Selenite Targets eIF4E-Binding Protein-1 to Inhibit Translation Initiation and Induce the Assembly of Non-Canonical Stress Granules

Stress granules (SGs) are large cytoplasmic ribonucleoprotein complexes that are assembled when cells are exposed to stress. SGs promote the survival of stressed cells by contributing to the reprogramming of protein expression as well as by blocking pro-apoptotic signaling cascades. These cytoprotective effects implicated SGs in the resistance of cancer cells to radiation and chemotherapy. We have found that sodium selenite, a selenium compound with chemotherapeutic potential, is a potent inducer of SG assembly. Selenite-induced SGs differ from canonical mammalian SGs in their morphology, composition and mechanism of assembly. Their assembly is induced primarily by eIF4E-binding protein1 (4EBP1)-mediated inhibition of translation initiation, which is reinforced by concurrent phosphorylation of eIF2α. Selenite-induced SGs lack several classical SG components, including proteins that contribute to pro-survival functions of canonical SGs. Our results reveal a new mechanism of mammalian SG assembly and provide insights into how selenite cytotoxicity may be exploited as an anti-neoplastic therapy

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal

Cigarette smoke extract impairs gingival epithelial barrier function

We previously showed that junctional adhesion molecule 1 (JAM1) and coxsackievirus and adenovirus receptor (CXADR), tight junction-associated proteins, have important roles to maintain epithelial barrier function in gingival tissues. Smoking is considered to be a significant risk factor for periodontal disease. The present study was conducted to examine the effects of cigarette smoke extract (CSE) on JAM1 and CXADR in human gingival epithelial cells. CSE was found to cause translocation of JAM1 from the cellular surface to EGFR-positive endosomes, whereas CXADR did not. Using a three-dimensional multilayered gingival epithelial tissue model, CSE administration was found to increase permeability to lipopolysaccharide and peptidoglycan, whereas overexpression of JAM1 in the tissue model prevented penetration by those substrates. Furthermore, vitamin C increased JAM1 expression, and inhibited penetration of LPS and PGN induced by CSE. These findings strongly suggest that CSE disrupts gingival barrier function via dislocation of JAM1, thus allowing bacterial virulence factors to penetrate into subepithelial tissues. Furthermore, they indicate that vitamin C increases JAM1 expression and prevents disruption of gingival barrier function by CSE.Yamaga S., Tanigaki K., Nakamura E., et al. Cigarette smoke extract impairs gingival epithelial barrier function. Scientific Reports 13, 9228 (2023); https://doi.org/10.1038/s41598-023-36366-z

SOCS3 Is Essential in the Regulation of Fetal Liver Erythropoiesis

AbstractSOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12–16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis

Feminizing Wolbachia endosymbiont disrupts maternal sex chromosome inheritance in a butterfly species

Wolbachia is a maternally inherited ubiquitous endosymbiotic bacterium of arthropods that displays a diverse repertoire of host reproductive manipulations. For the first time, we demonstrate that Wolbachia manipulates sex chromosome inheritance in a sexually reproducing insect. Eurema mandarina butterfly females on Tanegashima Island, Japan, are infected with the wFem Wolbachia strain and produce all‐female offspring, while antibiotic treatment results in male offspring. Fluorescence in situ hybridization (FISH) revealed that wFem‐positive and wFem‐negative females have Z0 and WZ sex chromosome sets, respectively, demonstrating the predicted absence of the W chromosome in wFem‐infected lineages. Genomic quantitative polymerase chain reaction (qPCR) analysis showed that wFem‐positive females lay only Z0 eggs that carry a paternal Z, whereas females from lineages that are naturally wFem‐negative lay both WZ and ZZ eggs. In contrast, antibiotic treatment of adult wFem females resulted in the production of Z0 and ZZ eggs, suggesting that this Wolbachia strain can disrupt the maternal inheritance of Z chromosomes. Moreover, most male offspring produced by antibiotic‐treated wFem females had a ZZ karyotype, implying reduced survival of Z0 individuals in the absence of feminizing effects of Wolbachia. Antibiotic treatment of wFem‐infected larvae induced male‐specific splicing of the doublesex (dsx) gene transcript, causing an intersex phenotype. Thus, the absence of the female‐determining W chromosome in Z0 individuals is functionally compensated by Wolbachia‐mediated conversion of sex determination. We discuss how Wolbachia may manipulate the host chromosome inheritance and that Wolbachia may have acquired this coordinated dual mode of reproductive manipulation first by the evolution of female‐determining function and then cytoplasmically induced disruption of sex chromosome inheritance

A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-$^{32}$P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC$_{50}$ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation

TGF-β-dependent reprogramming of amino acid metabolism induces epithelial–mesenchymal transition in non-small cell lung cancers

Epithelial–mesenchymal transition (EMT)—a fundamental process in embryogenesis and wound healing—promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer