Marc-Édouard Mirault

We report here that both kappa B-dependent transactivation of a reporter gene and NF-kappa B activation in response to tumor necrosis factor (TNF alpha) or H2O2 treatments are deficient in human T47D cell transfectants that overexpress seleno-glutathione peroxidase (GSHPx). These cells feature low reactive oxygen species (ROS) levels and decreased intracellular ROS burst in response to TNF alpha treatment. Decreased ROS levels and NF-kappa B activation were likely to result from GSHPx increment since these phenomena were no longer observed when GSHPx activity was reduced by selenium depletion. The cellular contents of the two NF-kappa B subunits (p65 and p50) and of the inhibitory subunit I kappa B-alpha were unaffected by GSHPx overexpression, suggesting that increased GSHPx activity interfered with the activation, but not the synthesis or stability, of Nf-kappa B. Nuclear translocation of NF-kappa B as well as I kappa B-alpha degradation were inhabited in GSHPx-overexpressing cells exposed to oxidative stress. Moreover, in control T47D cells exposed to TNF alpha, a time correlation was observed between elevated ROS levels and I kappa B-alpha degradation. We also show that, in growing T47D cells, GSHPx overexpression altered the isoform composition of I kappa B-alpha, leading to the accumulation of the more basic isoform of this protein. GSHPx overexpression also abolished the TNF alpha-mediated transient accumulation of the acidic and highly phosphorylated I kappa B-alpha isoform. These results suggest that intracellular ROS are key elements that regulate the phosphorylation of I kappa B-alpha, a phenomenon that precedes and controls the degradation of this protein, and then NF-kappa B activation.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.

When Drosophila melanogasteris exposed to 37~ a series of specific genes is activated, whereas most of the other genes, active at 25 ~ before this heat shock, appear to be repressed. The induction of about eight or nine new puffs on the salivary gland chromosomes occurs very shortly after the temperature shift to 37 ~ and at the same time most of the puffs active at 25 ~ before the heat shock rapidly regress at 37 ~ (Ritossa 1962; Ashburner 1970). The same new puffs are induced under a variety of other stress conditions unrelated to temperature (Berendes 1972; Lewis et al. 1975). The heat shock also induces the rapid synthesis of a small number of proteins, whereas the rate of syn-thesis of most cellular proteins, normally made at 25 ~ is strongly reduced (Tissi~res et al. 1974; Lewis et al. 1975; McKenzie et al. 1975). This phenomenon is observed in different issues (Tissi~res et al. 1974), in many different wild-type strains (L. Moran and A. P. Arrigo, unpubl.), and in different Drosophila rnelanogaster tissue-culture cell lines (McKenzie et