Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family.

Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth ß-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

Poly-γ-glutamic acid production of Bacillus subtilis (natto) in the absence of DegQ: A gain-of-function mutation in yabJ gene.

Poly-γ-glutamic acid (γPGA) production by Bacillus subtilis is regulated by the quorum sensing system where DegQ transmits the cell density signal to a DNA-binding protein DegU. A mutation suppressing the γPGA-negative phenotype of degQ gene knock-out mutant (ΔdegQ) was identified through whole genome sequencing. The mutation conferred an amino acid substitution of Ser103 to phenylalanine (S103F) in yabJ that belongs to the highly conserved YjgF/YER057c/UK114 family. Genetic experiments including LacZ-fusion assay of γPGA synthetic operon confirmed that the suppressor mutation (yabJS103F) was responsible for the recovery of γPGA production. The yabJ itself was not essential for the γPGA production and the mutant allele enabled γPGA production of the ΔdegQ strain even in the presence of wild type yabJ. Thus, yabJS103F was a dominant positive allele. degU-lacZ fusion gene was hyper-expressed in cells carrying the yabJS103F, but disruption of yabJ did not affect the transcription level of the degU-lacZ. These observations suggested that YabJ acquired a function to stimulate expression of degU by the S103F mutation which is involved in the regulation of γPGA synthesis.

Histone deacetylase inhibitors and aspirin interact synergistically to induce cell death in ovarian cancer cells.

Histone deacetylase inhibitors (HDIs) as well as non-steroidal anti-inflammatory drugs including aspirin show promise as antineoplastic agents. The treatment with both HDIs and aspirin can result in hyperacetylation of proteins. In this study, we investigated whether HDIs and aspirin interacted in inducing anticancer activity and histone acetylation. We found that the HDIs, suberoylanilide hydroxamic acid and sodium butyrate, and aspirin cooperated to induce cell death in the ovarian cancer cell line, A2780. The effect was synergistic, as evidenced by CI-isobologram analysis. However, aspirin had no effect on histone acetylation, neither in the absence nor presence of HDIs. To gain insight into the mechanism underlying the synergistic action of HDIs and aspirin, we employed the deacetylated metabolite of aspirin, salicylic acid, and the cyclooxygenase-1- and -2-selective inhibitors, SC-560 and NS-398, respectively. We found that HDIs and salicylic acid interacted synergistically, albeit less efficiently than HDIs and aspirin, to induce cancer cell death, suggesting that the acetyl and the salicyl moiety contributed to the cooperative interaction of aspirin with HDIs. SC-560 and NS-398 had little effect both when applied alone or in conjunction with HDIs, indicating that the combinatorial effect of HDIs and aspirin was not the result of cyclo-oxygenase inhibition. In conclusion, our study demonstrates that HDIs and aspirin synergize to induce cancer cell death and, thus, provides a rationale for a more in-depth exploration into the potential of combining HDIs and aspirin as a strategy for anticancer therapy.

Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing's sarcoma cells.

PURPOSE: The present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing's sarcoma. METHODS: Cytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot. RESULTS: All three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewing's sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis. CONCLUSIONS: These results suggest that HDIs may be considered as a novel treatment strategy for Ewing's sarcoma either applied as monotherapy or in combination with TRAIL.

Histone deacetylase inhibitors, but not vincristine, cooperate with radiotherapy to induce cell death in medulloblastoma.

BACKGROUND: Though ionising radiation (IR) is an efficient means of postoperative treatment for children with medulloblastoma, the disease is incurable in about a third of them. Thus, multimodality regimens have been introduced, typically combining IR with vincristine. MATERIALS AND METHODS: The combination of IR and vincristine was compared to the combination of IR and histone deacetylase inhibitors (HDIs) for their anticancer activity against medulloblastoma cells in vitro. Cytotoxic activities were assessed by measuring propidium iodide uptake and by cell cycle analysis. RESULTS: HDIs augmented the cytotoxic effect of IR, while the combination of vincristine and IR was significantly less cytotoxic than vincristine alone. Cell cycle analyses revealed that vincristine did not interfere with IR-induced G2/M arrest, whereas HDIs abolished the latter. CONCLUSION: These in vitro findings indicate a favourable interaction of IR and HDIs, but an unfavourable one of IR and vincristine, in medulloblastoma, and provide a rationale for comparing the combination of IR with either vincristine or HDIs in vivo.