Phosphorylation of eukaryotic initiation factor-2? in response to endoplasmic reticulum and nitrosative stress in the human protozoan parasite, Entamoeba histolytica.
ABSTRACT: Entamoeba histolytica is an intestinal parasite infecting over 50 million people worldwide and is the causative agent of amebic dysentery and amoebic liver abscess. In the human host, E. histolytica experiences stress brought on by nutrient deprivation and the host immune response. To be a successful parasite, E. histolytica must counter the stress; therefore, understanding the stress response may uncover new drug targets. In many systems, the stress response includes down-regulation of protein translation, which is regulated by phosphorylation of eukaryotic initiation factor (eIF-2?). Previous work has demonstrated that phosphorylation of the E. histolytica eIF-2? (EheIF-2?) increases significantly when exposed to long-term serum starvation, oxidative stress, and long-term heat shock. However, the effects of reagents that are known to induce nitrosative or endoplasmic reticulum (ER) stresses, on EheIF-2? have yet to be evaluated. Nitrosative stress is part of the host's immune response and ER stress can be caused by several physiological or pathological factors. We treated E. histolytica cells with various reagents known to induce nitrosative stress (DPTA-NONOate and SNP) or ER stress (BFA and DTT). We examined the morphology of the ER, tracked phosphorylation of EheIF-2?, and assessed protein translation in control and stressed cells. While all four stress-inducing reagents caused a global reduction in protein translation, only DTT was capable of also inducing changes in the morphology of the ER (consistent with ER stress) and phosphorylation of EheIF-2?. This suggests that DTT authentically induces ER stress in E. histolytica and that this stress is managed by the eIF-2?-based system. This was supported by the observation that cells expressing a non-phosphorylatable version of eIF-2? were also highly sensitive to DTT-stress. Since protein translation decreased in the absence of phosphorylation of eIF-2? (after treatment with DPTA-NONOate, SNP or BFA), the data also indicate that there are alternative protein-translational control pathways in E. histolytica. Overall, our study further illuminates the stress response to nitrosative stress and ER stress in E. histolytica.
Project description:E. histolytica of strains HM1:IMSS (virulent) and Rahman (avirulent) were examined for response to oxidative and nitrosative stress. Overall design: Response to oxidative stress in E. histolytica was assayed by treating Eh (HM1:IMSS) and Eh (Rahman) with 1mM H2O2 for 60 minutes or 90 minutes and comparing transcription to parasites grown in TYI. In addition, response to nitosative stress was assayed in Eh (HM1:IMSS) by treatment for 60 minutes with 200 µM DPTA-NONOate.
Project description:E. histolytica of strains HM1:IMSS (virulent) and Rahman (avirulent) were examined for response to oxidative and nitrosative stress. Response to oxidative stress in E. histolytica was assayed by treating Eh (HM1:IMSS) and Eh (Rahman) with 1mM H2O2 for 60 minutes or 90 minutes and comparing transcription to parasites grown in TYI. In addition, response to nitosative stress was assayed in Eh (HM1:IMSS) by treatment for 60 minutes with 200 µM DPTA-NONOate.
Project description:RNA sequencing was performed on Candida albicans wild type cells (JC50) grown to exponential phase on YPD , YPD plus Nitrosative Stress 2.5mM DPTA NONOate, and compared to exponential Candida albicans hog1 deletion mutant cells grown on on YPD , YPD plus Nitrosative Stress 2.5mM DPTA NONOate. Three independent experiments were performed.
Project description:Because multiple myeloma (MM) cells are at risk for endoplasmic reticulum (ER) stress, they require a carefully regulated mechanism to promote protein translation of selected transcripts when proliferation is stimulated. MAPK-interacting kinases (MNKs) may provide this mechanism by enhancing cap-dependent translation of a small number of critical transcripts. We, thus, tested whether MNKs played a role in MM responses to the myeloma growth factor interleukin-6 (IL-6). IL-6 activated MNK1 phosphorylation and induced phosphorylation of its substrate, eIF-4E, in MM lines and primary specimens. MNK paralysis, achieved pharmacologically or by shRNA, prevented MM expansion stimulated by IL-6. A phosphodefective eIF-4E mutant also prevented the IL-6 response, supporting the notion that MNK's role was via phosphorylation of eIF-4E. Both pharmacological MNK inhibition and expression of the phosphodefective eIF-4E mutant inhibited MM growth in mice. Although critical for IL-6-induced expansion, eIF-4E phosphorylation had no significant effect on global translation or Ig expression. Deep sequencing of ribosome-protected mRNAs revealed a repertoire of genes involved in metabolic processes and ER stress modulation whose translation was regulated by eIF-4E phosphorylation. These data indicate MM cells exploit the MNK/eIF-4E pathway for selective mRNA translation without enhancing global translation and risking ER stress.
Project description:Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasite's response to oxidative and nitrosative stresses, probing the transcriptional changes of trophozoites of a pathogenic strain after a 60 min exposure to H2O2 (1 mM) or a NO donor (dipropylenetriamine-NONOate, 200 microM), using whole-genome DNA microarrays. Genes encoding reactive oxygen and nitrogen species detoxification enzymes had high transcriptional levels under basal conditions and upon exposure to both stresses. On a whole-genome level, there was significant modulation of gene expression by H2O2 (286 genes regulated) and dipropylenetriamine-NONOate (1036 genes regulated) with a significant overlap of genes modulated under both conditions (164 genes). A number of transcriptionally regulated genes were in signalling/regulatory and repair/metabolic pathways. However, the majority of genes with altered transcription encode unknown proteins, suggesting as yet unraveled response pathways in E. histolytica. Trophozoites of a non-pathogenic E. histolytica strain had a significantly muted transcriptional response to H2O2 compared with the pathogenic strain, hinting that differential response to oxidative stress may be one factor that contributes to the pathogenic potential of E. histolytica.
Project description:In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). Recently, we identified a new family member, pancreatic eIF-2alpha kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2alpha phosphorylation was coincident with increased PEK autophosphorylation and eIF-2alpha kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2alpha and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2alpha kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.
Project description:The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging.
Project description:The endoplasmic reticulum (ER) unfolded protein response (UPR) restores equilibrium to the ER, but prolonged expression of the UPR effector CHOP (GADD153) is cytotoxic. We found that CHOP expression induced by ER stress was suppressed by prior engagement of toll-like receptor (TLR) 3 or 4 through a TRIF-dependent pathway. TLR engagement did not suppress phosphorylation of PERK or eIF-2alpha, which are upstream of CHOP, but phospho-eIF-2alpha failed to promote translation of the CHOP activator ATF4. In mice subjected to systemic ER stress, pretreatment with low dose lipopolysaccharide (LPS), a TLR4 ligand, suppressed CHOP expression and apoptosis in splenic macrophages, renal tubule cells and hepatocytes, and prevented renal dysfunction and hepatosteatosis. This protective effect of LPS did not occur in Trif(-/-) mice or in wild-type mice in which CHOP expression was genetically restored. Thus, TRIF-mediated signals from TLRs selectively attenuate translational activation of ATF4 and its downstream target gene CHOP. We speculate that this mechanism evolved to promote survival of TLR-expressing cells that experience prolonged levels of physiological ER stress in the course of the host response to invading pathogens.
Project description:Protein translation is inhibited by the unfolded protein response (UPR)-induced eIF-2? phosphorylation to protect against endoplasmic reticulum (ER) stress. In addition, we found additional inhibition of protein translation owing to diminished mTORC1 (mammalian target of rapamycin complex1) activity in ER-stressed multiple myeloma (MM) cells. However, c-myc protein levels and myc translation was maintained. To ascertain how c-myc was maintained, we studied myc IRES (internal ribosome entry site) function, which does not require mTORC1 activity. Myc IRES activity was upregulated in MM cells during ER stress induced by thapsigargin, tunicamycin or the myeloma therapeutic bortezomib. IRES activity was dependent on upstream MAPK (mitogen-activated protein kinase) and MNK1 (MAPK-interacting serine/threonine kinase 1) signaling. A screen identified hnRNP A1 (A1) and RPS25 as IRES-binding trans-acting factors required for ER stress-activated activity. A1 associated with RPS25 during ER stress and this was prevented by an MNK inhibitor. In a proof of principle, we identified a compound that prevented binding of A1 to the myc IRES and specifically inhibited myc IRES activity in MM cells. This compound, when used alone, was not cytotoxic nor did it inhibit myc translation or protein expression. However, when combined with ER stress inducers, especially bortezomib, a remarkable synergistic cytotoxicity ensued with associated inhibition of myc translation and expression. These results underscore the potential for targeting A1-mediated myc IRES activity in MM cells during ER stress.