Structure and activity of the septal peptidoglycan hydrolysis machinery crucial for bacterial cell division.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

Crystal structure of the lipopolysaccharide outer core galactosyltransferase WaaB involved in pathogenic bacterial invasion of host cells.

Lipopolysaccharide (LPS) is essential for most gram-negative bacteria and plays an important role in serum resistance, pathogenesis, drug resistance, and protection from harsh environments. The outer core oligosaccharide of LPS is involved in bacterial recognition and invasion of host cells. The D-galactosyltransferase WaaB is responsible for the addition of D-galactose to the outer core oligosaccharide of LPS, which is essential for Salmonella typhimurium invasion. Here we report the first crystal structures of WaaB and WaaB in complex with UDP to resolutions of 1.8 and 1.9 Å, respectively. Mutagenesis and enzyme activity assays confirmed that residues V186, K195, I216, W243, E276, and E269 of WaaB are essential for the binding and hydrolysis of UDP-galactose. The elucidation of the catalytic mechanism of WaaB is of great importance and could potentially be used for the design of novel therapeutic reagents.

[Effect of a novel EZH2 inhibitor GSK126 on prostate cancer cells].

Objective: To investigate the effect of a novel EZH2 inhibitor GSK126 on cell growth, apoptosis and migration of prostate cancer cells. Methods: Prostate cancer PC-3 and DU145 cells were treated with GSK126 at different doses. Cell growth was detected by sulforhodamine assay. Cell apoptosis was assayed by Annexin V-/PI kit. Transwell chamber and wound healing assays were conducted to detect cell migration. The mRNA level was detected by quantitative PCR, and protein expression was detected by Western blot analysis. Results: GSK126 showed significant effect on cell growth and apoptosis when the dose was higher than 50 µmol/L. Wound healing assay revealed that scratch space in PC-3 cells was significantly increased in a dose-dependent manner in GSK126-treated groups[(247.2±24.4),(347.2±19.2) and (410.5±18.1) µm in low, medium and high dose (5.0, 20.0, 50.0 µmol/L), respectively] as compared with the control group[(171.3±17.8) µm](all P<0.05). Transwell assay showed that migrated PC-3 cells in control group was 322.0±17.9,while those in GSK126-treated groups were 198.3±15.4 (low),82.7±6.2 (medium) and 30.2±4.1 (high), and the differences between the control group and GSK126-treated groups were significant(all P<0.05). In addition, GSK126 up-regulated E-cadherin mRNA expression and down-regulated N-cadherin and Vimentin mRNA expression, whereas had no significant effect on Snail, Fibronectin and VEGF-A mRNA expression. The protein expression of E-cadherin was elevated but VEGF-A protein did not change in GSK126-treated groups. Similar results were exhibited in DU145 cell. Conclusion: GSK126 can significantly inhibit cell migration and invasion in prostate cancer PC-3 and DU145 cells, which may be resulted from its effect on epithelial-mesenchymal transition. GSK126 may be used as a potential anti-prostate cancer dug in clinic.

The novel EZH2 inhibitor, GSK126, suppresses cell migration and angiogenesis via down-regulating VEGF-A.

PURPOSE: To explore the effects and mechanisms of GSK126, a novel inhibitor of histone methyltransferase enhancer of zeste homologue 2, on cancer cell migration. METHODS: Gastric cancer cell line MGC803 and human lung adenocarcinoma cell line A549 were treated with GSK126 at three doses. Transwell and wound healing assays were conducted to detect cell migration. Human umbilical vein endothelial cells tube formation assay and chick embryo chorioallantoic membrane assay were performed to assess the effects of GSK126 on angiogenesis in vitro and in vivo, respectively. The mRNA level of VEGF-A was detected by quantitative PCR, and the protein levels of VEGF-A were detected both by western blot analysis and immunohistochemistry. Epi-fluorescent intensity was obtained by in vivo imaging. RESULTS: GSK126 inhibited cell migration in both MGC803 and A549 in a dose-dependent manner, as revealed by transwell and wound healing assays. The effects of GSK 126 were similar to those of gefitinib at the same doses. Moreover, GSK126 at doses of 20 and 50 µM inhibited angiogenesis both in vitro and in vivo. GSK126 reduced both the mRNA and protein expression of VEGF-A in a dose-dependent manner. Finally, in vivo imaging assay revealed that GSK126 at 200 mg/kg significantly inhibited cancer cell migration. CONCLUSIONS: GSK126 inhibits cell migration and angiogenesis in solid tumor cell lines through down-regulation of VEGF-A expression. Thus, it may be considered as a novel anticancer drug candidate for solid tumor.

[Comparison of seizure induced by different drugs in ICR Mice].

OBJECTIVE: To compare seizure induced by different epileptic drugs in ICR mice. METHODS: Male adult ICR mice were injected with pilocarpine (Pilo), kainic acid (KA) and pentylenetetrazole (PTZ) to induce status epilepticus (SE). After 2 h of SE, seizures were terminated by injection of diazepam. Mice were sacrificed and sectioned for assessment of neuronal cell death by Fluro-Jade B staining after 7 d and mossy fiber sprouting by Timm staining after 28 d, respectively. Spontaneous seizures were detected by video for 28 d. RESULTS: Pilo and KA induced typical SE in ICR mice, which was identical to those observed in rats and C57/BL6 mice. Timm staining showed evident mossy fiber sprouting in both Pilo and KA treated mice. The incidences of spontaneous seizure were 57.1% and 35.7% in Pilo and KA treated mice, respectively. Mice treated with PTZ represented kindling model. No mossy fiber sprouting and spontaneous seizures were observed. No cell death was detected in all three groups. CONCLUSION: Similar seizure pattern is observed in ICR mice as in rats and C57/BL6 mice. Both Pilo and KA model are the ideal models for chronic temporal lobe epilepsy. ICR mice can be widely used as a cheaper substitute in epilepsy research.