Alternative glucose uptake mediated by ß-catenin/RSK1 axis under stress stimuli in mammalian cells.

Cells adapt to stress conditions by increasing glucose uptake as cytoprotective strategy. The efficiency of glucose uptake is determined by the translocation of glucose transporters (GLUTs) from cytosolic vesicles to cellular membranes in many tissues and cells. GLUT translocation is tightly controlled by the activation of Tre-2/BUB2/CDC16 1 domain family 4 (TBC1D4) via its phosphorylation. The mechanisms of glucose uptake under stress conditions remain to be clarified. In this study, we surprisingly found that glucose uptake is apparently increased for the early response to three stress stimuli, glucose starvation and the exposure to lipopolysaccharide (LPS) or deoxynivalenol (DON). The stress-induced glucose uptake was mainly controlled by the increment of ß-catenin level and the activation of RSK1. Mechanistically, ß-catenin directly interacted with RSK1 and TBC1D4, acting as the scaffold protein to recruit activated RSK1 to promote the phosphorylation of TBC1D4. In addition, ß-catenin was further stabilized due to the inhibition of GSK3ß kinase activity which is caused by activated RSK1 phosphorylating GSK3ß at Ser9. In general, this triple protein complex consisting of ß-catenin, phosphorylated RSK1, and TBC1D4 were increased in the early response to these stress signals, and consequently, further promoted the phosphorylation of TBC1D4 to facilitate the translocation of GLUT4 to the cell membrane. Our study revealed that the ß-catenin/RSK1 axis contributed to the increment of glucose uptake for cellular adaption to these stress conditions, shedding new insights into cellular energy utilization under stress.

The Specific Binding and Promotion Effect of Azoles on Human Aldo-Keto Reductase 7A2.

Human AKR 7A2 broadly participates in the metabolism of a number of exogenous and endogenous compounds. Azoles are a class of clinically widely used antifungal drugs, which are usually metabolized by CYP 3A4, CYP2C19, and CYP1A1, etc. in vivo. The azole-protein interactions that human AKR7A2 participates in remain unreported. In this study, we investigated the effect of the representative azoles (miconazole, econazole, ketoconazole, fluconazole, itraconazole, voriconazole, and posaconazole) on the catalysis of human AKR7A2. The steady-state kinetics study showed that the catalytic efficiency of AKR7A2 enhanced in a dose-dependent manner in the presence of posaconazole, miconazole, fluconazole, and itraconazole, while it had no change in the presence of econazole, ketoconazole, and voriconazole. Biacore assays demonstrated that all seven azoles were able to specifically bind to AKR7A2, among which itraconazole, posaconazole, and voriconazole showed the strongest binding. Blind docking predicted that all azoles were apt to preferentially bind at the entrance of the substrate cavity of AKR7A2. Flexible docking showed that posaconazole, located at the region, can efficiently lower the binding energy of the substrate 2-CBA in the cavity compared to the case of no posaconazole. This study demonstrates that human AKR7A2 can interact with some azole drugs, and it also reveals that the enzyme activity can be regulated by some small molecules. These findings will enable a better understanding of azole-protein interactions.

T-2 toxin and deoxynivalenol (DON) exert distinct effects on stress granule formation depending on altered activity of SIRT1.

The T-2 toxin and deoxynivalenol (DON), as the most concerned members of trichothecenes, induce cellular stress responses and various toxic effects. Stress granules (SGs) are rapidly formed in response to stress and play an important role in the cellular stress response. However, it is not known whether T-2 toxin and DON induce SG formation. In this study, we found that T-2 toxin induces SG formation, while DON surprisingly suppresses SG formation. Meanwhile, we discovered that SIRT1 co-localized with SGs and regulated SG formation by controlling the acetylation level of the SG nucleator G3BP1. Upon T-2 toxin, the acetylation level of G3BP1 increased, but the opposite change was observed upon DON. Importantly, T-2 toxin and DON affect the activity of SIRT1 via changing NAD+ level in a different manner, though the mechanism remains to be clarified. These findings suggest that the distinct effects of T-2 toxin and DON on SG formation are caused by changes in the activity of SIRT1. Furthermore, we found that SGs increase the cell toxicity of T-2 toxin and DON. In conclusion, our results reveal the molecular regulation mechanism of TRIs on SG formation and provide novel insights into the toxicological mechanisms of TRIs.

Cell fate determined by the activation balance between PKR and SPHK1.

Double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) activation via autophosphorylation is the central cellular response to stress that promotes cell death or apoptosis. However, the key factors and mechanisms behind the simultaneous activation of pro-survival signaling pathways remain unknown. We have discovered a novel regulatory mechanism for the maintenance of cellular homeostasis that relies on the phosphorylation interplay between sphingosine kinase 1 (SPHK1) and PKR during exogenous stress. We identified SPHK1 as a previously unrecognized PKR substrate. Phosphorylated SPHK1, a central kinase, mediates the activation of PKR-induced pro-survival pathways by the S1P/S1PR1/MAPKs/IKKα signal axis, and antagonizes PKR-mediated endoplasmic reticulum (ER) stress signal transduction under stress conditions. Otherwise, phosphorylated SPHK1 also acts as the negative feedback factor, preferentially binding to the latent form of PKR at the C-terminal kinase motif, inhibiting the homodimerization of PKR, suppressing PKR autophosphorylation, and reducing the signaling strength for cell death and apoptosis. Our results suggest that the balance of the activation levels between PKR and SPHK1, a probable hallmark of homeostasis maintenance, determines cell fate during cellular stress response.

