OXIDATIVE INACTIVATION OF THIOREDOXIN AS A CELLULAR GROWTH-FACTOR ANDPROTECTION BY A CYS(73)-]SER MUTATION

Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the pr oliferation of both normal and tumor cells. We have found that when st ored in the absence of reducing agents, human recombinant Trx undergoe s spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thiore doxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reducta se and that does not stimulate cell proliferation. Both conversions ca n be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx u ndergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer th at is stabilized by an intermolecular Cys(73)-Cys(73) disulfide bond. A Cys(73)-->Ser mutant Trx (C73S) was prepared to determine the role o f Cys(73) in oxidative stability and growth stimulation. C73S was as e ffective as Trx in stimulating cell growth and was a comparable substr ate for thioredoxin reductase. C73S did not show spontaneous or oxidan t-induced loss of activity and did not form a dimer. The results sugge st that Trx can exist in monomeric forms, some of which are mediated b y Cys(73) that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys(73) is also involved in formation of an enzymatically inactive homodimer, which occurs on long term storage or by chemical oxidation. Thus, although clearly involved in protein ina ctivation, Cys(73) is not necessary for the growth stimulating activit y of Trx. Copyright (C) 1996 Elsevier Science Inc.