ShRNA TMEM258 knockdown in HeLa
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE89542 | GEO | 2016/11/05
SECONDARY ACCESSION(S): PRJNA352509
REPOSITORIES: GEO
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Project description:Oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of OST complex remains incompletely understood. Here, we show that OST complex in C. elegans is crucial for ER protein homeostasis and enhances resistance to pathogenic bacteria, Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase.
2020-01-30 | GSE134687 | GEO
Project description:Multiple myeloma (MM) is an incurable malignancy characterized by mutated plasma cell clonal expansion in the bone marrow, leading to severe clinical symptoms. Thus, identifying new therapeutic targets for MM is crucial. We identified the oligosaccharyltransferase (OST) complex as a novel vulnerability in MM cells. Elevated expression of this complex is associated with relapsed, high-risk MM, and poor prognosis. Disrupting the OST complex suppressed MM cell growth, inducing cell cycle arrest and apoptosis. Combined inhibition with bortezomib synergistically eliminated MM cells in vitro and in vivo, via suppressing genes related to bortezomib-resistant phenotypes. Mechanistically, OST complex disruption downregulated pathological gene signatures (NF-kB signaling, mTORC1 pathway, glycolysis, MYC targets, and cell cycle), induced TRAIL-mediated apoptosis and inflammatory pathways. MYC translation was robustly suppressed upon inhibiting the OST complex. Collectively, the OST complex presents a novel target for MM treatment, and combining its inhibition with bortezomib offers a promising approach for relapsed MM patients.
2025-04-16 | GSE269271 | GEO
Project description:Investigating gene expression patterns in mice with conditional expression of the most common RVCL mutation, V235fs, and another mice expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosacchryltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE).
2018-03-15 | GSE96687 | GEO
Project description:The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called âER stressâ which induces the unfolded protein response (UPR) which is a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B-cells and then measured gene expression at 10 time-points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in ER stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE which showed significant increase in expression following ER stress in B-cells and in primary fibroblasts. To study the links between unfolded protein response and disease susceptibility, we identified ER stress responsive genes that are associated with human diseases and assessed individual differences in ER stress response. Many of the UPR genes are associated with Mendelian disorders such as Wolfram syndrome and complex human diseases including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility. Keywords: array-based gene expression We measured gene expression levels in immortalized B cells from members of 60 unrelated CEPH-Utah grandparents. Each individual was treated for 8 hours with either DMSO or with 4 ug/ml of tunicamycin. Gene expression was measured to identify tunicamycin-responsive genes. To assess whether there is a genetic component to the individual variation in gene expression response to ER stress, we used microarrays to measure expression of genes in 26 monozygotic twin pairs treated with either DMSO or 500 nM thapsigargin for 4 hours.
2010-04-08 | E-GEOD-19519 | biostudies-arrayexpress
Project description:The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called “ER stress” which induces the unfolded protein response (UPR) which is a complex cellular process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B-cells and then measured gene expression at 10 time-points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in ER stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE which showed significant increase in expression following ER stress in B-cells and in primary fibroblasts. To study the links between unfolded protein response and disease susceptibility, we identified ER stress responsive genes that are associated with human diseases and assessed individual differences in ER stress response. Many of the UPR genes are associated with Mendelian disorders such as Wolfram syndrome and complex human diseases including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility. Keywords: array-based gene expression
2010-03-30 | GSE19519 | GEO
Project description:Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy.
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Project description:Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress.
2015-05-12 | GSE68718 | GEO
Project description:We have identified a potential selective autophagy receptor protein in Arabidopsis thaliana, C53/AT5G06830/ERP1, that is recruited to ER upon ER-stress activation and induces autophagosome formation. By affinity proteomics IP/MS with ATG8A and E, we identified this new ER-phagy receptor, which is also conserved in metazoans. With biophysical characterization, we revealed its unprecedent binding mode to ATG8 (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. However, its cargo targeted for degradation is still elusive. Initial quantitative proteomics analyses (TMT) of wild type and Atc53 mutant lines revealed that Atc53 mediates degradation of ER resident proteins as well as proteins passaging the ER to the cell wall, apoplast, and lipid droplets.
2021-09-09 | PXD019988 | Pride
Project description:HeLa cells transduced with shRNA from MISSION library (TRCN0000289677) using lentiviral delivery system. HeLa cells transduced with scrambled shRNA, gifted from Dr. Mauricio Reginato. Sequence is 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′ Peptidyl-prolyl isomerase H (PPIH) is a cyclophilin, an enzyme that interconverts cis and trans isomers of proline. PPIH associates with the human spliceosome, the complex and dynamic machinery that removes intronic sequence from pre-messenger RNA (pre-mRNA). Although PPIH affects the ability of the spliceosome to assemble and catalyze splicing in vitro, little is known about what PPIH does to splicing in vivo. To understand the function of PPIH in the nucleus, we knocked down PPIH in human cells. We characterized a set of alternative splicing and transcriptional events that are PPIH-responsive. We used these splicing and transcriptional bioassays to show first that PPIH-responsive events are largely specific, even within the cyclophilin family. We then identified a role for proline isomerization in transcriptional regulation, but not in regulation of alternative splicing. The development of a bioassay for PPIH function can be used to answer fundamental questions about the role of spliceosomal proteins in regulating splicing and other nuclear functions.
2017-12-31 | GSE103648 | GEO
Project description:BackgroundRNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.ResultsTo determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.ConclusionThe use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
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