Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the 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 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.
Departments of Pharmaceutical and Biomedical Sciences and Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA.
Danyelle M
DM
Townsend
Yefim
Y
Manevich
Lin
L
He
Ying
Y
Xiong
Robert R
RR
Bowers
Steven
S
Hutchens
Kenneth D
KD
Tew
D010129:4-Aminobenzoic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000494:pharmacology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003545:Cysteine / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D017510:Protein Folding; D040901:Proteomics / Q000379:methods; D013329:Structure-Activity Relationship; D062366:para-Aminobenzoates
D001391:Azo Compounds; C485370:O(2)-(2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate; D062366:para-Aminobenzoates; D009569:Nitric Oxide; D019704:Protein Disulfide-Isomerases; D005978:Glutathione; D003545:Cysteine; D010129:4-Aminobenzoic Acid