Identification of thioredoxin-binding protein-2/vitamin D-3 up-regulated protein 1 as a negative regulator of thioredoxin function and expression

Recent works have shown the importance of reduction/oxidation (redox) regul ation in various biological phenomena. Thioredoxin (TRX) is one of the majo r components of the thiol reducing system and plays multiple roles in cellu lar processes such as proliferation, apoptosis, and gene expression. To inv estigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D-3 up-r egulated protein 1 (VDUP1). The association of TRX with TBP-2/ VDUP1 was ob served in vitro and in vivo, TBP-2/VDUP1 bound to reduced TRX but not to ox idized TRX nor to mutant TRX, in which two redox active cysteine residues a re substituted by serine, Thus, the catalytic center of TRX seems to be imp ortant for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and H EK293 cells transiently transfected with TBP-2/VDUP1 expression vector, dec rease of insulin reducing activity of TRX and diminishment of TRX expressio n was observed. These results suggested that TBP-2/VDUP1 serves as a negati ve regulator of the biological function and expression of TRX, Treatment of HL-60 cells with la,25-dihydroxyvitamin D-3 caused an increase of TBP-2/VD UP1 expression and down-regulation of the expression and the reducing activ ity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.