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

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:34481949", "kind": "science-record", "revision": 6, "content_hash": "sha256:88aedb74c15b1cdb64ccbd152eab869c454eaee3f1b98103f6dca77e0aa4e087", "created_at": "2026-11-01T15:41:50Z", "updated_at": "2026-11-01T21:26:09Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.", "doi": "10.1016/j.mcpro.2021.100144", "pmid": "34481949", "authors": ["Eva Pauwels", "Claudia Rutz", "Becky Provinciael", "Joren Stroobants", "Dominique Schols", "Enno Hartmann", "Eberhard Krause", "Heike Stephanowitz", "Ralf Schülein", "Kurt Vermeire"], "journal": "Molecular & cellular proteomics : MCP", "year": 2021, "abstract": "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 these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) 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). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences.", "claims": [{"id": "claim-method", "statement": "To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (80, 81, 82) partner repository with the dataset identifier PXD027712.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:34481949", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4."}, {"asset": "source:pmid:34481949", "locator": "sections[22] \"Data Availability\" paragraph 2", "quote": "The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (80, 81, 82) partner repository with the dataset identifier PXD027712."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (80, 81, 82) partner repository with the dataset identifier PXD027712. 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Western blot and flow cytometry analysis of ERLEC1, PTK7,", "both experiments. For the flow cytometry and Western blotting validation experiments, at least three indep", "tential CADA Targets With Flow Cytometry and Western Blotting To confirm the potential CADA hits from the", "experiments. Values are mean ± SD; n ≥ 3. B, Western blot images of cell lysates from nontransfected ("], "order_basis": "paper_text_order:1351", "id": "assay:1"}, {"role": "data_generation", "definition_id": "flow_cytometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["family C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein", "higher than 2.0 in both experiments. For the flow cytometry and Western blotting validation experiments,", "sion Analysis of Potential CADA Targets With Flow Cytometry and Western Blotting To confirm the potentia", "treated or DMSO-treated SUP-T1 cells. First, flow cytometry was used to analyze the natural cell surface"], "order_basis": "paper_text_order:1367", "id": "assay:2"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.77, "evidence": ["were performed using a different passage of cultured cells. For the in vitro translation/transloc"], "order_basis": "paper_text_order:4729", "id": "assay:3"}, {"role": "data_generation", "definition_id": "transfection", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["of huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected cells.A, concentration–response curves of CA", "5, SORT-V5, PTK7-V5, ERLEC1-V5, or DNAJC3-V5 transfected HEK293T cells treated for 24 h with differen", "for huCD4, SORT, PTK7, ERLEC1, and DNAJC3 in transfected HEK293T cells. Samples from (B) were quantif", "proteins SORT and huCD4. HEK293T cells were transfected with the plasmids, treated with increasing c"], "order_basis": "paper_text_order:6194", "id": "assay:4"}]}