Oxidative inactivation of thioredoxin as a cellular growth factor and protection by a Cys73-->Ser mutation.
Thioredoxin (Trx) is a widely distributed redox protein that regulates several intracellular redox-dependent processes and stimulates the proliferation of both normal and tumor cells. We have found that when stored in the absence of reducing agents, human recombinant Trx undergoes spontaneous oxidation, losing its ability to stimulate cell growth, but is still a substrate for NADPH-dependent reduction by human thioredoxin reductase. There is a slower spontaneous conversion of Trx to a homodimer that is not a substrate for reduction by thioredoxin reductase and that does not stimulate cell proliferation. Both conversions can be induced by chemical oxidants and are reversible by treatment with the thiol reducing agent dithiothreitol. SDS-PAGE suggests that Trx undergoes oxidation to monomeric form(s) preceding dimer formation. We have recently shown by X-ray crystallography that Trx forms a dimer that is stabilized by an intermolecular Cys73-Cys73 disulfide bond. A Cys73-->Ser mutant Trx (C73S) was prepared to determine the role of Cys73 in oxidative stability and growth stimulation. C73S was as effective as Trx in stimulating cell growth and was a comparable substrate for thioredoxin reductase. C73S did not show spontaneous or oxidant-induced loss of activity and did not form a dimer. The results suggest that Trx can exist in monomeric forms, some of which are mediated by Cys73 that do not stimulate cell proliferation but can be reduced by thioredoxin reductase. Cys73 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 inactivation, Cys73 is not necessary for the growth stimulating activity of Trx.
Gasdaska
J R
JR
Arizona Cancer Center, University of Arizona Health Services Center, Tucson 85724-5024, USA.
Kirkpatrick
D L
DL
Montfort
W
W
Kuperus
M
M
Hill
S R
SR
Berggren
M
M
Powis
G
G
D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D017354:Point Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D006133:Growth Substances; D011994:Recombinant Proteins; D013879:Thioredoxins