In vivo functions of mammalian glutaredoxin 2

Oxygen is essential for all respirating life forms. However, the use of molecular oxygen as terminal electron acceptor of the respiratory electron chain leads to the generation of reactive oxygen species (ROS) as product of incomplete oxygen reduction. To different extends, ROS can react with and damage DNA, lipids and proteins. To cope with and recover from ROS-induced damage, cells have developed several antioxidant systems. Oxidative stress was once defined as an imbalance between ROS and the antioxidant systems. Today we know that several independent redox circuits exist and look at oxidative stress as an imbalance in these redox signaling and control pathways. In this respect, cysteine residues in proteins are the key regulatory units, because they are highly susceptible to oxidative modifications that can affect both activity and function of proteins. Thiol-disulfide oxidoreductases are proteins that catalyze either the formation or reduction of disulfide bonds. The focus of this thesis was the characterization of mammalian Glutaredoxin 2 (Grx2), a member of this family of proteins.

We have identified human Grx2 as the first thiol-disulfide oxidoreductase that can complex an iron-sulfur cluster. The dimeric holo-enzyme complex was enzymatically inactive. Monomerization induced by both oxidants and reductants activated the protein. Based on these findings, we have proposed a role as redox-sensor for the cluster: under normal conditions the majority of Grx2 in the cell is present in the inactive dimeric form. During oxidative stress conditions, the holo-complex dissociates and active monomeric Grx2 is released. These results were coherent with our finding that knock-down of Grx2 does not affect the viability of HeLa cells per se. However, loss of Grx2 dramatically sensitized the cells towards oxidative stress-induced cell death when they were challenged with doxorubicin and phenylarsine oxide. These results imply an important role of Grx2 in the response of cells to oxidative stress.

We have investigated the expression pattern of mammalian Grx2 mRNA variants and identified three different isoforms of human Grx2: the mitochondrial isoform (Grx2a) was ubiquitously expressed in all tissues and two cytosolic/nuclear isoforms (Grx2b and Grx2c) were restricted to testis in healthy tissue, but also present in several cancer cell lines, indicating a potential function in malignant transformation. In mouse we have identified five transcript variants, encoding three different protein isoforms: (1) mitochondrial Grx2a corresponds to human Grx2a, (2) Grx2c is a cytosolic protein homologous to human Grx2c but derived from different transcript variants and (3) Grx2d, an enzymatically inactive protein that lacks structurally important amino acid residues, with no counterpart in human cells. In contrast to human, mouse Grx2c was not exclusively expressed in testis, but expressed in specific cells of many organs, for instance, in enteroendocrine cells of the fundic gland and in the white pulpa of the spleen.

List of scientific papers

I. Lillig CH, Lönn ME, Enoksson M, Fernandes AP, Holmgren A (2004). Short interfering RNA-mediated silencing of glutaredoxin 2 increases the sensitivity of HeLa cells toward doxorubicin and phenylarsine oxide. Proc Natl Acad Sci USA. 101(36): 13227-32. Epub 2004 Aug 24
https://pubmed.ncbi.nlm.nih.gov/15328416

II. 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 U S A. 102(23): 8168-73. Epub 2005 May 25
https://pubmed.ncbi.nlm.nih.gov/15917333

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, Avval FZ, Capani F, Holmgren A, Lillig CH (2008). Identification, Expression Pattern, and Characterization of Mouse Glutaredoxin 2 Isoforms. Antioxid Redox Signal. Aug 15: Epub ahead of print
https://pubmed.ncbi.nlm.nih.gov/18707224