Christoph Hudemann

Thiol redox control via thioredoxin and glutaredoxin systems

The Trx (thioredoxin) and Grx (glutaredoxin) systems control cellular redox potential, keeping a reducing thiol-rich intracellular state, which on generation of reactive oxygen species signals through thiol redox control mechanisms. Here, we give a brief overview of the human Trx and Grx systems. The main part focuses on our current knowledge about mitochondrial Grx2, which facilitates mitochondrial redox homoeostasis during oxidative stress-induced apoptosis.

Identification, Expression Pattern, and Characterization of Mouse Glutaredoxin 2 Isoforms

Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase involved in the maintenance of mitochondrial redox homeostasis. Grx2 was first characterized as mitochondrial protein, but alternative mRNA variants lacking the transit peptide-encoding first exon were demonstrated for human and proposed for mouse. We systematically screened for alternative transcript variants of mouse Grx2. We identified a total of six exons, three constitutive (II, III, and IV), two alternative first exons (exons Ia and Ic), and one single-cassette exon (exon IIIb) located between exons III and IV. Exons Ic and IIIb are not present in the human genome; mice lack human exon Ib. The six exons give rise to five transcript variants that encode three protein isoforms: mitochondrial Grx2a, a cytosolic isoform that is homologous to the cytosolic/nuclear human Grx2c and present in specific cells of many tissues and the testis-specific isoform Grx2d that is unique to mice. Mouse Grx2c can form an iron/sulfur cluster-bridged dimer, is enzymatically active as a monomer, and can donate electrons to ribonucleotide reductase. Testicular cells lack mitochondrial Grx2a but contain cytosolic Grx2. Prominent immunostaining was detected in spermatogonia and spermatids. These results provide evidence for additional functions of Grx2 in the cytosol, in cell proliferation, and in cellular differentiation.

Both Thioredoxin 2 and Glutaredoxin 2 Contribute to the Reduction of the Mitochondrial 2-Cys Peroxiredoxin Prx3 *

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (Km, 23.8 μmol·liter−1; Vmax, 1.2 μmol·(mg·min)−1) similar to Trx2 (Km, 11.2 μmol·liter−1; Vmax, 1.1 μmol·(mg·min)−1). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.

Identification of potential protein dithiol-disulfide substrates of mammalian Grx2.

BACKGROUND Glutaredoxins (Grxs) catalyze the reduction of protein disulfides via the dithiol mechanism and the de-/glutathionylation of substrates via the monothiol mechanism. These rapid, specific, and generally also reversible modifications are part of various signaling cascades regulating for instance cell proliferation, differentiation and apoptosis. Even though crucial functions of the conserved, mitochondrial Grx2a and the cytosolic/nuclear Grx2c isoforms have been proposed, only a few substrates have been identified in vitro or in vivo. The significance of redox signaling is emerging, yet a general lack of methods for the time-resolved analysis of these distinct and rapid modifications in vivo constitutes the biggest challenge in the redox signaling field. METHODS AND RESULTS Here, we have identified potential interaction partners for Grx2 isoforms in human HeLa cells and mouse tissues by an intermediate trapping approach. Some of the 50 potential substrates are part of the cytoskeleton or act in protein folding, cellular signaling and metabolism. Part of these interactions were further verified by immunoprecipitation or a newly established 2-D redox blot. CONCLUSIONS Our study demonstrates that Grx2 catalyzes both the specific oxidation and the reduction of cysteinyl residues in the same compartment at the same time and without affecting the global cellular thiol-redox state. GENERAL SIGNIFICANCE The knowledge of specific targets will be helpful in understanding the functions of Grx2. The 2-D redox blot may be useful for the analysis of the overall thiol-redox state of proteins with high molecular weight and numerous cysteinyl residues, that evaded analysis by previously described methods.