Regulation of human thioredoxin and glutaredoxin systems by oxidation and s-nitrosylation

Abstract: The homeostasis of intracellular redox status has a crucial role in the cell survival and different processes such as DNA synthesis, gene expression, enzymatic activity, etc. This homeostasis is achieved via an intense balance between pro-oxidants and anti-oxidants. Among anti-oxidants, there are two enzymatic systems which are also the main intracellular general protein disulfide reductases, the thioredoxin (Trx) and glutaredoxin (Grx) systems. The Trx system contains Trx, Trx reductase (TrxR) and NADPH, while the Grx system is composed of glutathione (GSH), glutathione reductase (GR), Grx and NADPH. The upregulation of Trx, TrxR, and Grx have been reported in various tumor cells that are directly correlated to the resistance to chemotherapy, aggressive tumor growth and poor prognosis. Therefore, these two systems are potential targets for anti-tumor therapy as well as some other clinical applications. Hence, it is important to know how the activity and structure of these proteins are affected via chemical compounds or by post-translational modifications. Motexafin gadolinium (MGd), a new chemotherapeutic drug, was an NADPH oxidizing substrate for mammalian TrxRs with a Km-value of 8.65 ?M (kcat/Km of 4.86 × 104 M-1s-1). The reaction involved redox cycling of MGd by oxygen producing superoxide and hydrogen peroxide. MGd acted as a non-competitive inhibitor (IC50 of 6 ?M) for rat TrxR. MGd was a better substrate (kcat/Km of 2.23 × 105 M-1 s-1) for TrxR from E. coli and a strong inhibitor of Trx-dependent protein disulfide reduction. Direct reaction between MGd and reduced Trx from either human or E. coli was negligible. Ribonucleotide reductase (RNR) is an essential enzyme for DNA synthesis. MGd inhibited recombinant mouse RNR activity with either 3 ?M reduced human Trx (IC50 2 ?M) or 4 mM dithiothreitol (IC50 6 ?M) as electron donors. Our results may explain the effects of the drug on cancer cells, which often overproduce TrxR and have induced RNR for replication and repair. Besides two cysteines in the active site, human cytosolic Grx1 and mitochondrial Grx2 contain three and two additional structural cysteines, respectively. We analyzed the redox state and disulfide pairing of Cys residues upon GSSG oxidation and S-nitrosylation. Grx1 was partly inactivated both by S-nitrosylation and oxidation. Inhibition by nitrosylation was reversible under anaerobic conditions; aerobically it was stronger and irreversible, due to nitration. Oxidation of Grx1 induced a complex pattern of disulfide bonded dimers and oligomers formed between Cys 8 and either Cys 79 or Cys 83. An intramolecular disulfide between Cys 79 and Cys 83 was also identified. Grx2 retains activity upon oxidation, did not form dimers or oligomers and could not be S-nitrosylated. The dimeric iron sulfur cluster coordinating inactive form of Grx2 dissociated upon nitrosylation, leading to activation of the protein. Besides active site cysteines in the conserved motif of -Cys-Gly-Pro-Cys-, human cytosolic Trx1 has three structural Cys residues in positions 62, 69 and 73 which upon diamide oxidation induce a second Cys 62-Cys 69 disulfide as well as dimers and multimers. After incubation with H2O2, only monomeric two-disulfide molecules are generated which are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. We found that nitrosylation by GSNO at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys 69 and Cys 73 were nitrosylated and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. The reversible inhibition of human Trx1 activity by H2O2 and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.