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

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.

• Authors: Danyelle M Townsend, Yefim Manevich, Lin He, Ying Xiong, Robert R Bowers Jr, Steven Hutchens, Kenneth D Tew
• Journal: Cancer research (Cancer Res) Vol. 69 Issue 19 Pg. 7626-34 (Oct 01 2009) ISSN: 1538-7445 [Electronic] United States
• PMID: 19773442 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Azo Compounds
• O(2)-(2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate
• para-Aminobenzoates
• Nitric Oxide
• Protein Disulfide-Isomerases
• Glutathione
• Cysteine
• 4-Aminobenzoic Acid

Topics

• 4-Aminobenzoic Acid (pharmacology)
• Amino Acid Sequence
• Azo Compounds (pharmacology)
• Catalytic Domain
• Cell Line, Tumor
• Cysteine (metabolism)
• Female
• Glutathione (metabolism)
• HL-60 Cells
• Humans
• Models, Molecular
• Molecular Sequence Data
• Neoplasms (drug therapy, enzymology, metabolism)
• Nitric Oxide (metabolism)
• Ovarian Neoplasms (drug therapy, enzymology, metabolism)
• Protein Disulfide-Isomerases (metabolism)
• Protein Folding
• Proteomics (methods)
• Structure-Activity Relationship
• para-Aminobenzoates