Affymetrix whole transcriptome gene expression analysis of ER-stressed Jurkat cells
ABSTRACT: We induced ER stress in Jurkat T-cells with dithiothreitol (DTT), which reduces the protein disulfide bonds and causes the accumulation of misfolded proteins in the ER lumen. For assessment of DTT effects on cells, we performed Affymetrix whole transcriptome gene expression analysis. The cells were treated for 6 hours with 2.5 mM Dithiothreitol (Sigma-Aldrich). Total RNA was isolated from DTT-treated (2 replicates) and control (2 replicates) Jurkat cells and hybridized to a gene expression arrays (GeneChip Gene 1.0 ST Array System; Affymetrix, Santa Clara, CA).
Project description:We induced ER stress in Jurkat T-cells with dithiothreitol (DTT), which reduces the protein disulfide bonds and causes the accumulation of misfolded proteins in the ER lumen. For assessment of DTT effects on cells, we performed Affymetrix whole transcriptome gene expression analysis. The cells were treated for 6 hours with 2.5 mM Dithiothreitol (Sigma-Aldrich). Total RNA was isolated from DTT-treated (2 replicates) and control (2 replicates) Jurkat cells and hybridized to a gene expression arrays (GeneChip Gene 1.0 ST Array System; Affymetrix, Santa Clara, CA).
Project description:Secretory proteins are subjected to stringent quality control systems in the endoplasmic reticulum (ER) which include the targeting of misfolded proteins for proteasomal destruction via the ER-associated degradation (ERAD) pathway. Since deletion of ERAD genes in the filamentous fungus Aspergillus niger had hardly any effect on growth, this study investigates whether autophagy might function as an alternative process to eliminate misfolded proteins from the ER. We generated A. niger double mutants by deleting genes essential for ERAD (derA) and autophagy (atg1 or atg8), and assessed their growth both under normal and ER stress conditions. Sensitivity toward ER stress was examined by treatment with dithiothreitol (DTT) and by expressing a mutant form of glucoamylase (mtGlaA::GFP) in which disulfide bond sites in GlaA were mutated. Misfolding of mtGlaA::GFP was confirmed, as mtGlaA::GFP accumulated in the ER. Expression of mtGlaA::GFP in ERAD and autophagy mutants resulted in a twofold higher accumulation in ?derA and ?derA?atg1 strains compared to ?atg1 and wild type. As ?derA?atg1 mutants did not show increased sensitivity toward DTT, not even when mtGlaA::GFP was expressed, the results indicate that autophagy does not act as an alternative pathway in addition to ERAD for removing misfolded proteins from the ER in A. niger.
Project description:Dithiothreitol (DTT) is the standard reagent for reducing disulfide bonds between and within biological molecules. At neutral pH, however, >99% of DTT thiol groups are protonated and thus unreactive. Herein, we report on (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), a dithiol that can be synthesized from l-aspartic acid in a few high-yielding steps that are amenable to a large-scale process. DTBA has thiol pK(a) values that are ~1 unit lower than those of DTT and forms a disulfide with a similar E°' value. DTBA reduces disulfide bonds in both small molecules and proteins faster than does DTT. The amino group of DTBA enables its isolation by cation-exchange and facilitates its conjugation. These attributes indicate that DTBA is a superior reagent for reducing disulfide bonds in aqueous solution.
Project description:For fifty years, dithiothreitol (DTT) has been the preferred reagent for the reduction of disulfide bonds in proteins and other biomolecules. Herein we report on the synthesis and characterization of 2,3-bis(mercaptomethyl)pyrazine (BMMP), a readily accessible disulfide-reducing agent with reactivity under biological conditions that is markedly superior to DTT and other known reagents.
Project description:The data described here provide an analysis of the dynamic response of HeLa cell proteome to dithiothreitol (DTT) inducing stress of the endoplasmic reticulum (ER). During ER stress, accumulation of misfolded and unfolded proteins in the lumen of the ER initiates the Unfolded Protein Response (UPR), resulting in a large-scale redistribution of proteins. We used label-free mass spectrometry to monitor the proteomic changes of HeLa cells during a 30-h time course, monitoring eight time points (0, 0.5, 1, 2, 8, 16, 24, and 30 h). The data are associated with the research article "Differential dynamics of the mammalian mRNA and protein expression response to misfolding stress" , which discusses a core dataset of 1237 proteins. Here, we present the extended dataset of 2131 proteins. The raw mass spectrometry data and the analysis results have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PRIDE: PXD002039.
