Modulation of p53 dependent gene expression and cell death through thioredoxin-thioredoxin reductase by the Interferon-Retinoid combination.

We have shown earlier that the IFN-beta and all-trans retinoic acid (RA) combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic technique we have identified several Genes associated with Retinoid-IFN induced Mortality (GRIM). One of the GRIMs was human thioredoxin reductase (TR), a redox enzyme. Since the overexpressed TR augments IFN/RA stimulated cell death, we explored the mechanisms of TR-mediated death. Here we show that TR augments cell death by upregulating the transcriptional activity of p53 tumor suppressor. This process does not involve a physical increase in levels of p53. Using redox inactive mutants of TR and its substrate, thioredoxin (Trx), we demonstrate that IFN/RA-induced regulation of p53 dependent gene expression requires TR and Trx. In contrast-over-expression of wildtype TR or Trx augment the p53 dependent gene expression in response to IFN/RA treatment. Consistent with these results an increased DNA binding activity of p53 was noted in the presence of TR. These studies identify a novel mechanism of p53 mediated cell death regulation involving redox enzymes.

Thioredoxin reductase as a pathophysiological factor and drug target.

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2-based oxidative stress. Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nM. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjögren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance. TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.

Purification and functional analysis of the Mycobacterium leprae thioredoxin/thioredoxin reductase hybrid protein.

In Mycobacterium leprae, thioredoxin and thioredoxin reductase are expressed from a single gene. This results in the expression of a hybrid protein with subunits attached to each other by a hydrophilic peptide linker. In all other organisms studied so far, thioredoxin (Trx) and thioredoxin reductase (TR) are expressed as two separate proteins. This raises the question of whether the hybrid protein is enzymatically active and, if so, whether TR reduces its own Trx partner or alternatively a heterologous Trx subunit. To address this question, the hybrid TR/Trx protein of M. leprae as well as the individual parts of the hybrid gene coding for either TR or Trx were overexpressed in Escherichia coli and purified. The purified proteins were tested for their ability to catalyze NADPH-dependent insulin disulfide reduction. Here we show that the M. leprae hybrid protein is indeed enzymatically active. Compared with the enzymatic activity of the separately expressed Trx and TR proteins, the hybrid protein is shown to be more efficient, particularly at low equimolar concentrations. This suggests that the hybrid protein of M. leprae is active by itself and that its activity involves intramolecular interactions between the TR and Trx domains. The activity of the hybrid protein increases when exogenous TR or Trx is added, indicating an additional role for intermolecular interactions.