Identification of cysteine, methionine and tryptophan residues of actin oxidized in vivo during oxidative stress.
Increased levels of reactive oxygen species (ROS) cause oxidative stress and are believed to play a key role in the development of age-related diseases and mammalian aging in general by oxidizing proteins, lipids, and DNA. In this study, we have investigated the effects of ROS on actin in an established rat model of acute oxidative stress using short-term X-ray irradiation. Relative to the control, the actin functions studied in vitro were reduced for (i) actin polymerization to a minimum of 33% after 9 h and (ii) actin activated Mg(2+)-ATPase activity of myosin to 55% after 9 h. At 24 h, the activities had partially recovered to 64 and 80% of the control sample, respectively. The underlying oxidative modifications were also studied at the molecular level. The content of reactive carbonyl-groups increased 4-fold within the studied 24 h period. Among the five cysteine residues of actin, Cys(239) and Cys(259) were oxidized to sulfenic (Cys-SOH), sulfinic (Cys-SO(2)H), or sulfonic (Cys-SO(3)H) acids by increasing amounts over the time periods studied. The content of methionine sulfoxides also increased for 15 of the 16 methionine residues, with Met(44), Met(47), and Met(355) having the highest sulfoxide contents. Met(82) was also further oxidized to the sulfone. Among the four tryptophan residues present in actin, only Trp(79) and Trp(86) appeared to undergo oxidation. The relative contents of hydroxy-tryptophan, N-formyl-kynurenine, and kynurenine increased after irradiation, reaching a maximum in the 9 h sample.
Fedorova
Maria
M
Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.
Kuleva
Nadezda
N
Hoffmann
Ralf
R
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism / Q000528:radiation effects; D018384:Oxidative Stress / Q000528:radiation effects*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D050050:Protein Carbonylation / Q000528:radiation effects; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014965:X-Rays
D000199:Actins; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D014364:Tryptophan; D008715:Methionine; D014357:Trypsin; D003545:Cysteine