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Abstract Background Human renal cell carcinoma (RCC) is very resistant to chemotherapy. ABT-737 is a novel inhibitor of anti-apoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. Results We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposide-treatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. Conclusions Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells.

Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD[sup.+] leukemia cells. This synergized with the allogeneic CD8[sup.+] T cell response, leading to long-term survival in six mouse models of FLT3-ITD[sup.+] AML. Sorafenib-related IL-15 production caused an increase in CD8[sup.+]CD107a[sup.+]IFN-[gamma][sup.+] T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD[sup.+] AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8[sup.+] T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT., Author(s): Nimitha R Mathew [1, 2]; Francis Baumgartner [1]; Lukas Braun [1]; David O'Sullivan [3]; Simone Thomas [4]; Miguel Waterhouse [1]; Tony A Müller [1]; Kathrin Hanke [1, 2]; Sanaz [...]

Escherichia coli lives in the mammalian gastrointestinal tract anaerobically at high osmolarity as well as in the soil aerobically at varying osmolarities. Adaptation to these varying environmental conditions is crucial for growth and survival of E. coli. Two-dimensional protein gels were used to visualize global time-dependent changes (10 to 60 min) in the proteome of cells responding to osmotic stress (0.4 M NaCl or 0.7 M sorbitol) under aerobic or anaerobic conditions. The protein profiles revealed an induction of 12 proteins (Dps, HchA, HdhA, InfB, OsmC, OsmY, ProX, KatE, PspA, TalA, TktB, and TreF) under osmotic stress in an aerobic milieu. Eleven additional proteins (OtsB, YceI, YciE, YciF, YgaU, YjbJ, AcnA, MetL, PoxB, Ssb, and YhbO) were induced by osmotic stress imposed by NaCl. Most of the accumulated proteins were cross-protecting proteins (e.g., OsmY, OsmC, Dps, and KatE) which are regulated at the transcriptional level predominantly by RpoS and other regulators (e.g., integration host factor, OxyR, H-NS, LRP, and FIS). Comparative analysis of the proteome of E. coli grown under aerobic or anaerobic conditions under osmotic stress (NaCl) revealed an overlap of the up-regulated proteins of more than 50%. Ten proteins (PoxB, AcnA, TalA, TktB, KatE, PspA, Ssb, TreF, MetL, and YhbO) were detectable only under aerobic, high-osmolality conditions. Time-dependent alterations of the proteome were monitored, allowing classification of the up-regulated proteins into early, middle, and long-term phases of adaptation. Only a few proteins were found to be down-regulated upon osmotic stress.

Results demonstrate that an osmotic upshift alters mRNA levels of 152 genes during early transcription in Escherichia coli as determined by DNA microarray technology.

Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis. [ABSTRACT FROM AUTHOR]

A study focusing on universal stress proteins (Usp)G (UP12) of Escherchia coli, which belongs to the UspFG subfamily, was conducted. The results by the resolution of the UspG by two-dimensional gel electrophoresis uncovered a posttranslational modification during its overexpression in Escherichia coli.

Background: Human renal cell carcinoma (RCC) is very resistant to chemotherapy. ABT-737 is a novel inhibitor of antiapoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. Results: We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposidetreatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. Conclusions: Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells. [ABSTRACT FROM AUTHOR]

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and antiapoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim[sub S]. Regulated expression of Bim[sub S] in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim[sub S] showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim[sub S] enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting. [ABSTRACT FROM AUTHOR]

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein Bim(S) to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated Bim(S)-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of Bim(S) was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of Bim(S)-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.