Biochemical properties of human glutaredoxins

Abstract: Glutaredoxins (Grxs) are highly conserved thiol-disulfide oxidoreductases that utilize electrons from the tripeptide glutathione (GSH) to catalyze thiol-disulfide exchange reactions. Bacteria, yeast and plants contain multiple dithiol Grxs, which are involved in different cellular processes like DNA synthesis, defense against oxidative stress, apoptosis, and regulation of transcription factor binding activity. Since only one Grx (Grx1) was known in mammalian cells the aim of this thesis project was to identify and characterize additional mammalian Grxs. Two isoforms of a novel human Grx with putative mitochondrial or nuclear localization signals (Grx2a and Grx2b, respectively) were identified in the expressed sequence tag database. Analysis of the genomic sequence localized Grx2 to chromosome 1, and suggested that alternative first exon splicing generated the two isoforms. Both isoforms of human Grx2 encoded proteins of approximately 18 kDa with 34 % sequence identity to the previously characterized cytosolic Grx1. While all the regions characteristic of Grx was conserved in Grx2, the active site (CSYC) contained a serine replacing the proline in the usual consensus CPYC motif Northern and Western blot analysis revealed ubiquitous expression of Grx2 in human tissues. Recombinant Grx2 differed significantly in its biochemical properties from Grx1. Grx2 exhibited a tenfold lower GSH-dependent thiol-disulfide reducing specific activity than Grx1. However, given the higher apparent affinity of Grx2 for glutathionylated substrates the efficiencies for the enzymes were almost the same. Mutant analysis showed further that the catalytic properties of Grx2 were linked to the Pro to Ser exchange in the non-conserved active site sequence. Unlike all previously characterized eukaryotic glutaredoxins, Grx2 was a substrate for both the cytosolic and mitochondrial isoforms of thioredoxin reductase. This electron transfer pathway enabled Grx2 to reduce GSH-mixed disulfides and low molecular weight disulfides, including GSSG. This activity, in conjunction with a high specificity towards glutathionylated substrates endows Grx2 with properties suitable to remove and control the levels of glutathionylated proteins and GSSG. When considering other possible cellular reductants for Grxs we also found dihydrolipoamide to catalyze Grx-dependent reduction of GSSG, which demonstrated that Grxs are able to work in a reverse electron flow. In contrast to Grx1, Grx2 displayed features typical of glutathione S-transferases, such as conjugation of GSH to electrophilic compounds and binding to S-linked GSH-Sepharose. Unexpectedly, the latter property was dependent on Grx2 being in the oxidized state. A Grx2S38P active site mutant completely abolished the binding to the Sepharose, demonstrating a crucial role of the Pro to Ser exchange also for GSH binding. This suggested that Grx2 might form rather stable complexes with glutathionylated. proteins during oxidative conditions. Also, Grx1 and Grx2 revealed completely different patterns upon treatment with oxidants and reductants. Whereas Grx1 formed disulfide bonded dimers and oligomers and was partly inactivated upon oxidation, Grx2 retained activity and revealed no tendency to oligomerize. Instead the precence of a second structural disulfide was strongly indicated in Grx2, which stabilized the protein. In summary, the significant differences between the two mammalian Grxs might be an adaptation to different redox environments or reactions catalyzed. The unusual properties of Grx2 implicate this enzyme in cellular redox regulation and defense against oxidative stress in mitochondria.