An energy transduction mechanism used in bacterial flagellar type III protein export

Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a proton–protein antiporter that uses the two components of proton motive force, Δψ and ΔpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliH–FliI complex, which turns the export gate into a highly efficient, Δψ-driven protein export apparatus

Additional file 1: of Tubulin is a molecular target of the Wnt-activating chemical probe

Supplemental results of cell-based assays and tubulin polymerization assay. Figure S1. Measurements of fold changes in the cellular area. Figure S2. Inhibitory activity on the tubulin polymerization and the cellular microtubule network. Figure S3. The absorbance and fluorescence profiles of AMBMP. Figure S4. Measurements of growth inhibitory activity, cell cycle distribution, and mitotic spindle of MDA-MB231 cells treated with AMBMP. (DOCX 6384 kb

Feedback activation of AMPK-mediated autophagy acceleration is a key resistance mechanism against SCD1 inhibitor-induced cell growth inhibition

<div><p>Elucidating the bioactive compound modes of action is crucial for increasing success rates in drug development. For anticancer drugs, defining effective drug combinations that overcome resistance improves therapeutic efficacy. Herein, by using a biologically annotated compound library, we performed a large-scale combination screening with Stearoyl-CoA desaturase-1 (SCD1) inhibitor, T-3764518, which partially inhibits colorectal cancer cell proliferation. T-3764518 induced phosphorylation and activation of AMPK in HCT-116 cells, which led to blockade of downstream fatty acid synthesis and acceleration of autophagy. Attenuation of fatty acid synthesis by small molecules suppressed the growth inhibitory effect of T-3764518. In contrast, combination of T-3764518 with autophagy flux inhibitors synergistically inhibited cellular proliferation. Experiments using SCD1 knock-out cells validated the results obtained with T-3764518. The results of our study indicated that activation of autophagy serves as a survival signal when SCD1 is inhibited in HCT-116 cells. Furthermore, these findings suggest that combining SCD1 inhibitor with autophagy inhibitors is a promising anticancer therapy.</p></div

Growth inhibition mode of action (MOA) summary for T-3764518.

<p>Autophagy activation functions as a survival signal in cells treated with T-3764518. Dual inhibition of SCD1 and autophagy may be an effective strategy to combat cancer.</p

Combinatorial effects of autophagy inducers and inhibitors with T-3764518 in HCT-116 cells.

<p>(A) Combinatorial effects of serially diluted AZD8055 or STA5326 with T-3764518 (100 nM) in HCT-116 cells after 72 h. Data was expressed as the mean ± standard deviation of representative of more than two independent experiments. Each experiment contains at least four replicates. (B) Drug matrix heatmap illustrating ΔBliss values. HCT-116 cells were treated with AZD8055 or STA5326 alone or in combination with T-3764518 at the indicated concentrations. Bliss sum scores >0 indicate a synergistic effect; Bliss sum scores <0 indicate an antagonistic effect.</p

Inhibition of fatty acid synthesis cascade acted antagonistically to T-3764518.

<p>(A) Dose-response analysis of T-3764518 in HCT-116 cells treated with serial dilutions of T-3764518 for 72 h. Percent inhibition was normalized to wells treated with DMSO or no cells as 0% and 100% growth inhibition controls, respectively. Data was expressed as the mean ± standard deviation of representative of more than two independent experiments. Each experiment contains at least four replicates. (B) Summary of combination screening. Vertical and horizontal axes represent percent growth inhibition in the presence or absence of T-3764518, respectively. Red, ACC inhibitors; Green, Bax channel blocker. Based on the Bliss model, compounds were classified as synergistic, additive/independent, or antagonistic. (C) Western blot of HCT-116 cells treated with DMSO or T-3764518 (100 nM) for the indicated times; actin was used as a loading control. (D) Combination and single condition effects of compound 7a (10 μM) and GSK2194069 (10 μM) with T-3764518 (100 nM) on HCT-116 cells 72-h after treatment. Data was expressed as the mean ± standard deviation of representative of more than two independent experiments. Each experiment contains at least four replicates. NS, not significant by unpaired t-test.</p

Chemical structure of compounds used.

<p>The reported MOA of each compound was also described.</p

Validation of the results obtained by T-3764518 with SCD1-KO cells.

<p>(A) Western blot analysis of SCD1-KO cell lysates; actin was used as a loading control. (B) Growth inhibitory effect of T-3764518 (100 nM) on SCD1-KO and SCD1-WT cells after 72 h of treatment. Data was expressed as the mean ± standard deviation of representative of more than two independent experiments. Each experiment contains at least four replicates. NS, not significant by unpaired t-test.</p