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A proteomic study on the membrane protein fraction of T cells confirms high substrate selectivity for the ER translocation inhibitor cyclotriazadisulfonamide

ABSTRACT: Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of type I integral membrane protein human CD4 (huCD4) across the ER in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared to huCD4. We performed a quantitative proteomic study on the membrane fraction of human T-cells to analyze how many integral membrane proteins are sensitive to CADA. We employed stable isotope labelling by amino acids in cell culture (SILAC) technique in combination with quantitative mass spectrometry on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA-treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, Endoplasmic Reticulum Lectin 1 (ERLEC1), Inactive Tyrosine-Protein Kinase 7 (PTK7), and DnaJ Homolog Subfamily C member 3 (DNAJC3). The sensitivity of these proteins for the translocation inhibitor CADA was confirmed by biochemical experiments including Western blots, flow cytometry and cell free in vitro translation/translocation experiments.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell

SUBMITTER: Fan Liu

LAB HEAD: Fan Liu

PROVIDER: PXD027712 | Pride | 2021-09-08

REPOSITORIES: Pride

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Publications

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Pauwels Eva E Rutz Claudia C Provinciael Becky B Stroobants Joren J Schols Dominique D Hartmann Enno E Krause Eberhard E Stephanowitz Heike H Schülein Ralf R Vermeire Kurt K

Molecular & cellular proteomics : MCP 20210902

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for thes ...[more]

PMID: 34481949

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Project description:Prostaglandin E2 (PGE2) is involved in several inflammatory conditions including periodontitis. The aim of this study was to investigate the global gene expression profile of tumor necrosis factor alpha (TNFalpha) stimulated human gingival fibroblasts, focusing on signal pathways related to PGE2 production and the new PGE2-synthesizing enzymes, prostaglandin E synthases (PGES). The expression of microsomal prostaglandin E synthase-1 (mPGES-1) as well as the upstream cyclooxygenase-2 (COX-2) was up-regulated by TNFalpha, accompanied by increased PGE2 production. In contrast, the expression of microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFalpha. Using microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, we identified differentially expressed genes in response to TNFalpha treatment. Enrichment analysis of microarray data identified two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappaB (NF-kappaB). We used specific inhibitors and phosphorylation analysis to confirm their role in PGE2 regulation. Both JNK and NF-kappaB inhibitors reduced the TNFalpha-stimulated up-regulation of mPGES-1 and COX-2 as well as subsequent PGE2 production. The novel finding that TNFalpha-stimulated mPGES-1 is regulated by JNK suggests this kinase as a potential future target for treatment strategies in inflammatory disorders, including periodontitis. Keywords: Time course, gene expression, factorial design. Three human gingival fibroblast cell lines were established from gingival biopsies obtained from 3 healthy patients, 3 to 12 years of age, with no clinical signs of periodontal disease. Cells were incubated with or without TNF-alpha (20 ng/ml) for 1, 3 or 6h. After incubation for 1, 3 or 6 h in the two conditions, the cells were immediately frozen in liquid nitrogen and then stored at -70°C for subsequent isolation of total RNA. The experimental design of the microarray study was set up as a time-course factorial design, to best observe the TNF-alpha induced gene expression changes over time. C++ program (G.F. Glonek, P.J. Solomon, Factorial and time course designs for cDNA microarray experiments, Biostatistics 5 (2004) 89-111) was used to determine the exact layout of the design in order to estimate the interaction effect between treatment and time, i.e. genes that are differentially expressed over time, with optimal statistical efficiency. Cyanine 5 and cyanine 3 were used for labeling. For each cell line 12 hybridizations were performed in a time-course factorial design. In total, 36 hybridizations were performed.

Dataset Information

A proteomic study on the membrane protein fraction of T cells confirms high substrate selectivity for the ER translocation inhibitor cyclotriazadisulfonamide

Publications

A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Similar Datasets

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