior studies have shown that protein disulfide isomerase (PDI) is a molecular target of PABA/NO treatment in cancer cells [2, 5, 7–9]. PDI is the most abundant chaperone/isomerase in the endoplasmic reticulum and plays a pivotal role in protein folding through isomerase and chaperone activity. The active site cysteine residues are S-glutathionylated (PDI-SSG) following PABA/NO treatment. The functional consequences are reduced isomerase activity, accumulation of unfolded/misfolded proteins, and
References
[1]
K. D. Tew, “Glutathione-associated enzymes in anticancer drug resistance,” Cancer Research, vol. 54, no. 16, pp. 4313–4320, 1994.
[2]
D. M. Townsend, V. L. Findlay, and K. D. Tew, “Glutathione S-transferases as regulators of kinase pathways and anticancer drug targets,” Methods in Enzymology, vol. 401, article 19, pp. 287–307, 2005.
[3]
Y. Xiong, J. D. Uys, K. D. Tew, and D. M. Townsend, “S-glutathionylation: from molecular mechanisms to health outcomes,” Antioxidants and Redox Signaling, vol. 15, no. 1, pp. 233–270, 2011.
[4]
K. D. Tew, “TLK-286: a novel glutathione S-transferase-activated prodrug,” Expert Opinion on Investigational Drugs, vol. 14, no. 8, pp. 1047–1054, 2005.
[5]
D. M. Townsend, V. J. Findlay, F. Fazilev et al., “A glutathione S-transferase pi-activated prodrug causes kinase activation concurrent with S-glutathionylation of proteins,” Molecular Pharmacology, vol. 69, no. 2, pp. 501–508, 2006.
[6]
D. M. Townsend, “S-glutathionylation: indicator of cell stress and regulator of the unfolded protein response,” Molecular Interventions, vol. 7, no. 6, pp. 313–324, 2008.
[7]
J. E. Saavedra, A. Srinivasan, G. S. Buzard et al., “PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity,” Journal of Medicinal Chemistry, vol. 49, no. 3, pp. 1157–1164, 2006.
[8]
D. M. Townsend, Y. Manevich, H. Lin et al., “Nitrosative stress-induced S-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response,” Cancer Research, vol. 69, no. 19, pp. 7626–7634, 2009.
[9]
J. D. Uys, Y. Xiong, and D. M. Townsend, “Nitrosative stress-induced S-glutathionylation of protein disulfide isomerase,” Methods in Enzymology, vol. 490, pp. 321–332, 2011.
[10]
X. Fu, P. Wang, and B. T. Zhu, “Protein disulfide isomerase is a multifunctional regulator of estrogenic status in target cells,” Journal of Steroid Biochemistry and Molecular Biology, vol. 112, no. 1–3, pp. 127–137, 2008.
[11]
C. Turano, S. Coppari, F. Altieri, and A. Ferraro, “Proteins of the PDI family: unpredicted non-ER locations and functions,” Journal of Cellular Physiology, vol. 193, no. 2, pp. 154–163, 2002.
[12]
C. C. Landel, P. J. Kushner, and G. L. Greene, “The interaction of human estrogen receptor with DNA is modulated by receptor-associated proteins,” Molecular Endocrinology, vol. 8, no. 10, pp. 1407–1419, 1994.
[13]
J. R. Schultz-Norton, W. H. McDonald, J. R. Yates, and A. M. Nardulli, “Protein disulfide isomerase serves as a molecular chaperone to maintain estrogen receptor α structure and function,” Molecular Endocrinology, vol. 20, no. 9, pp. 1982–1995, 2006.
[14]
T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” Journal of Immunological Methods, vol. 65, no. 1-2, pp. 55–63, 1983.
[15]
T. Uehara, T. Nakamura, D. Yao et al., “S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration,” Nature, vol. 441, no. 7092, pp. 513–517, 2006.
[16]
D. M. Townsend, Y. Manevich, L. He, S. Hutchens, C. J. Pazoles, and K. D. Tew, “Novel role for glutathione S-transferase pi. Regulator of protein S-glutathionylation following oxidative and nitrosative stress,” The Journal of Biological Chemistry, vol. 284, no. 1, pp. 436–445, 2009.
[17]
A. Holmgren, “Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide,” The Journal of Biological Chemistry, vol. 254, no. 19, pp. 9627–9632, 1979.
[18]
V. J. Findlay, D. M. Townsend, J. E. Saavedra et al., “Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug,” Molecular Pharmacology, vol. 65, no. 5, pp. 1070–1079, 2004.
[19]
S. Hutchens, Y. Manevich, L. He, K. D. Tew, and D. M. Townsend, “Cellular resistance to a nitric oxide releasing glutathione S-transferase P-activated prodrug, PABA/NO,” Investigational New Drugs, vol. 29, no. 5, pp. 719–729, 2010.
[20]
Y. Manevich, D. M. Townsend, S. Hutchens, and K. D. Tew, “Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and s-glutathionylation of nitric oxide synthetase,” PLoS ONE, vol. 5, no. 11, Article ID e14151, 2010.
[21]
J. C. M. Tsibris, L. T. Hunt, G. Ballejo, W. C. Barker, L. J. Toney, and W. N. Spellacy, “Selective inhibition of protein disulfide isomerase by estrogens,” The Journal of Biological Chemistry, vol. 264, no. 24, pp. 13967–13970, 1989.
[22]
F. Hatahet and L. W. Ruddock, “Protein disulfide isomerase: a critical evaluation of its function in disulfide bond formation,” Antioxidants and Redox Signaling, vol. 11, no. 11, pp. 2807–2850, 2009.
[23]
G. Tian, S. Xiang, R. Noiva, W. J. Lennarz, and H. Schindelin, “The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites,” Cell, vol. 124, no. 1, pp. 61–73, 2006.
