Resolution of oxidative stress by thioredoxin reductase: Cysteine versus selenocysteine

Abstract

Thioredoxin reductase (TR) catalyzes the reduction of thioredoxin (TRX), which in turn reduces mammalian typical 2-Cys peroxiredoxins (PRXs 1–4), thiol peroxidases implicated in redox homeostasis and cell signaling. Typical 2-Cys PRXs are inactivated by hyperoxidation of the peroxidatic cysteine to cysteine-sulfinic acid, and regenerated in a two-step process involving retro-reduction by sulfiredoxin (SRX) and reduction by TRX. Here transient exposure to menadione and glucose oxidase was used to examine the dynamics of oxidative inactivation and reactivation of PRXs in mouse C10 cells expressing various isoforms of TR, including wild type cytoplasmic TR1 (Sec-TR1) and mitochondrial TR2 (Sec-TR2) that encode selenocysteine, as well as mutants of TR1 and TR2 in which the selenocysteine codon was changed to encode cysteine (Cys-TR1 or Cys-TR2). In C10 cells endogenous TR activity was insensitive to levels of hydrogen peroxide that hyperoxidize PRXs. Expression of Sec-TR1 increased TR activity, reduced the basal cytoplasmic redox state, and increased the rate of reduction of a redox-responsive cytoplasmic GFP probe (roGFP), but did not influence either the rate of inactivation or the rate of retro-reduction of PRXs. In comparison to roGFP, which was reduced within minutes once oxidants were removed reduction of 2-Cys PRXs occurred over many hours. Expression of wild type Sec-TR1 or Sec-TR2, but not Cys-TR1 or TR2, increased the rate of reduction of PRXs and improved cell survival after menadione exposure. These results indicate that expression levels of TR do not reduce the severity of initial oxidative insults, but rather govern the rate of reduction of cellular factors required for cell viability. Because Sec-TR is completely insensitive to cytotoxic levels of hydrogen peroxide, we suggest TR functions at the top of a redox pyramid that governs the oxidation state of peroxiredoxins and other protein factors, thereby dictating a hierarchy of phenotypic responses to oxidative insults.

Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma

“…Interestingly, under normal conditions disulfide-bonded PRX3 dimers appear to be relatively long lived. After cessation of acute oxidative stress, disulfide-bonded PRX3 dimers persist for several hours in mouse lung epithelial cells, and their rate of reduction is dictated by the activity of TR2 [ 59 ]. Thus, the presumed catalytic intermediate targeted by TS is both present and persistent in MM tumor cells.…”

Repurposing drugs as pro‐oxidant redox modifiers to eliminate cancer stem cells and improve the treatment of advanced stage cancers

“…Here, gold was bound at three different sites, not directly involving the C‐terminal SeC residue. The TrxRs are very insensitive or slow responders to the direct or rapid effects of cytotoxic cellular levels of H 2 O 2, based upon the insensitivity of their SeC to inactivation by this peroxide, but rather, the TrxR/Trx system preferentially acts on the more rapid acting Prxs, suggesting that TrxR functions at the top of the redox pyramid (Figure A and B) by regulating the oxidation state of the Prxs and other protein factors to dictate a hierarchy of phenotypic responses to oxidative stress …”

“…The TrxRs are very insensitive or slow responders to the direct or rapid effects of cytotoxic cellular levels of H 2 O 2, based upon the insensitivity of their SeC to inactivation by this peroxide, but rather, the TrxR/Trx system preferentially acts on the more rapid acting Prxs, suggesting that TrxR functions at the top of the redox pyramid ( Figure 5A and 5B) by regulating the oxidation state of the Prxs and other protein factors to dictate a hierarchy of phenotypic responses to oxidative stress. 156,167 TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous ROS when GSH synthesis is inhibited and TrxR2 knockout is embryonic lethal, establishing that TrxR2 plays an essential role in cell survival. 119 Moreover, studies of isolated heart mitochondria 168 respiring on glutamate/malate showed a 10-fold decrease in the ratio of oxidized to reduced TrxR2.…”

Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression

“…Hepatocytes were transfected with TXNRD1-targeting siRNA (sense, 5′-CCAUAGAGGGCGAAUUUAAUU-3′; antisense, 5′-UUAAAUUCGCCCUCUAUGGUU-3′) [22] using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. A control siRNA (sense, 5′-ACGUGACACGUUCGGAGAAUU-3′; antisense, 5′-UUCUCCGAACGUGUCACGUUU-3′) was also used.…”