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The heme oxygenase‐1 protein is susceptible to oxidative modification through protein carbonyl formation
Author(s) -
Yang Guang,
Hu Angela,
Bordner Jessica,
Dennery Phyllis A,
Dennery Phyllis A
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a816
Subject(s) - protein carbonylation , chemistry , lysine , biochemistry , heme oxygenase , endoplasmic reticulum , immunoprecipitation , streptavidin , heme , oxidative phosphorylation , microbiology and biotechnology , oxidative stress , biotin , amino acid , biology , enzyme , lipid peroxidation , gene
Heme oxygenase‐1 (HO‐1) is an integral membrane of the smooth endoplasmic reticulum. This hydrophobic membrane‐binding domain is located at the c‐terminus adjacent to a PEST sequence that can be cleaved by proteases. Recently we have found that an HO‐1 protein lacking the C‐terminus localized to the nucleus under oxidative stress. Protein carbonyl formation is a hallmark of oxidative stress and carbonylation at specific sites can alter protein function. Because HO‐1 is a stress response protein we wanted to know whether HO‐1 protein was susceptible to carbonyl formation and whether carbonylation at specific sites explained HO‐1 nuclear localization. Cultured fibroblasts were incubated with hemin at 0–60 μM for 8 h, with 0–200 μM H 2 O 2 or with a mixture of 100 μM FeCl 3 + 25 mM ascorbic acid (MCO) for 4 h. Cell lysates were immunoprecipitated with HO‐1 antibodies and were subjected to protein carbonyl detection using a commercially available kit. In other experiments, lysates were incubated with 5 mM biotin hydrazide and then immunoprecipitated with streptavidin and loaded onto an acrylamide gel for HO‐1 detection using Western analysis. Protein carbonyl formation on HO‐1 was observed with all oxidant incubations. Because hydrazide labels specific carbonyls from the side chains of lysine or arginine, this suggests that carbonyls on HO‐1 are more likely to target lysine or arginine residues.