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The Ebola virus (EBOV) genome encodes a partly conserved 40-residue nonstructural polypeptide, called the delta peptide, that is produced in abundance during Ebola virus disease (EVD). The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full-length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey, and human origin; increase ion permeability across confluent cell monolayers; and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin and that it may be a novel, targetable aspect of Ebola virus disease pathology.IMPORTANCE During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved nonstructural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology and as a possible target for novel countermeasures.

Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.

Uterine leiomyomas, benign uterine smooth muscle tumors that affect 30% of reproductive-aged women, are a significant health concern. The initiation event for these tumors is unclear, but 17beta-estradiol (E2) is an established promoter of leiomyoma growth. E2 not only alters transcription of E2-regulated genes but also can rapidly activate signaling pathways. The aim of our study is to investigate the role of rapid E2-activated cytoplasmic signaling events in the promotion of leiomyomas. Western blot analysis revealed that E2 rapidly increases levels of phosphorylated protein kinase C alpha (PKC alpha) in both immortalized uterine smooth muscle (UtSM) and leiomyoma (UtLM) cell lines, but increases levels of phosphorylated ERK1/2 only in UtLM cells. Our studies demonstrate a paradoxical effect of molecular and pharmacological inhibition of PKC alpha on ERK1/2 activation and cellular proliferation in UtLM and UtSM cells. PKC alpha inhibition decreases levels of phosphorylated ERK1/2 and proliferation in UtLM cells but raises these levels in UtSM cells. cAMP-PKA signaling is rapidly activated only in UtSM cells with E2 and inhibits ERK1/2 activation and proliferation. We therefore propose a model whereby E2's rapid activation of PKC alpha and cAMP-PKA signaling plays a central role in the maintenance of a low proliferative index in normal uterine smooth muscle via its inhibition of the MAPK cascade and these pathways are altered in leiomyomas to promote MAPK activation and proliferation. These studies demonstrate that rapid E2-signaling pathways contribute to the promotion of leiomyomas.

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Phytochemicals bind to and regulate the human estrogen receptors (ERalpha and ERbeta), mimicking actions of the endogenous estrogen, 17beta-estradiol, and known antiestrogens such as ICI 182,780. Recently, however, some of these estrogenic phytochemicals have been shown to affect other signal transduction pathways, such as receptor tyrosine kinases and mitogen-activated protein kinases (MAPK). Previously, we found that certain phytochemicals, such as flavone, apigenin, kaempferide and chalcone, have potent antiestrogenic activity. However, the antiestrogenicity of these compounds does not correlate with their ER binding capacity, suggesting alternative signaling as a mechanism for their antagonistic effects. In this study, we examined the effects of these compounds on the transcription factor activator protein-1 (AP-1). Using AP-1-luciferase stable human endometrial adenocarcinoma Ishikawa and human embryonic kidney (HEK) 293 cells, chalcone, flavone and apigenin all stimulated AP-1 activity. Additionally, we determined the effects of the phytochemicals on transcription factors that are downstream targets of various MAPK pathways. To test this, we used HEK 293 cells stably cointegrated with GAL4 transcriptional activation systems of Elk-1, c-Jun or C/EBP homologous protein (CHOP). Chalcone was the only phytochemical that activated all three transcription factors [Elk-1, 2.7-fold (P < 0.001); c-Jun, 2.7-fold (P = 0.025); CHOP, 3.0-fold (P = 0.002)], whereas apigenin stimulated CHOP (3.9-fold; P < 0.001), but inhibited phorbol myristoyl acetate-induced c-Jun activity (71%;P = 0.006). This work suggests that phytochemicals affect multiple signaling pathways that converge at the level of transcriptional regulation. The ability of flavonoids to regulate MAPK-responsive pathways in a selective manner indicates a mechanism by which phytochemicals may influence human health and disease.

Growth factor activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway has been shown to activate the estrogen receptor (ER) A and to mediate tamoxifen resistance in breast cancer. Here, we investigated the regulation of the transcriptional activity of the newer ERB by PI3K-AKT signaling. Tissue arrays of breast cancer specimens showed a positive association between the expressions of AKT and ERB in the clinical setting. Reporter gene assays using pharmacologic and molecular inhibitors of AKT and constitutively active AKT revealed for the first time the ability of AKT to (a) potentiate ERB activity and (b) target predominantly the activation function-2 (AF2) domain of the receptor, with a requirement for residue K269. Given the importance of coactivators in ER transcriptional activity, we further investigated the possible involvement of steroid receptor coactivator 1 (SRC1) and glucocorticoid receptor-interacting protein 1 (GRIP1) in AKT regulation of ERB. Mammalian two-hybrid assays revealed that AKT enhanced both SRC1 and GRIP1 recruitment to the ERB-AF2 domain, and reporter gene analyses revealed that AKT and GRIP1 cooperatively potentiated ERB-mediated transcription to a level much greater than either factor alone. Investigations into AKT regulation of GRIP with mammalian one-hybrid assays showed that AKT potentiated the activation domains of GRIP1 itself, and in vitro kinase assays revealed that AKT directly phosphorylated GRIP1. The cross-talk between the PI3K-AKT and ERB pathways, as revealed by the ability of AKT to regulate several components of ERBmediated transcription, may represent an important aspect that may influence breast cancer response to endocrine therapy. (Cancer Res 2006; 66(17): 8373-81)