Cysteine 343 in the substrate binding domain is the primary <em>S</em>-Nitrosylated site in protein disulfide isomerase
Ogura, Jiro; Ruddock, Lloyd W.; Mano, Nariyasu (2020-08-05)
Jiro Ogura, Lloyd W. Ruddock, Nariyasu Mano, Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase, Free Radical Biology and Medicine, Volume 160, 2020, Pages 103-110, ISSN 0891-5849, https://doi.org/10.1016/j.freeradbiomed.2020.07.029
© 2020 Elsevier B.V. This manuscript version is made available under the CC-BY-NC-ND 4.0 license by http://creativecommons.org/licenses/by-nc-nd/4.0/.
https://creativecommons.org/licenses/by-nc-nd/4.0/
https://urn.fi/URN:NBN:fi-fe202103056643
Tiivistelmä
Abstract
Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b’ domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b’ domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.
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