Cloning and Expression of a Novel Human Glutaredoxin (Grx2) with Mitochondrial and Nuclear Isoforms

Abstract

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSHdisulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2-31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5 region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

Targeted disruption of the glutaredoxin 1 gene does not sensitize adult mice to tissue injury induced by ischemia/reperfusion and hyperoxia

“…The second Glrx (Glrx2) gene encodes two proteins as a result of alteration of RNA splicing. One of the Grx2 isoforms is expressed with a N-terminal leader sequence that directs it to the mitochondria, and both have C-terminal sequences that suggest nuclear localization [24,25]. As expected, cells overexpressing Grx1 are more resistant to doxorubicin-induced toxicity compared to control cells [26].…”

20 S Proteasome from Saccharomyces cerevisiae Is Responsive to Redox Modifications and IsS-Glutathionylated

“…Although it has been demonstrated already that GSH is distributed inside the mammalian nucleus at concentrations as high as in the cytoplasm (32), its presence inside the yeast nucleus is not clear, neither is the distribution of glutarredoxin isoforms inside yeast cells clear thus far. A mammalian nuclear isoform was described already (33,34) whereas information on yeast nuclear isoforms is still lacking. Because glutaredoxin activity is coupled to direct GSH consumption, its presence inside the nucleus would be strong evidence of GSH distribution in the nucleus.…”

Mitochondrial Thioredoxin System

“…A second antioxidant defense, the mitochondrial thioredoxin system, which includes Trx2, thioredoxin reductase-2, and NADPH (see below), is a potential source of disulfide reductase activity required for maintaining mitochondrial proteins in their reduced state; thioredoxins catalyze reduction of protein disulfides at much higher rates than GSH (19,20). An additional mitochondrial thiol/disulfide oxidoreductase, glutaredoxin-2 (Grx2), was recently discovered, which relies on GSH and not Trx as electron donor (21,22). Little is known on the function(s) of the mitochondrial thioredoxin system and its potential role in the regulation of cell survival.…”