Elena Hidalgo

An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.‐) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe‐SoxR contained non‐heme iron (up to 1.6 atoms per monomer); apo‐SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe‐SoxR indicated that it contains a redox active iron‐sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe‐SoxR and apo‐SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe‐SoxR stimulated transcription initiation at soxS in vitro > 100‐fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe‐SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress.

Binuclear 2Fe-2S Clusters in the Escherichia coli SoxR Protein and Role of the Metal Centers in Transcription

SoxR protein of Escherichia coli is activated by superoxide-generating agents or nitric oxide as a powerful transcription activator of the soxS gene, whose product activates ∼10 other promoters. SoxR contains non-heme iron essential for abortive initiation of transcription in vitro. Here we show that this metal dependence extends to full-length transcription in vitro. In the presence of E. coli σ70 RNA polymerase, iron-containing SoxR mediates open complex formation at the soxS promoter, as determined using footprinting with Cu-5-phenyl-1,10-phenanthroline. We investigated the nature of the SoxR iron center by chemical analyses and electron paramagnetic resonance spectroscopy. Dithionite-reduced Fe-SoxR exhibited an almost axial paramagnetic signature with g values of 2.01 and 1.93 observable up to 100 K. These features, together with quantitation of spin, iron, and S2−, and hydrodynamic evidence that SoxR is a homodimer in solution, indicate that (SoxR)2 contains two [2Fe-2S] clusters. Treatment of Fe-SoxR with high concentrations of dithiothreitol caused subtle changes in the visible absorption spectrum and blocked transcriptional activity without generating reduced [2Fe-2S] centers, but was also associated with the loss of iron from the protein. However, lowering the thiol concentration by dilution allowed spontaneous regeneration of active Fe-SoxR.

The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor.

SoxR protein is a redox-responsive transcription factor that governs a regulon of oxidative stress and antibiotic resistance genes in Escherichia coli. Purified SoxR contains oxidized [2Fe-2S] clusters and stimulates in vitro transcription of its target gene soxS up to 100-fold. SoxR transcriptional activity, but not DNA binding, is completely dependent on the [2Fe-2S] clusters; apo-SoxR prepared in vitro binds the soxS promoter with unchanged affinity but does not have transcription activity. Thus, modulation of the SoxR [2Fe-2S] clusters was proposed to control the proteins function in transcription. Here, we provide evidence that SoxR with reduced [2Fe-2S] clusters is inactive. Redox titration of purified SoxR revealed a midpoint potential of −285 ± 10 mV (pH 7.6). In vitro transcription assays showed that SoxR was inactivated when the [2Fe-2S] cluster was reduced (−380 mV), and full activity was restored upon reoxidation (+100 mV). The results suggest that one-electron oxidation and reduction of the [2Fe-2S] cluster regulate SoxR transcriptional activity.

Redox Signal Transduction: Mutations Shifting [2Fe-2S] Centers of the SoxR Sensor-Regulator to the Oxidized Form

SoxR is a [2Fe-2S] transcription factor triggered by oxidative stress and activated in vitro by one-electron oxidation or assembly of the iron-sulfur centers. To distinguish which mechanism operates in cells, we studied constitutively active SoxR (SoxRc) proteins. Three SoxRc proteins contained [2Fe-2S] centers required for in vitro transcription and, like wild-type SoxR, were inactivated by chemical reduction. However, in vivo spectroscopy showed that even without oxidative stress, the three SoxRc proteins failed to accumulate with reduced [2Fe-2S] (< or = 4% compared to > or = 40% for wild type). One SoxRc protein had a redox potential 65 mV lower than wild type, consistent with its accumulation in the oxidized (activated) form in vivo. These results link in vitro and in vivo approaches showing novel redox regulation that couples an iron-sulfur oxidation state to promoter activation.

Redox signal transduction via iron-sulfur clusters in the SoxR transcription activator

Protein iron-sulfur (FeS) centers have recently been implicated in the regulation of gene expression. In the redox-sensing SoxR protein, the oxidation state of [2Fe-2S] centers controls its activity as a transcription activator independent of DNA-binding ability. Thus, FeS centers allosterically link cellular oxidative stress to the expression of defense genes.

