Tumor necrosis factor receptor-associated factor (TRAF)2 represses the T helper cell type 2 response through interaction with NFAT-interacting protein (NIP45).

Recently we have identified a novel protein NIP45 (nuclear factor of activated T cells [NFAT]-interacting protein) which substantially augments interleukin (IL)-4 gene transcription. The provision of NIP45 together with NFAT and the T helper cell type 2 (Th2)-specific transcription factor c-Maf to cells normally refractory to IL-4 production, such as B cells or Th1 clones, results in substantial IL-4 secretion to levels that approximate those produced by primary Th2 cells. In studies designed to further our understanding of NIP45 activity, we have uncovered a novel facet of IL-4 gene regulation. We present evidence that members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins, generally known to function as adapter proteins that transduce signals from the tumor necrosis factor receptor superfamily, contribute to the repression of IL-4 gene transcription and that this effect is mediated through their interaction with NIP45.

Regulatory locus soxRS partially protects Escherichia coli against ozone.

Ozone is one of the major city air pollutants. Since it is known to induce the overexpression of superoxide-dismutase in various models, and is also a powerful oxidant, we tested if ozone can induce the expression of the soxRS regulon of Escherichia coli, which is activated by superoxide and nitric oxide. A sub-lethal exposure to ozone was unable to activate the expression of soxS'::lacZ transcriptional fusions. However, cells lacking the soxRS locus were more susceptible than wild-type to ozone-mediated killing. Constitutive expression of the soxRS regulon did not increase the resistance to ozone. Ozone might be exerting a selective pressure upon oxidative-stress defense mechanisms in airborne bacteria.

The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator.

The SoxR protein of Escherichia coli responds to redox signals by activating the transcription of soxS, which encodes another transcription activator that directly stimulates oxidative stress genes. We show here that transcription of the soxR gene, which is positioned head-to-head with soxS in the chromosome, initiates in the intergenic region and is itself repressed by SoxR protein in in vitro transcription experiments. Analysis of single-copy operon fusions to soxR, combined with the results of Northern blotting experiments, verified this regulation and the transcription start site in vivo. The structure of the overlapping promoters is such that the single SoxR-binding site is located in the -10/-35 spacer of the soxS promoter, but just downstream of the -10 element of the soxR promoter. Activated and non-activated SoxR bind this site equally well, exerting nearly constant repression of soxR; activated SoxR simultaneously stimulates the soxS promoter >/=30-fold. The functional soxR promoter depresses soxS transcription when SoxR is not activated and enhances soxS transcription when SoxR is activated, as shown by comparing the expression of soxS'::lacZ fusions with and without the soxR -35 element (induction ratio only approximately 7-fold). SoxR thus represents a highly polar, redox-regulated transcriptional switch that maximizes the change in expression of soxS.

Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the [2Fe-2S] centers in transcriptional activation.

The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages. SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters. Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro . The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR. Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR. The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent. However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold. Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat. These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.