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Cancer Res. 2009 Oct 1;69(19):7626-34. doi: 10.1158/0008-5472.CAN-09-0493. Epub 2009 Sep 22.

Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response.

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  • 1Departments of Pharmaceutical and Biomedical Sciences and Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA.

Abstract

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 O(2)-[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 alpha-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.

PMID:
19773442
[PubMed - indexed for MEDLINE]
PMCID:
PMC2756322
Free PMC Article
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