Inhibition of EZH2 and activation of ERRγ synergistically suppresses gastric cancer by inhibiting FOXM1 signaling pathway.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related mortality worldwide, because of the low efficacy of current therapeutic strategies. Estrogen-related receptor γ (ERRγ) was previously showed as a suppressor of GC. However, the mechanism and effective therapeutic method based on ERRγ is yet to be developed. METHODS: The expression levels of ERRγ, EZH2, and FOXM1 were detected by immunohistochemistry, qRT-PCR, and western blot. The regulatory mechanisms of ERRγ and FOXM1 were analyzed by ChIP, EMSA, and siRNA. The effects of EZH2 inhibitor (GSK126) or/and ERRγ agonist (DY131) on the tumorigenesis of gastric cancer cell lines were examined by cell proliferation, transwell migration, wound healing, and colony formation assays. Meanwhile, the inhibitory effects of GSK126 or/and DY131 on tumor growth were analyzed by xenograft tumor growth assay. RESULTS: The expression of ERRγ was suppressed in tumor tissues of GC patients and positively correlated with prognosis, as opposed to that of EZH2 and FOXM1. EZH2 transcriptionally suppressed ERRγ via H3K27me3, which subsequently activated the expression of master oncogene FOXM1. The combination of GSK126 and DY131 synergistically activated ERRγ expression, which subsequently inhibited the expression of FOXM1 and its regulated pathways. Synergistic combination of GSK126 and DY131 significantly inhibited the tumorigenesis of GC cell lines and suppressed the growth of GC xenograft. CONCLUSION: The FOXM1 signaling pathway underlying the ERRγ-mediated gastric cancer suppression was identified. Furthermore, combined treatment with EZH2 inhibitor and ERRγ agonist synergistically suppressed GC progression by inhibiting this signaling pathway, suggesting its high potential in treating GC patients.

Phe-125 and Phe-226 of pig cytochrome P450 1A2 stabilize the binding of aflatoxin B1 and 7-ethoxyresorufin through the key CH/π interactions.

Cytochrome P450 1A2 (CYP1A2) plays important roles in the metabolism of many planar and aromatic drugs and also contributes to the bioactivation of aflatoxin B1 (AFB1) in vivo. To date, the structural basis for CYP1A2's preference to the planar substrates remains unclear. Herein, we investigated the structure-activity relationships for pig CYP1A2 catalyzing AFB1 and 7-ethoxyresorufin (7-ER). A molecular docking study was performed based on a constructed model of pig CYP1A2, which predicted the contributions of Thr-118, Thr-124, Phe-125, Phe-226, Leu-260, and Asp-313 to the substrate catalysis. Site-directed mutagenesis and kinetic analyses exhibited the common grounds: Phe-125, Phe-226 and Asp-313 were vital to AFB1 oxidation (including exo-epoxidation and 9A-hydroxylation) and ethoxyresorufin O-deethylation. Meanwhile, Phe-125 and Phe-226 formed CH/π interactions with AFB1/7-ER, and Asp-313 formed hydrogen bonds with them. Based on other published reports, this study further emphasizes the critical roles of Phe-125 and Phe-226 in recognizing the planar substrates. Our findings highlight the structural basis of pig CYP1A2 specifically catalyzing AFB1 and 7-ER, and may help to elucidate the underlying mechanism of CYP1A2's metabolic preference to the planar and aromatic substrates.

Multiple CH/π Interactions Maintain the Binding of Aflatoxin B1 in the Active Cavity of Human Cytochrome P450 1A2.

Human cytochrome P450 1A2 (CYP1A2) is one of the key CYPs that activate aflatoxin B1 (AFB1), a notorious mycotoxin, into carcinogenic exo-8,9-epoxides (AFBO) in the liver. Although the structure of CYP1A2 is available, the mechanism of CYP1A2-specific binding to AFB1 has not been fully clarified. In this study, we used calculation biology to predict a model of CYP1A2 with AFB1, where Thr-124, Phe-125, Phe-226, and Phe-260 possibly participate in the specific binding. Site-directed mutagenesis was performed to construct mutants T124A, F125A, F226A, and F260A. Escherichia coli-expressed recombinant proteins T124A, F226A, and F260A had active structures, while F125A did not. This was evidenced by Fe2+∙Carbon monoxide (CO)-reduced difference spectra and circular dichroism spectroscopy. Mutant F125A was expressed in HEK293T cells. Steady kinetic assays showed that T124A had enhanced activity towards AFB1, while F125A, F226A, and F260A were significantly reduced in their ability to activate AFB1, implying that hydrogen bonds between Thr-124 and AFB1 were not important for substrate-specific binding, whereas Phe-125, Phe-226, and Phe-260 were essential for the process. The computation simulation and experimental results showed that the three key CH/π interactions between Phe-125, Phe-226, or Phe-260 and AFB1 collectively maintained the stable binding of AFB1 in the active cavity of CYP1A2.