title: A Proteomic Survey Indicates Sortilin as a Secondary Substrate of the ER Translocation Inhibitor Cyclotriazadisulfonamide (CADA).
abstract: The small molecule CADA was shown to down-modulate the expression of human CD4 in a signal peptide-dependent way through inhibition of its cotranslational translocation across the ER membrane. Previous studies characterizing general glycoprotein levels and the expression of 14 different cell surface receptors showed selectivity of CADA for human CD4. Here, a PowerBlot Western Array was used as a screen to analyze the proteome of CADA-treated SUP-T1 human CD4+T lymphocytes. This high-throughput monoclonal antibody panel-based immunoblotting assay of cellular signaling proteins revealed that only a small subset of the 444 detected proteins was differentially expressed after treatment with CADA. Validation of these proteomic data with optimized immunoblot analysis confirmed the CADA-induced change in expression of the cell cycle progression regulator pRb2 and the transcription factor c-Jun. However, the up-regulation of pRb2 or down-modulation of c-Jun by CADA had no impact on cell cycle transition. Also, the reduced protein level of human CD4 did not inhibit T cell receptor signaling. Interestingly, the signal peptide-containing membrane protein sortilin was identified as a new substrate for CADA. Both cellular expression and in vitro cotranslational translocation of sortilin were significantly reduced by CADA, although to a lesser extent as compared with human CD4. Our data demonstrate that a small signal peptide-binding drug is able to down-modulate the expression of human CD4 and sortilin, apparently with low impact on the cellular proteome.
authors.affiliation: From the ‡KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
authors.forename: Victor
authors.initials: V
authors.lastname: Van Puyenbroeck
authors.affiliation: From the ‡KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
authors.forename: Elisa
authors.initials: E
authors.lastname: Claeys
authors.affiliation: From the ‡KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Schols
authors.affiliation: §Department of Chemistry, University of Nevada, Reno, NV, USA.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Bell
authors.affiliation: From the ‡KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium; kurt.vermeire@kuleuven.be.
authors.forename: Kurt
authors.identifier: 0000-0003-1123-1907
authors.initials: K
authors.lastname: Vermeire
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006571:Heterocyclic Compounds / Q000494:pharmacology*; D006801:Humans; D021381:Protein Transport / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D015704:CD4 Antigens; D018797:Cell Cycle Proteins; D006571:Heterocyclic Compounds; D000099183:Sortilin; C498074:9-benzyl-3-methylene-1,5-di-p-toluenesulfonyl-1,5,9-triazacyclododeane