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Nitrosative Stress–Induced S-Glutathionylation of Protein Disulfide Isomerase Leads to Activation of the Unfolded Protein Response

Departments of ¹ Pharmaceutical and Biomedical Sciences and ² Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina

Requests for reprints: Kenneth D. Tew, Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250505, Charleston, SC 29425. Phone: 843-792-2514; Fax: 843-792-2475; E-mail: tewk{at}musc.edu.

Key Words: glutathione • protein disulfide isomerase • unfolded protein response

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively called the unfolded protein response (UPR). Protein disulfide isomerase (PDI) is one of the most abundant ER proteins and maintains a sentinel function in organizing accurate protein folding. Treatment of cells with O²-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) resulted in a dose-dependent increase in intracellular nitric oxide that caused S-glutathionylation of various proteins. Within 4 h, PABA/NO activated the UPR and led to translational attenuation as measured by the phosphorylation and activation of the ER transmembrane kinase, pancreatic ER kinase, and its downstream effector eukaryotic initiation factor 2 in human leukemia (HL60) and ovarian cancer cells (SKOV3). Cleavage of the transcription factor X-box protein 1 and transcriptional activation of the ER resident proteins BiP, PDI, GRP94, and ERO1 (5- to 10-fold induction) also occurred. Immunoprecipitation of PDI showed that whereas nitrosylation was undetectable, PABA/NO treatment caused S-glutathionylation of PDI. Mass spectroscopy analysis showed that single cysteine residues within each of the catalytic sites of PDI had a mass increase [+305.3 Da] consistent with S-glutathionylation. Circular dichroism confirmed that S-glutathionylation of PDI results in alterations in the -helix content of PDI and is concurrent with inhibition of its isomerase activity. Thus, it appears that S-glutathionylation of PDI is an upstream signaling event in the UPR and may be linked with the cytotoxic potential of PABA/NO. [Cancer Res 2009;69(19):7626–34]