ABSTRACT: Transcriptional profiling of ER stress condition comparing control (untreated), dithiothreitol (DTT) treated, or tunicamycin (TM) treated conditions on P. angusta DL1 strain. Six-condition experiment: normal condition vs. ER stress condition (TM 30, 60, 120min; DTT 30, 60, 120min) in P. angusta DL1 strain. Independently grown and harvested. Two replicates per array.
Project description:Transcriptional profiling of hac1 deletion mutant compared to wild type (control). Wild type vs. hac1 deletion mutant of Ogataea angusta DL1 strain in exponential growth. Two biological replicates, with dye-swaps.
Project description:BACKGROUND: NR4A3/NOR-1 is a member of the NR4A orphan nuclear receptor subfamily, which contains early response genes that sense and respond to a variety of stimuli in the cellular environment. The role of NR4A3 in insulin expression in pancreatic beta cells remains unknown. METHODS: Dynamic changes in NR4A3 were examined in a pancreatic beta-cell line, MIN6, treated with thapsigargin (TG), palmitate (PA), tunicamycin (TM), and dithiothreitol (DTT), chemicals that produce cell stress and even apoptosis. We exploited virus infection techniques to induce expression of NR4A3 or three deletion mutants, and determined expression of insulin and insulin regulatory genes in MIN6 cells. RESULTS: TG and PA, two endoplasmic reticulum (ER) stress inducers, were able to induce unfolded protein response (UPR) activation and elevation of NR4A3 expression in MIN6 cells, whereas TM and DTT, two other ER stress inducers, were able to induce UPR activation but not NR4A3 elevation. MIN6 cells over-expressing NR4A3 protein after adenoviral infection exhibited reduced transcription of the insulin genes Ins1 and Ins2, and reduced insulin protein secretion, which were negatively correlated with NR4A3 expression levels. Functional analysis of different deletion mutants of NR4A3 showed that deleting the activation domain AF1 or the DNA-binding domain abolished the down-regulation of insulin transcription by NR4A3 in MIN6 cells, indicating that this down-regulative role was closely related to the NR4A3 trans-activation activity. Over-expression of NR4A3 in MIN6 cells resulted in reduced mRNA transcription of the insulin positive-regulation genes, Pdx1 and NeuroD1. CONCLUSION: Some ER stress inducers, such as TG or PA, are able to elevate NR4A3 expression in MIN6 cells, while others, such as TM or DTT, are not. Over-expression of NR4A3 in MIN6 cells results in down-regulation of insulin gene transcription and insulin secretion. NR4A3 reduces insulin gene expression by modulating the expression of Pdx1 and NeuroD1.
Project description:The effects of bovine bile and SDS on transcriptional events were studied by means of genome wide microarrays in Enterococcus faecalis V583. Transcriptional profiles were obtained through time series experiments over periods of 120min for cells treated with bile and the combination of SDS and bile, and 360min. for SDS.
Project description:Aggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5' untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1Δ) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1Δ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1Δ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha.
Project description:The unfolded protein response (UPR) is central to endoplasmic reticulum (ER) homeostasis by controlling its size and protein folding capacity. When activated by unfolded proteins in the ER-lumen or aberrant lipid compositions, the UPR adjusts the expression of hundreds of target genes to counteract ER stress. The proteotoxic drugs dithiothreitol (DTT) and tunicamycin (TM) are commonly used to induce misfolding of proteins in the ER and to study the UPR. However, their potential impact on the cellular lipid composition has never been systematically addressed. Here, we report the quantitative, cellular lipid composition of Saccharomyces cerevisiae during acute, proteotoxic stress in both rich and synthetic media. We show that DTT causes rapid remodeling of the lipidome when used in rich medium at growth-inhibitory concentrations, while TM has only a marginal impact on the lipidome under our conditions of cultivation. We formulate recommendations on how to study UPR activation by proteotoxic stress without interferences from a perturbed lipid metabolism. Furthermore, our data suggest an intricate connection between the cellular growth rate, the abundance of the ER, and the metabolism of fatty acids. We show that Saccharomyces cerevisiae can produce asymmetric lipids with two saturated fatty acyl chains differing substantially in length. These observations indicate that the pairing of saturated fatty acyl chains is tightly controlled and suggest an evolutionary conservation of asymmetric lipids and their biosynthetic machineries.
Project description:To understand the relationship between gene expression and capsule formation, H99 cells were cultured overnight at 37ºC in the following eight conditions: low iron medium with or without both 500 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM bathophenanthroline disulfonate (BPDS); phosphate-buffered saline (PBS) with or without 10% v/v fetal bovine serum; Dulbecco's Modified Eagle's Medium (from Sigma) in ambient air or 5% CO2; and Littman's medium with either 0.01 µg/ml or 1 µg/ml thiamine. All samples were hybridized against a common reference pool of total RNA. Three biological replicates were performed for each growth condition, with the exception of growth in DMEM.
Project description:We discovered that vitamin C (ascorbic acid) is required for differentiation of mouse bone marrow-derived progenitor cells in T cell cultures based on OP9-DL1 stromal cells. This microarray dataset evaluates differential expression of genes in OP9-DL1 stromal cells as well as in lymphocytes developing in the cultures when a stabilized form of ascorbic acid (l-ascorbic acid 2-phosphate; pAsc) is added. Cultures were maintained in the absence of pAsc for 13 days, at which time 800 micromolar pAsc was added. Cells were harvested after 24 and 72 hours (24 hours only in the case of OP9-DL1 cells) and solubilized in Trizol. Two-condition experiment, differentiating T lymphoid cells with or without pAsc, 2 timepoints, OP9-DL1 cells with or without pAsc, 1 timepoint, no technical replicates
Project description:The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.