The role of the mitochondrial thioredoxin-2 system in cell function

Abstract

The hypothesis upon which this research is based is that the mitochondrial thioredoxin-2 system, which consists of mitochondrial thioredoxin-2 (Trx-2), mitochondrial thioredoxin reductase (TrxR-2) and mitochondrial thioredoxin peroxidase (Prdx-3), protects cells against apoptosis and regulates cell growth via mitochondrial redox homeostasis. Trx-2 mRNA was found expressed in a panel of cancer cell lines and Trx-2 protein is localized exclusively to the mitochondria. An alternate splice form of Trx-2 lacking exon 2 was identified that does not yield protein. Forced overexpression of Trx-2 in cell lines by using constitutive and inducible promoter systems increased mRNA levels, but did not yield an increase in Trx-2 protein. The role of Trx-2 in mammalian development was examined by generating a mouse deficient in Trx-2. Massive apoptosis, exencephaly and early embryonic lethality was seen in the Trx-2(-/-) homozygous embryos. Trx-2(-/-) embryonic fibroblasts were not viable under normal growth conditions, but could be rescued with maintenance in hypoxia. Trx-2(-/-) cells were also lacking mature cytochrome c, implicating Trx-2 in cytochrome c biogenesis. The Trx-2(+/-) heterozygous mice, which appear normal, had an increased sensitivity to some forms of oxidative toxicity (eg. acute doxorubicin) compared to the wild-type mice. To investigate the role of Prdx-3, WEHI7.2 cells with overexpression of Prdx-3 were generated by stable transfection. Overexpression of Prdx-3 inhibited cell proliferation, reduced cellular hydrogen peroxide levels and altered the mitochondrial membrane potential. Prdx-3 overexpression protected the cell from various drug-induced and hypoxia-induced apoptosis. Prdx-3 protein degradation was decreased during hypoxia, leading to a longer half-life under hypoxic conditions. Additionally, Prdx-3 protein levels were increased in a RCC4 cells expressing wild-type VHL compared to RCC4 cells with mutant VHL.