Biochemical, structural, and biological properties of human thioredoxin active site peptides.
The human redox protein thioredoxin is an autocrine growth factor for some cancer cells. Redox activity is essential for this function but other required structural features of thioredoxin are not known. Two 8-mer peptides (I and II) and one 14-mer peptide (III) were designed based on the amino acid sequence of the redox active site of thioredoxin. Peptide I and peptide III contained the wild-type sequence of thioredoxin while peptide II contained serine residues in place of the catalytically active cysteines. Circular dichroism spectroscopy indicated that all three peptides were comprised mainly of random coil, with peptide III containing slightly more ordered secondary structure. Peptides I and III were substrates for thioredoxin reductase with KM values of 890 and 265 microM, respectively. The redox inactive peptide II could not compete with thioredoxin for reduction by thioredoxin reductase in a coupled insulin reduction assay. However, peptide II was a competitive inhibitor for the reduction of 5, 5'-dithiobis-(2-nitrobenzoic acid) by thioredoxin reductase. All three peptides gave only background levels of stimulation of the proliferation of Swiss 3T3 murine fibroblasts when compared to the stimulation caused by thioredoxin. These results suggest that while the ability of thioredoxin to stimulate cellular proliferation is redox-dependent, more information than that contained in the redox active site domain alone defined by 14 amino acids is required.
Oblong
J E
JE
Arizona Cancer Center, Tucson 85724.
Berggren
M
M
Powis
G
G
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase