Transcriptome analysis of HeLa cells knocked down for EXT1 or FBXW7 gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE138030 | GEO | 2020/09/25
REPOSITORIES: GEO
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Project description:Exostosin-1 (EXT1) is an ER-resident glycosyltransferase known to polymerize heparan sulfate chain by sequential addition of glucuronic acid and N-acetylglucosamine molecules. This reaction results in heavily glycosylated proteoglycans (PG) at the plasma membrane, which form cell-type specific complexes and play vital roles in cell-cell and cell-pathogens interactions, cell growth, plasticity and proliferation in the local tissue environment. Different evolutionary forces drive the structure and function of protein glycosylation between the inside and outside of the plasma membrane, and an understanding of how the proteome composition of internal cell membranes is regulated remains a fundamental question in biology. In addition, potential interplays between whole cell proteome and membrane composition are completely unknown. We performed EXT1 knockdown in HeLa cells using shRNA, isolated ER microsomes, performed label-free mass spectrometry, and processed the data with byonic 3.5 software to identify glycoproteins. We identified a total of 1080 proteins, 226 with altered expression, 51 phosphoproteins, 25 O-glycosylated and 97 N-glycosylated membrane proteins. We also performed stable isotope labeling amino acids in cell culture comparing proteomes from HeLa knocked down for EXT1 and control cells. We identified a total 5215 proteins, with 3713 proteins having a SILAC ratio and 396 proteins out of total with a significant p-value (p-value <0.05). Our study demonstrate that the glycosyltransferase enzyme contributes to the heterogeneity of intracellular membrane proteins, particularly proteins controlling ER membrane morphologies in response to different cell types and states.
2021-05-10 | PXD015660 | Pride
Project description:Lacritin targets its coreceptor 'syndecan-1' in lacritin-dependent production of the important dry eye mucin 'MUC16'. Real-time PCR analysis suggests that this effect is post-transcriptional. In contrast, serum stimulation of MUC16 by these cells is transcriptionally dependent. Our goal is to determine whether this regulation is global to other heavily O-glycosylated proteins, and if so, if it is dependent on transcription of a Golgi glycosyltransferases. If not, the mechanism might be microRNA dependent. Among other glycogenes, expression of heparanases, sulfotransferases and epimerase are also of interest. Heparanase is required for lacritin binding to its co-receptor syndecan-1 and this binding site may require a sulfated iduronic acid. In request 1705, we recently asked core D whether they could generate this sulfate iduronic acid-containing glycan (iduronic acid (2-O-sulfated)-N-acetylglucosamine-glucuronic acid-N-acetylglucosamine-[glucuronic acid-N-acetylglucosamine]5-glucuronic acid-galactose-galactose-SERINE) to test as a competitive inhibitor in binding studies. Our experiment is to compare Glyco-gene expression by human corneal epithelial cells in response to cell communication glycoprotein 'lacritin' versus negative control deletion construct 'C-25 lacritin' and versus positive control serum.
2011-09-05 | E-GEOD-31930 | biostudies-arrayexpress
Project description:Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating Nacetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Human EXT1 and EXT2, lacking the N-terminal transmembrane helix, were co-expressed as a secreted complex using human embryonic kidney cells (FreeStyle 293-F). We purified 0.5 mg of the complex from 900 mL suspension culture using two immobilized metal affinity chromatography steps and an interspersed protease treatment to cleave off the alkaline phosphatase fusion protein serving as a secretion signal. Mass spectrometry-based proteomic analysis confirmed the quality of the sample with EXT1 and EXT2 found to be the most abundant proteins, and being present in comparable amounts.
2022-12-09 | PXD034871 | Pride
Project description:Exostosin 1 (Ext1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of Ext1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos showed that Ext1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as Ext1 regulated genes. This analysis investigating the role of Ext1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. Exostosin 1 (EXT1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of EXT1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. EXT1 MO embryos showed that EXT1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as EXT1 regulated genes. This analysis investigating the role of EXT1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. The embryos were injected with mispair or Ext1 MO were collected at St. 10.5. The RNA was extracted and for probe synthesis and the hybridization was performed on Affymetrix Xenopus laevis genome v2.0 array. The experiments were repeated for three times. Results of paired control and experimental samples were compared.
2013-10-23 | E-GEOD-51562 | biostudies-arrayexpress
Project description:Exostosin 1 (Ext1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of Ext1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. Ext1 MO embryos showed that Ext1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as Ext1 regulated genes. This analysis investigating the role of Ext1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development. Exostosin 1 (EXT1) is a glycosyltransferase involved in the biosynthesis of the extracellular matrix Heparan Sulfate Proteoglycan (HSPG). Knockdown of EXT1 caused gastrulation defects and formation of an abnormal body axis. Since ext1 has been implicated as an indirect contributor to multiple signaling pathways in vertebrate development, microarray was used to identify genes expressed in gastrulae that would be affected by a reduction in ext1 expression. Microarray-based comparisons of gene expression in control vs. EXT1 MO embryos showed that EXT1 is involved in regulating genes that are related to metabolic process, development and signaling pathways. Half of the hits from the microarray are uncharacterized genes. Approximately forty-five percent of genes are related to metabolic process and thirty percent of genes are belonged to signaling and developmental process categories. Ten percent of each up-regulated and down-regulated gene set is predicted to function in establishment of localization by GO, which is consistent with EXT1 being involved in the movement of extracellular substances. The transcription factors or signaling protein, Isl1, Pitx2, TBX5A, Wnt5A, Wnt7A, WT1, Pax3, Wnt1, and Xbra were identified as EXT1 regulated genes. This analysis investigating the role of EXT1 during gastrulation and provide the information that EXT1 plays an important role in Xenopus early development.
2013-10-23 | GSE51562 | GEO
Project description:We assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. This experiment assesses the role of the FBXW7-KMT2D axis in regulating gene expression in OPM-1 cells.
2020-04-07 | GSE148131 | GEO
Project description:Purpose: FBXW7 is one of the most frequently mutated tumor suppressors, the deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatic analyses and genome-wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multi-drug resistance (MDR), to a bigger extent than well-established MDR-drivers such as ABCB1. Proteomic data from FBXW7-deficient cells and human cancer samples identify the upregulation of mitochondrial function as a hallmark of FBXW7 deficiency, which has been previously linked to an increased resistance to chemotherapy. Accordingly, genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7-deficient cells. For instance, targeting mitochondrial translation with the antibiotic Tigecycline efficiently kills FBXW7-deficient cells in vitro and in vivo, by a mechanism that involves activation of the Integrated Stress Response (ISR). Searching for additional drugs that overcome MDR in FBXW7-deficient cells, we found several targeted therapies such as Erlotinib, Dasatinib or Vemurafenib which unexpectedly also activate the ISR. Together, our study reveals that one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, and identifies a general principle to overcome such resistance. Methods: This study was performed in 2 groups of DLD1 cells with duplicate samples per group. Both control (Wild Type, WT) and experimental (FBXW7 -/-, KO) were exposed to Tigecycline (10µM). DMSO was used as a control for the treatment. After 24h, the RNA was extracted and the transcriptome was profiled with RNA-Sequencing (RNA-seq) analysis. Results: our study reveals that one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, and identifies a general principle to overcome such resistance.
2022-06-09 | GSE189499 | GEO
Project description:In order to examine the role of macrophage heparan sulfate proteoglycans in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages. In order to determine the impact of altering Ndst1 expression, we crossbred mice bearing a conditional loxP-flanked (M-bM-^@M-^\floxedM-bM-^@M-^]) allele of Ndst1 (Ndst1f/f) to transgenic mice expressing the bacterial Cre recombinase under control of the lysozyme 2 promoter (LysMCre) to drive inactivation of the gene in myeloid cells. We compared the transcriptome of bone marrow derived macrophages from Ndst1f/fLysMCre- and Ndst1f/fLysMCre+ macrophages in basal growth medium and after foam cell conversion using 50M-BM-5g/ml of aggregated LDL (agLDL) to asses the impact of altered macrophage heparan sulfate sulfation on gene expression. Total RNA obtained from bone marrow derived macrophages (BMDM) derived form two wild-type and two Ndfst1-deficient mice before and after foam cell conversion and analyzed in diplicate (n=4 for each conditions; total of 4 conditions)
2014-11-04 | E-GEOD-51009 | biostudies-arrayexpress
Project description:In order to examine the role of macrophage heparan sulfate proteoglycans in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages. In order to determine the impact of altering Ndst1 expression, we crossbred mice bearing a conditional loxP-flanked (“floxed”) allele of Ndst1 (Ndst1f/f) to transgenic mice expressing the bacterial Cre recombinase under control of the lysozyme 2 promoter (LysMCre) to drive inactivation of the gene in myeloid cells. We compared the transcriptome of bone marrow derived macrophages from Ndst1f/fLysMCre- and Ndst1f/fLysMCre+ macrophages in basal growth medium and after foam cell conversion using 50µg/ml of aggregated LDL (agLDL) to asses the impact of altered macrophage heparan sulfate sulfation on gene expression.
2014-11-04 | GSE51009 | GEO