Jianqiang Xu

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-β receptor tyrosine kinase signaling

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1−/−) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-β receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-β receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

Significance Several functions in cells require reductive processes, i.e., the enzymatic catalysis of a transfer of electrons to specific cellular substrates. One major reductive system in the cytosol of human cells depends upon thioredoxin 1 (Trx1), which in turn is kept reduced by thioredoxin reductase 1 (TrxR1) using NADPH. In the present study it is shown that another protein in addition to Trx1, called thioredoxin-related protein of 14 kDa (TRP14), is highly efficient together with TrxR1 in catalyzing reduction of L-cystine or nitric oxide-derivatized cysteine residues. It is also shown that TRP14, in contrast to Trx1, is resistant to inactivation by hydrogen peroxide. These findings reveal that several TrxR1-dependent functions in cells may not be propelled solely by Trx1, but instead relate to activities of TRP14. Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min−1⋅µM−1) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min−1⋅µM−1). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

Multilevel Regulation of 2-Cys Peroxiredoxin Reaction Cycle by S-Nitrosylation

Background: S-Nitrosylation may regulate 2-Cys peroxiredoxin systems to affect cellular metabolism of hydrogen peroxide. Results: Nitrosylation impeded the peroxiredoxin-1 catalytic cycle by directly inhibiting the activity of peroxiredoxin-1 and by interfering with the recycling of oxidized peroxiredoxin-1 by the thioredoxin system. Conclusion: Nitrosylation exerts multilevel control of the thioredoxin-peroxiredoxin system. Significance: Through its multiple effects on the thioredoxin-peroxiredoxin system, nitrosylation may influence cellular redox homeostasis. S-Nitrosothiols (SNOs), formed by nitric oxide (NO)-mediated S-nitrosylation, and hydrogen peroxide (H2O2), a prominent reactive oxygen species, are implicated in diverse physiological and pathological processes. Recent research has shown that the cellular action and metabolism of SNOs and H2O2 involve overlapping, thiol-based mechanisms, but how these reactive species may affect each others fate and function is not well understood. In this study we investigated how NO/SNO may affect the redox cycle of mammalian peroxiredoxin-1 (Prx1), a representative of the 2-Cys Prxs, a group of thioredoxin (Trx)-dependent peroxidases. We found that, both in a cell-free system and in cells, NO/SNO donors such as S-nitrosocysteine and S-nitrosoglutathione readily induced the S-nitrosylation of Prx1, causing structural and functional alterations. In particular, nitrosylation promoted disulfide formation involving the pair of catalytic cysteines (Cys-52 and Cys-173) and disrupted the oligomeric structure of Prx1, leading to loss of peroxidase activity. A highly potent inhibition of the peroxidase catalytic reaction by NO/SNO was seen in assays employing the coupled Prx-Trx system. In this setting, S-nitrosocysteine (10 μm) effectively blocked the Trx-mediated regeneration of oxidized Prx1. This effect appeared to be due to both competition between S-nitrosocysteine and Prx1 for the Trx system and direct modulation by S-nitrosocysteine of Trx reductase activity. Our findings that NO/SNO target both Prx and Trx reductase may have implications for understanding the impact of nitrosylation on cellular redox homeostasis.

A Txnrd1-dependent metabolic switch alters hepatic lipogenesis, glycogen storage, and detoxification