Project description:The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll-like receptors, and the low-density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N-terminal signal sequences and C-terminal ER-retention signals and that mammals have an additional member CNPY5, also known as plasma cell-induced ER protein 1/marginal zone B cell-specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha-helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin-like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse-chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.
Project description:In <i>Arabidopsis thaliana,</i> the evolutionary conserved N-terminal acetyltransferase (Nat) complexes NatA and NatB co-translationally acetylate 60% of the proteome. Both have recently been implicated in the regulation of plant stress responses. While NatA mediates drought tolerance, NatB is required for pathogen resistance and the adaptation to high salinity and high osmolarity. Salt and osmotic stress impair protein folding and result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER). The ER-membrane resident E3 ubiquitin ligase DOA10 targets misfolded proteins for degradation during ER stress and is conserved among eukaryotes. In yeast, DOA10 recognizes conditional degradation signals (Ac/N-degrons) created by NatA and NatB. Assuming that this mechanism is preserved in plants, the lack of Ac/N-degrons required for efficient removal of misfolded proteins might explain the sensitivity of NatB mutants to protein harming conditions. In this study, we investigate the response of NatB mutants to dithiothreitol (DTT) and tunicamycin (TM)-induced ER stress. We report that NatB mutants are hypersensitive to DTT but not TM, suggesting that the DTT hypersensitivity is caused by an over-reduction of the cytosol rather than an accumulation of unfolded proteins in the ER. In line with this hypothesis, the cytosol of NatB depleted plants is constitutively over-reduced and a global transcriptome analysis reveals that their reductive stress response is permanently activated. Moreover, we demonstrate that <i>doa10</i> mutants are susceptible to neither DTT nor TM, ruling out a substantial role of DOA10 in ER-associated protein degradation (ERAD) in plants. Contrary to previous findings in yeast, our data indicate that N-terminal acetylation (NTA) does not inhibit ER targeting of a substantial amount of proteins in plants. In summary, we provide further evidence that NatB-mediated imprinting of the proteome is vital for the response to protein harming stress and rule out DOA10 as the sole recognin for substrates in the plant ERAD pathway, leaving the role of DOA10 in plants ambiguous.
Project description:In mutant INS gene-induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production.
Project description:The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.
Project description:A key element in the quality control of glycoprotein folding is the UDP-Glc:glycoprotein glucosyltransferase (GT), which in cell-free assays exclusively glucosylates misfolded glycoproteins. In order to test if such a protein conformation is a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT, a Schizosaccharomyces pombe double mutant (gls2/alg6) was constructed. With this mutant, Man9GlcNAc2 is transferred to proteins and no removal of glucose units added by GT occurs as it lacks glucosidase II. The same proportion of glucosylated (Glc1Man9GlcNAc2) and unglucosylated (Man9GlcNAc2 and Man8GlcNAc2) endoplasmic reticulum (ER)-specific compounds was produced when cells were pre-incubated for 10, 20 or 30 min and further incubated with [14C]glucose for 10 min at 28 degrees C with or without 5 mM dithiothreitol (DTT), thus indicating not only that DTT did not affect protein glucosylation but also that no increased glucosylation of glycoproteins occurred in the presence of the drug. Monitoring Golgi-specific modifications of oligosaccharides after pulse-chase experiments performed in the presence or absence of 5 mM DTT showed that exit of the bulk of glycoproteins synthesized from the ER and thence their proper folding had been prevented by the drug. Cells pulse-chase labeled at 37 degrees C in the absence of DTT also yielded glucosylated and unglucosylated protein-linked oligosaccharides without Golgi-specific modifications. It was concluded that a misfolded protein conformation is not a sufficient condition for in vivo glucosylation of all N-linked oligosaccharides by GT.