Isoforms of mammalian glutaredoxin 2

Iron-sulfur proteins are characterized by the presence of iron-sulfur clusters containing sulfide linked iron centers in variable oxidation states. Glutaredoxins (Grx) are, among other redoxins, increasingly recognized to be involved at the interface between redox regulation, iron metabolism and iron-cluster biosynthesis. These versatile oxidoreductases are part of a so-called thioredoxin family acting as facilitators against redox dependent dysregulation of numerous cellular pathways (e.g. protein folding and metabolic pathways). They can generally either reduce or oxidize cysteine residues of proteins, the prime target of reactive oxygen speciesinduced post-translational alteration that affects protein structure and activity. These modifications occur in nearly all subcellular compartments, raising the necessity for practically every oxidoreductase to be present in most compartments.

Previous reports about Grx2 indicated the presence of additional forms, the focus of this thesis lies upon the question how Grx2 activity is regulated as well as how many isoforms of mammalian glutaredoxin 2 there are. Here, we have characterized human Grx2 as the first iron sulfur centercontaining thiol-disulfide oxidoreductase of the thioredoxin fold protein family. It forms a [2Fe-2S] cluster-bridged dimer coordinated by two N-terminal active site thiols of two Grx2 monomers and two molecules of non-covalently bound glutathione. Addition of glutathione disulfide or other oxidants led to the formation of the enzymatic monomer. We therefore proposed that the [2Fe-2S] cluster serves as redox sensor for the activation of Grx2 upon oxidizing conditions.

Human and mouse both possess a generally expressed mitochondrial Grx2a, emphasizing the importance of oxidoreductases in mitochondria, the prime site for the formation of ROS. Alternative splicing gives rise to further isoforms. In human we confirmed the presence of Grx2b and identified a third variant named Grx2c, both located in the cytosol and nucleus in testis and cancer cells. Using the glutaredoxin specific HED assay, they essentially displayed the same activity while with thioredoxin reductase as electron donor Grx2b was only half as efficient as hGrx2c. Only Grx2a and Grx2c are able to form an enzymatically inactive iron-sulfur cluster upon normal cellular conditions, their sibling Grx2b is evidently constitutively active. High levels of Grx2 were found in spermatids, spermatogonia and Sertoli cells indicating unknown functions in cellular development and tumor progression.

Mouse expression pattern displayed similarities to human with some notable differences. The expression of the cytosolic mGrx2c is, unlike in humans, not restricted to testis. Similar to human, the iron-sulfur cluster was shown for the mitochondrial and the cytosolic Grx2a /2c. Mouse Grx2c displayed specific activity and was also able to donate electrons to ribonucleotide reductase. The putative isoform Grx2d is partially encoded by a single cassette exon IIIb which is unique to mouse. Glutaredoxin specific activityfor this protein could not be measured, its functions are to date unknown.

To conclude, here we present new forms of glutaredoxin 2 in human and mouse, a new model of regulation and potential functions in tumor progression and cell differentiation.

List of scientific papers

I. Lillig CH, Berndt C, Vergnolle O, Lönn ME, Hudemann C, Bill E, Holmgren A (2005). Characterization of human glutaredoxin 2 as iron-sulfur protein: a possible role as redox sensor. Proc Natl Acad Sci USA. 102(23): 8168-73. Epub 2005 May 25
https://pubmed.ncbi.nlm.nih.gov/15917333

II. Berndt C, Hudemann C, Hanschmann EM, Axelsson R, Holmgren A, Lillig CH (2007). How does iron-sulfur cluster coordination regulate the activity of human glutaredoxin 2? Antioxid Redox Signal. 9(1): 151-7
https://pubmed.ncbi.nlm.nih.gov/17115894

III. Lönn ME, Hudemann C, Berndt C, Cherkasov V, Capani F, Holmgren A, Lillig CH (2008). Expression pattern of human glutaredoxin 2 isoforms: identification and characterization of two testis/cancer cell-specific isoforms. Antioxid Redox Signal. 10(3): 547-57
https://pubmed.ncbi.nlm.nih.gov/18092940

IV. Hudemann C, Lönn ME, Godoy JR, Zahedi Avval F, Capani F, Holmgren A, Lillig CH (2009). Identification, expression pattern, and characterization of mouse glutaredoxin 2 isoforms. Antioxid Redox Signal. 11(1): 1-14
https://pubmed.ncbi.nlm.nih.gov/18707224