Besides helping to maintain a reducing intracellular environment, the thioredoxin (Trx) system impacts bioenergetics and drug metabolism. We show that hepatocyte-specific disruption of Txnrd1, encoding Trx reductase-1 (TrxR1), causes a metabolic switch in which lipogenic genes are repressed and periportal hepatocytes become engorged with glycogen. These livers also overexpress machinery for biosynthesis of glutathione and conversion of glycogen into UDP-glucuronate; they stockpile glutathione-S-transferases and UDP-glucuronyl-transferases; and they overexpress xenobiotic exporters. This realigned metabolic profile suggested that the mutant hepatocytes might be preconditioned to more effectively detoxify certain xenobiotic challenges. Hepatocytes convert the pro-toxin acetaminophen (APAP, paracetamol) into cytotoxic N-acetyl-p-benzoquinone imine (NAPQI). APAP defenses include glucuronidation of APAP or glutathionylation of NAPQI, allowing removal by xenobiotic exporters. We found that NAPQI directly inactivates TrxR1, yet Txnrd1-null livers were resistant to APAP-induced hepatotoxicity. Txnrd1-null livers did not have more effective gene expression responses to APAP challenge; however, their constitutive metabolic state supported more robust GSH biosynthesis, glutathionylation, and glucuronidation systems. Following APAP challenge, this effectively sustained the GSH system and attenuated damage.

Pyrroloquinoline quinone modulates the kinetic parameters of the mammalian selenoprotein thioredoxin reductase 1 and is an inhibitor of glutathione reductase

Pyrroloquinoline quinone (PQQ) is a redox active cofactor for bacterial quinoproteins. Dietary PQQ also has prominent physiological effects in mammals although no mammalian quinoprotein has yet been conclusively identified. Here we found that PQQ has substantial effects on the redox active mammalian selenoprotein thioredoxin reductase 1 (TrxR1). PQQ efficiently inhibited the activity of TrxR1 with its main native substrate thioredoxin and acted as a low efficiency substrate in a Sec-dependent TrxR1-catalyzed reduction. Interestingly, PQQ also stimulated redox cycling of TrxR1 with another quinone substrate, juglone, as much as 13-fold (k(cat)/K(m) increased from 105 min(-1) μM(-1) to 1331 min(-1) μM(-1) for juglone in the presence of 50 μM PQQ, mainly through a lowered apparent K(m) for juglone). Glutathione reductase was also inhibited by PQQ but in contrast to the effects of PQQ on TrxR1, its quinone reduction was not further stimulated. These results reveal that glutathione reductase and the mammalian selenoprotein TrxR1 are direct PQQ protein targets, although not being genuine quinoproteins. These findings may help explain several of the effects of PQQ seen in mammals.

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K(i)=2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k(inact)) of (0.73 ± 0.07) × 10⁻³ μM⁻¹ min⁻¹. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Thiophosphate and selenite conversely modulate cell death induced by glutathione depletion or cisplatin: effects related to activity and Sec contents of thioredoxin reductase

Thiophosphate (SPO(3)) was recently shown to promote cysteine insertion at Sec (selenocysteine)-encoding UGA codons during selenoprotein synthesis. We reported previously that irreversible targeting by cDDP [cis-diamminedichloroplatinum(II) or cisplatin] of the Sec residue in TrxR1 (thioredoxin reductase 1) contributes to cDDP cytotoxicity. This effect could possibly be attenuated in cells expressing less reactive Sec-to-cysteine-substituted TrxR1 variants, or pronounced in cells with higher levels of Sec-containing TrxR1. To test this, we supplemented cells with either SPO(3) or selenium and subsequently determined total as well as specific activities of cellular TrxR1, together with extent of drug-induced cell death. We found that cDDP became less cytotoxic after incubation of A549 or HCT116 cells with lower SPO(3) concentrations (100-300 μM), whereas higher SPO(3) (>300 μM) had pronounced direct cytotoxicity. NIH 3T3 cells showed low basal TrxR1 activity and high susceptibility to SPO(3) cytotoxicity, or to glutathione depletion. Supplementing NIH 3T3 cells with selenite, however, gave increased cellular TrxR1 activity with concomitantly decreased dependence on glutathione, whereas the susceptibility to cDDP increased. The results suggest molecular mechanisms by which the selenium status of cells can affect their glutathione dependence while modulating the cytotoxicity of drugs that target TrxR1.

Wobble decoding by the Escherichia coli selenocysteine insertion machinery

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.