SPACING OF PROMOTER ELEMENTS REGULATES THE BASAL EXPRESSION OF THE SOXS GENE AND CONVERTS SOXR FROM A TRANSCRIPTIONAL ACTIVATOR INTO A REPRESSOR

SoxR protein of Escherichia coli governs a global response against superoxide‐generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance. A redox signal activates SoxR post‐translationally to trigger transcription of a second regulatory gene, soxS. Activated and non‐activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription. SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near‐consensus −10 and −35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp). We have constructed and analyzed site‐specific deletions that alter the promoter element spacing. Reducing the spacer length to 16‐18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat. More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment. Gel mobility‐shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase. Thus, activated SoxR remodels the unusual configuration of the wild‐type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the −10 and the −35 elements.

Activation of the redox sensor Pap1 by hydrogen peroxide requires modulation of the intracellular oxidant concentration

The transcription factor Pap1 and the MAP kinase Sty1 are key regulators of hydrogen peroxide‐induced responses in Schizosaccharomyces pombe. Pap1 can be activated quickly at low, but not high, hydrogen peroxide concentrations. The MAP kinase Sty1 has been reported to participate in Pap1 activation by the oxidant. Here, we provide biochemical and genetic evidence for the in vivo formation of a hydrogen peroxide‐induced disulphide bond in Pap1, which precedes the rapid and reversible nuclear accumulation of the transcription factor. We show that activation of the Sty1 cascade before the oxidative insult, or overexpression of the Sty1‐regulated genes ctt1 (encoding catalase) or gpx1 (encoding glutathione peroxidase), can accelerate Pap1 entry even at high doses of hydrogen peroxide. In fact, the lack of Sty1 impedes Pap1 nuclear localization, but only at high doses of the oxidant. We propose that, whereas low doses of hydrogen peroxide lead directly to Pap1 oxidation‐activation, high concentrations of the oxidant initially activate the Sty1 pathway, with the consequent increase in scavenging enzymes, which in turn helps to decompose the excess of hydrogen peroxide and achieve an appropriate concentration for the subsequent activation of Pap1. Our results also suggest that activation of Sty1 at high doses of hydrogen peroxide may also be required to trigger other antioxidant activities such as those reverting the overoxidation of cysteine residues at the Pap1 pathway.

Oxidative stress in Schizosaccharomyces pombe: different H2O2 levels, different response pathways

Schizosaccharomyces pombe triggers different signalling pathways depending on the severity of the oxidative stress exerted, the main ones being the Pap1 and the Sty1 pathways. The Pap1 transcription factor is more sensitive to hydrogen peroxide (H2O2) than the MAP kinase Sty1 pathway, and is designed to induce adaptation, rather than survival, responses. The peroxiredoxin Tpx1 acts as a H2O2 sensor and the upstream activator of the Pap1 pathway. Therefore, sensitivity to H2O2 depends on this thioredoxin peroxidase. In order to achieve maximal activation of the MAP kinase pathway, the concentration of H2O2 needs to be at least fivefold higher than that to fully activate Pap1. Tpx1 is a H2O2 scavenger, thus its peroxidase activity is essential for aerobic growth. As described for other eukaryotic peroxiredoxins, high doses of H2O2 temporarily inactivate Tpx1 and delay Pap1 activation, whereas the Sty1 pathway remains fully functional under these conditions. As part of the Sty1-dependent transcriptional response, the expression of Srx1 is induced and this reductase re-activates the over-oxidised Tpx1. Therefore, the antioxidant pathways of the fission yeast are perfectly designed so that the transcriptional programs triggered by the different signalling pathways never overlap.

Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues

During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.

Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway

Either calorie restriction, loss‐of‐function of the nutrient‐dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient‐dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient‐dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress‐dependent Sty1 mitogen‐activated protein (MAP) kinase. During transition to stationary phase in glucose‐limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose‐rich conditions. Deletion of the genes coding for the SCH9‐homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose‐rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose‐rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.