Functional characterization of cytosolic and mitochondrial thioredoxin reductases

Mammalian thioredoxin reductases (TrxRs) are homodimeric selenoproteins belonging to the nucleotide oxidoreductase family. They contain a C-terminal penultimate selenocysteine residue, which is kept reduced by the N-terminal redox active site, CVNVGC, of the adjacent subunit. The low pKa, of the selenocysteine residue, in combination with its Cterminal accessibility, gives TrxRs broad substrate specificity. The main substrates of TrxRs are thioredoxins (Trxs), which are reduced using NADPH as an electron donor. Trx can then act as a general protein-disulfide reductase and reduce a variety of substrates. This constitutes the thioredoxin system. Apart from the classical cytosolic thioredoxin system, there exists a complete thioredoxin system exclusively in mitochondria with its own thioredoxin (Trx2) and thioredoxin reductase (TrxR2). In this thesis, we describe the functional characterization of the cytosolic and mitochondrial thioredoxin reductases.

It has been suggested that there exists a link between selenium and vitamin E in the protection against lipid membrane peroxidation. Here, we present evidence that TrxR1 is a major reducer of ubiquinone, a regenerator of vitamin E. This reduction is entirely selenium dependent, and puts forward TrxR1 as an important enzyme in the antioxidant defence of lipid membranes.

Furthermore, we show that cell lines overexpressing TrxR2 have a higher viability than control cells upon complex III inhibition. This effect may be ascribed to the reduction of cytochrome c by TrxR2, since this would allow electrons to bypass complex III via TrxR2. Indeed, we demonstrate that TrxR2 is a potent reducer of cytochrome c.

In addition, we show that cells overexpressing TrxRs have a surprising elevated expression of markers associated with differentiation, compared to control cells. This effect is evident for both the classic cytosolic form, TrxR1a, and its cytosolic splice variant TrxR1b. Expression of TrxR1a and TrxR1b apparently preceds the expression of the genes associated with differentiation, suggesting TrxRs to be involved in the early onset of differentiation. Furthermore, some genes are oppositely regulated by TrxR1a and TrxR1b, implying attentiveness in future TrxR1 gene silencing experiments.

In summary, the results presented in this thesis give better understanding of the functions of TrxRs. Our work illustrates the diverse roles of TrxRs, from mediators of redox homeostasis in distinct cellular compartments to their implications in gene expression pathways. The role of TrxRs in these redox regulatory mechanisms are far from resolved and, as reflected in this thesis, much more work is needed in this field of research.

List of scientific papers

I. Xia L, Nordman T, Olsson JM, Damdimopoulos A, Bjorkhem-Bergman L, Nalvarte I, Eriksson LC, Arner ES, Spyrou G, Bjornstedt M (2003). The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress. J Biol Chem. 278(4): 2141-6.
https://pubmed.ncbi.nlm.nih.gov/12435734

II. Nalvarte I, Damdimopoulos AE, Spyrou G (2004). Human mitochondrial thioredoxin reductase reduces cytochrome c and confers resistance to complex III inhibition. Free Radic Biol Med. 36(10): 1270-8.
https://pubmed.ncbi.nlm.nih.gov/15110392

III. Nalvarte I, Damdimopoulos AE, Nystom C, Nordman T, Miranda-Vizuete A, Olsson JM, Eriksson L, Bjornstedt M, Arner ES, Spyrou G (2004). Overexpression of enzymatically active human cytosolic and mitochondrial thioredoxin reductase in HEK-293 cells. Effect on cell growth and differentiation. J Biol Chem. 279(52): 54510-7.
https://pubmed.ncbi.nlm.nih.gov/15471857

IV. Nalvarte I, Guoveris A, Spyrou G (2006). Expression and activity of Thioredoxin Reductase 1a and 1b regulates the expression of genes associated with differentiation, adhesion and tumorgenesis. [Manuscript]