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Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6
- Comprehensive identification of novel post-translational modifications in cellular peroxiredoxin 6
- Jeong J.; Kim Y.; Kyung Seong J.; Lee K.-J.
- Ewha Authors
- SCOPUS Author ID
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- Journal Title
- vol. 12, no. 9, pp. 1452 - 1462
- SCI; SCIE; SCOPUS
- Peroxiredoxin 6 (PRDX6), a 1-Cys peroxiredoxin, is a bifunctional enzyme acting both as a glutathione peroxidase and a phospholipase A2. However, the underlying mechanisms and their regulation mechanisms are not well understood. Because post-translational modifications (PTMs) have been shown to play important roles in the function of many proteins, we undertook, in this study, to identify the PTMs in PRDX6 utilizing proteomic tools including nanoUPLC-ESI-q-TOF MS/MS employing selectively excluded mass screening analysis (SEMSA) in conjunction with MODi and MODmap algorithm. We chose PRDX6 obtained from liver tissues from two inbred mouse strains, C57BL/6J and C3H/HeJ, which vary in their susceptibility to high-fat diet-induced obesity and atherosclerosis, and a B16F10 melanoma cell line for this study. When PRDX6 protein samples were separated on 2D-PAGE based on pI, several PRDX6 spots appeared. They were purified and the low abundant PTMs in each PRDX6 spot were analyzed. Unexpected mass shifts (Δm = -34, +25, +64, +87, +103, +134, +150, +284 Da) observed at active site cysteine residue (Cys47) were quantified using precursor ion intensities. Mass differences of -34, +25, and +64 Da are presumed to reflect the conversion of cysteine to dehydroalanine, cyano, and Cys-SO2-SH, respectively. We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da) as well as unknown modifications (+134, +150, +284 Da). Comprehensive analysis of these PTMs revealed that the PRDX6 exists as a heterogeneous mixture of molecules containing a multitude of PTMs. Several of these modifications occur at cysteine residue in the enzyme active site. Other modifications observed, in PRDX6 from mouse liver tissues included, among others, mono- and dioxidation at Trp and Met, acetylation at Lys, and deamidation at Asn and Gln. Comprehensive identification of the diverse PTMs occurring in this bifunctional PRDX6 enzyme should help understand how PRDX6 plays key roles in oxidative stresses. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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