Cloning and expression of a cDNA for human thioredoxin.

Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide

“…This had equivalent activity to the wildtype thioredoxin in the DTT-coupled insulin disulfide reduction assays [21]. It should be noted that the human thioredoxin analyzed also contains the substitution Met74-->Thr [18,19,22]. Analysis of oxidized E. coli thioredoxin [3] was straightforward.…”

“…The high-resolution NMR work on thioredoxin in solution, initially focused on the reduced form of the human [16] and E. coli [17] proteins. Cloning of human thioredoxin resulted from a novel functional role of this protein as a secreted cytokine [18], isolated from either conditioned medium of human T-lymphotropic virus type I transformed T cells or from Epstein-Barr virus transformed B cells [19,20]. Like other mammalian thioredoxins [1], the human protein contains additional structural cysteine residues with sulfhydryl groups [11]; one of these, Cys73, is located on the surface close to the active site and in an equivalent position to Gly74 in E. coli thioredoxin [3,4].…”

Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NFκB

“…The aromatic ring of the S_4(Phe58) residue is stacked against the six-membered ring of Trp31; the aliphatic portion of the side chain of the S 3 (Arg59) residue is in contact with the methyl groups of Val59 and Ala66; the aromatic ring of the S_ 2 (Tyr6O) residue is in contact with the methyl groups of Val59, Ala66, Val71 and Thr74; the 3-methine of the S_ 1 (Val61 ) residue and the 13-and y-methylene groups of the Sl 1 (Glu63) residue are in contact with Ala73; the disulfide bridge involving the So(Cys62) residue is in contact with the methyl group of Ala35 and the side chains of Pro34 and Pro75; the S 2 (Gly64) residue is in contact with Pro34; and finally, the S+ 5 (Pro65) residue is in contact with Pro34, the C-methyl and Cy-methylene groups of Ile38, the C13-and Cy-methylene groups of Met37, and the methyl group of Ala92. It is worth noting that the variant of hTRX employed in this study has a threonine at position 74 [36], whereas another variant has a methionine at this position [18]. This difference should have no effect on the hydrophobic interaction between the residue at position 74 and the aromatic ring of the S_ 2 (Tyr60 ) residue of the peptide.…”

“…The coding sequence was verified by DNA sequencing [46]. Expression and purification of the mutant hTRX was performed by procedures identical to those used previously for the wild-type and [C62A, C69A, C73A] mutant proteins [36,47], and uniform labeling with either 5 N or with 15N and 1 3 C was carried out by growing the bacteria in minimal medium with 15 NH4Cl or both 5 NH 4 CI and 1 3 C 6 -glucose as the sole nitrogen and carbon sources, respectively. It is worth noting that the activity of the triple Cys->Ala mutant protein [C62A, C69A, C73A] in the spectrophotometric insulin reduction assay [48] is the same as that of the wild-type protein [47].…”

The elements of life and medicines

“…As an antioxidant, GSH participates in protection of the cell from oxidative stress either directly or as a cofactor of glutathione peroxidases [406]. Thioredoxins, with a dithiol-disulfide active site, are a class of redox proteins that participate in redox signalling in the body and are coded by the TXN gene [407]. Cysteine (Cys) is biosynthesized in humans and participates in enzymatic reactions as a nucleophile.…”