Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

International audienceA single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy

Scythe Regulates Apoptosis-inducing Factor Stability during Endoplasmic Reticulum Stress-induced Apoptosis.

International audienceScythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe(-/-) cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe(-/-) cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF

Sequential interplay between BAG6 and HSP70 upon heat shock.

International audienceBAG6/Scythe/Bat3 is a cochaperone of the heat shock protein HSP70 and is involved in various developmental processes, cellular stress and viability. BAG6 interferes with the protein-refolding activity of HSP70 but its precise involvement in proteotoxic stresses remains unknown. We show that BAG6 is required for the accumulation of HSP70 upon heat shock and that conversely, once accumulated, HSP70 leads to the massive and CHIP-independent degradation of BAG6 through the ubiquitin-proteasome system. These reciprocal influences between BAG6 and HSP70 upon heat shock suggest that BAG6 is a central regulator of the cellular content of HSP70. The HSP70-driven degradation of BAG6, following the BAG6-dependent accumulation of HSP70, could allow the protein-refolding activity of HSP70 and limit the extent of its induction

Cyclooxygenase-2 Inhibition Sensitizes Human Colon Carcinoma Cells to TRAIL-Induced Apoptosis through Clustering of DR5 and Concentrating Death-Inducing Signaling Complex Components into Ceramide-Enriched Caveolae

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The Reaper-Binding Protein Scythe Modulates Apoptosis and Proliferation during Mammalian Development

Scythe (BAT3 [HLA-B-associated transcript 3]) is a nuclear protein that has been implicated in apoptosis, as it can modulate Reaper, a central apoptotic regulator in Drosophila melanogaster. While Scythe can markedly affect Reaper-dependent apoptosis in Xenopus laevis cell extracts, the function of Scythe in mammals is unknown. Here, we report that inactivation of Scythe in the mouse results in lethality associated with pronounced developmental defects in the lung, kidney, and brain. In all cases, these developmental defects were associated with dysregulation of apoptosis and cellular proliferation. Scythe(−)(/)(−) cells were also more resistant to apoptosis induced by menadione and thapsigargin. These data show that Scythe is critical for viability and normal development, probably via regulation of programmed cell death and cellular proliferation

Plasmablasts derive from CD23-negative activated B cells after the extinction of IL-4/STAT6 signaling and IRF4 induction

International audienceThe terminal differentiation of B cells into antibody-secreting cells (ASCs) is a critical component of adaptive immune responses. However, it is a very sensitive process, and dysfunctions lead to a variety of lymphoproliferative neoplasias including germinal center-derived lymphomas. To better characterize the late genomic events that drive the ASC differentiation of human primary naive B cells, we used our in vitro differentiation system and a combination of RNA sequencing and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC sequencing). We discovered 2 mechanisms that drive human terminal B-cell differentiation. First, after an initial response to interleukin-4 (IL-4), cells that were committed to an ASC fate downregulated the CD23 marker and IL-4 signaling, whereas cells that maintained IL-4 signaling did not differentiate. Second, human CD23- cells also increased IRF4 protein to levels required for ASC differentiation, but they did that independently of the ubiquitin-mediated degradation process previously described in mice. Finally, we showed that CD23- cells carried the imprint of their previous activated B-cell status, were precursors of plasmablasts, and had a phenotype similar to that of in vivo preplasmablasts. Altogether, our results provide an unprecedented genomic characterization of the fate decision between activated B cells and plasmablasts, which provides new insights into the pathological mechanisms that drive lymphoma biology

Evaluation of the Potential Biological Effects of the 60-GHz Millimeter Waves Upon Human Cells

International audienceWe investigate potential biological effects of low-power millimeter-wave radiation on human cell viability and intracellular protein homeostasis. A specific exposure system allowing to perform far-field exposures with power densities close to those expected from the future wireless communications in the 60-GHz band has been developed and characterized. Specific absorption rate (SAR) values were determined for the biosamples under test using the FDTD method. It was shown that millimeter-wave radiation at 60.42 GHz and with a maximum incident power density of 1 mW/cm2 does not alter cell viability, gene expression, and protein conformation

Role of Heat Shock Factor 2 in proteasome subunits genes expression

Poster présenté par Sylvain LecomteHeat Shock Factors (HSF) form the main family of transcription factors involved in response to proteotoxic stress. Five HSF are currently described in vertebrate, but only HSF1 and HSF2 are for the moment, well documented. HSF1 is considered as the bona-fide stress-induced transcription factor, whereas HSF2 is more described for its implication in gametogenesis and embryonic development. However, recent studies have shown that HSF2 can have additional roles in response to protein denaturation1,2 and in bookmarking of several stress-related genes 3,4. We have studied the specific role of HSF1 and HSF2 in the cellular response to proteasome inhibition. Using immortalized Mouse Embryonic Fibroblast (iMEF) derived from knock-out Hsf1 and/or Hsf2 mice, we found that cells deleted for either HSF1 or HSF2 are more sensitive to proteasome inhibition than wild type cells. This result suggests that both HSFs are important for cellular response to proteasome inhibitor treatment. To establish the relationship between the proteasome and these transcription factors, several tests were performed using knock-out cells. First, we found a significant decrease of the chymotrypsin-like activity of the proteasome in Hsf2-/- and in Hsf1-/- & Hsf2-/- iMEF. Secondly, the basal expression of ten proteasome subunits was measured by real time PCR. In agreement with our measure of proteasome activity, we found that two catalytic subunits, PSMB2 and PSMB5 which support trypsin-like and chymotrypsin-like activity respectively, were present at lower level in HSF2 deleted iMEF. Moreover, PSMA1, PSMC4 and PSMD10 also display a lower expression in absence of HSF2, whereas the other proteasome subunits tested are not affected. In conclusion, our data showed that HSF2 is involved in some proteasome subunits basal expression. In HSF2 deficient iMEF, decrease expression of these subunits have a direct influence in cell physiology with a decrease of catalytic activity of the proteasome. These results could open a new field in cancer therapy: development of HSF2 inhibitors could improve the action and reduce chemoresistance to proteasome inhibitors such as bortezomib