Studies on ribonucleotide reductase, a target for anticancer therapy, with focus on electron donor aspects : thioredoxin and glutaredoxin systems

Faithful replication of DNA and its repair are fundamental processes in all living cells which utilize deoxyribonucleotides as DNA building blocks. Ribonucleotide reductase (RNR) is the essential enzyme for de novo synthesis of these precursors from ribonucleoside diphosphates. Each catalytic cycle of RNR, requires disulfide bond reduction which as shown in Escherichia coli,is catalyzed by thioredoxin (Trx) or glutaredoxin (Grx) systems. These are general protein disulfide reductases, and the presence of one system is essential. The Trx system comprises Trx, Trx reductase (TrxR), and NADPH; and the Grx system is composed of Grx, glutathione (GSH), glutathione reductase, and NADPH. This thesis studies mammalian RNR; in S-phase cells RNR comprises a weak complex of a catalytic R1 protein, containing redox active cysteine residues, and a radical generator subunit termed R2.

By analyzing recombinant mouse RNR with respect to electron donors, we found that Trx1 and Grx1 had similar catalytic efficiency (kcat/Km). With 4 mM GSH, Grx1 showed a higher affinity (apparent Km-value 0.18 µM) compared to Trx1 which displayed a higher apparent kcat suggesting its major role in S-phase DNA replication. Surprisingly, Grx activity was strongly dependent on GSH concentrations (apparent Km-value 3 mM), and a Grx2 Cys40Ser mutant was active despite only one cysteine residue in the active site. These results demonstrate a GSH-mixed disulfide mechanism for Grx catalysis, in contrast to the dithiol mechanism for Trx. We propose that this may be an advantage with the low levels of RNR for DNA repair or in tumor cells with high RNR and no or low Trx expression.

Different isoforms of mouse Grx2 were identified, which were further characterized with respect to subcellular localization, expression pattern, and enzymatic activity. Amoung three different isoforms (Grx2a, Grx2c, and Grx2d), mitochondrial Grx2a was expressed in all tissues, except testis. On the other hand, Grx2c and Grx2d were cytosolic and expressed in testis. Mouse Grx2c had general Grx-activity and could reduce RNR, but Grx2d lacked enzymatic activity. These data provide evidence for additional functions of Grx2 in the cytosol, in cell proliferation, and in cellular differentiation.

Motexafin gadolinium (MGd) is a new anticancer agent with promising results in clinical trials, which selectively targets tumor cells and works as a radiation enhancer. It mediates redox reactions generating reactive oxygen species (ROS) with oxidation of intracellular reducing molecules. MGd was an NADPH-oxidizing substrate for TrxR. The reaction involved redox cycling of MGd by oxygen producing superoxide. MGd acted as a non-competitive inhibitor for TrxR. In contrast, direct reaction between MGd and reduced Trx was negligible. MGd inhibited recombinant RNR activity with either the Trx system or dithiothreitol as electron donors. Overall, our results show that MGd induces generation of ROS by TrxR and is a powerful inhibitor of RNR.

Further studies on the mechanism of inhibition of RNR by MGd, revealed at least two different mechanisms for its effect: interruption in subunit oligomerization, and direct inhibition of the catalytic subunit (R1). Co-localization of MGd and RNR in the cytoplasm was shown particularly in the S-phase. These data elucidate another important effect of MGd on the cancer cells with overproduction of RNR, and highlights its efficacy as an anticancer agent.

List of scientific papers

I. Hashemy SI, Ungerstedt JS, Zahedi Avval F, Holmgren A (2006). "Motexafin gadolinium, a tumor-selective drug targeting thioredoxin reductase and ribonucleotide reductase." J Biol Chem 281(16): 10691-7. Epub 2006 Feb 14.
https://doi.org/10.1074/jbc.M511373200

II. 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://doi.org/10.1089/ars.2008.2068

III. Zahedi Avval F, Berndt C, Pramanik A, Holmgren A (2009). "Mechanism of inhibition of ribonucleotide reductase with motexafin gadolinium (MGd)." Biochem Biophys Res Commun 379(3): 775-9. Epub 2009 Jan 1.
https://doi.org/10.1016/j.bbrc.2008.12.128

IV. Zahedi Avval F, Holmgren A (2009). "Molecular mechanisms of thioredoxin and glutaredoxin as hydrogen donors for mammalian S-phase ribonucleotide reductase." J. Biol. Chem. [Accepted]
https://doi.org/10.1074/jbc.M809338200