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 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.

Keywords: CD4; ER cotranslational translocation; SILAC; cyclotriazadisulfonamide; signal peptide; small-molecule inhibitor.