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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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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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