The glucosinolate breakdown product indole-3-carbinol acts as an auxin antagonist in roots of Arabidopsis thaliana.

The glucosinolate breakdown product indole-3-carbinol functions in cruciferous vegetables as a protective agent against foraging insects. While the toxic and deterrent effects of glucosinolate breakdown on herbivores and pathogens have been studied extensively, the secondary responses that are induced in the plant by indole-3-carbinol remain relatively uninvestigated. Here we examined the hypothesis that indole-3-carbinol plays a role in influencing plant growth and development by manipulating auxin signaling. We show that indole-3-carbinol rapidly and reversibly inhibits root elongation in a dose-dependent manner, and that this inhibition is accompanied by a loss of auxin activity in the root meristem. A direct interaction between indole-3-carbinol and the auxin perception machinery was suggested, as application of indole-3-carbinol rescues auxin-induced root phenotypes. In vitro and yeast-based protein interaction studies showed that indole-3-carbinol perturbs the auxin-dependent interaction of Transport Inhibitor Response (TIR1) with auxin/3-indoleacetic acid (Aux/IAAs) proteins, further supporting the possibility that indole-3-carbinol acts as an auxin antagonist. The results indicate that chemicals whose production is induced by herbivory, such as indole-3-carbinol, function not only to repel herbivores, but also as signaling molecules that directly compete with auxin to fine tune plant growth and development.

Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells.

The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Epizootic hemorrhagic disease virus induces and benefits from cell stress, autophagy, and apoptosis.

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Combined genetic and epigenetic interferences with interferon signaling expose prostate cancer cells to viral infection.

Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling.

Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates.

Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern.

Neuregulin promotes incomplete autophagy of prostate cancer cells that is independent of mTOR pathway inhibition.

BACKGROUND: Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. CONCLUSIONS/SIGNIFICANCE: Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.