Biochemical properties of human glutaredoxins

摘要: Glutaredoxins (Grxs) are highly conserved thiol-disulfide oxidoreductases that utilize electrons from the tripeptide glutathione (GSH) to catalyze exchange reactions. Bacteria, yeast and plants contain multiple dithiol Grxs, which involved in different cellular processes like DNA synthesis, defense against oxidative stress, apoptosis, regulation of transcription factor binding activity. Since only one Grx (Grx1) was known mammalian cells aim this thesis project identify characterize additional Grxs. Two isoforms a novel human with putative mitochondrial or nuclear localization signals (Grx2a Grx2b, respectively) were identified expressed sequence tag database. Analysis genomic localized Grx2 chromosome 1, suggested alternative first exon splicing generated two isoforms. Both encoded proteins approximately 18 kDa 34 % identity previously characterized cytosolic Grx1. While all regions characteristic Grx2, active site (CSYC) contained serine replacing proline usual consensus CPYC motif. Northern Western blot analysis revealed ubiquitous expression tissues. Recombinant differed significantly its biochemical properties exhibited ten-fold lower GSH-dependent reducing specific activity than However, given higher apparent affinity for glutathionylated substrates efficiencies enzymes almost same. Mutant showed further catalytic linked Pro Ser non-conserved sequence. Unlike eukaryotic glutaredoxins, substrate both thioredoxin reductase. This electron transfer pathway enabled reduce GSH-mixed disulfides low molecular weight disulfides, including GSSG. activity, conjunction high specificity towards endows suitable remove control levels When considering other possible reductants Grxs we also found dihydrolipoamide Grx-dependent reduction GSSG, demonstrated able work reverse flow. In contrast Grx1, displayed features typical S-transferases, such as conjugation GSH electrophilic compounds S-linked GSH-Sepharose. Unexpectedly, latter property dependent on being oxidized state. A Grx2S38P mutant completely abolished Sepharose, demonstrating crucial role binding. might form rather stable complexes during conditions. Also, Grx1 patterns upon treatment oxidants reductants. Whereas formed disulfide bonded dimers oligomers partly inactivated oxidation, retained no tendency oligomerize. Instead precence second structural strongly indicated stabilized protein. summary, significant differences between be an adaptation redox environments reactions catalyzed. The unusual implicate enzyme stress mitochondria. LIST OF PUBLICATIONS is based following articles, will referred by their Roman numerals: I. Lundberg, M., Johansson C., Chandra J., Enoksson Jacobsson G., Ljung Holmgren A. (2001) Cloning glutaredoxin (Grx2) J. Biol. Chem. 276, 26269-26275 II. Porras P., Pedrajas J.R., Martinez-Galisteo E., Padilla C.A., A., Barcena J.A. (2002) efficiency. Biochem. Biophys. Res. Commun. 295, 1046-1051 III. Lillig H.C., (2004) Human reduces S-glutathionylated accepting either 279, 7537-7543 IV. Lundberg has S-transferase glutathione. Manuscript V. Elgan H.T., Berndt D.K., state oxidation glutaredoxins identification native 2.