title: Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation.
abstract: In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium; Institute of Biology, CSCM, University of Lübeck, Lübeck, Germany; MRC-Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
authors.forename: Kurt
authors.initials: K
authors.lastname: Vermeire
authors.affiliation: Department of Chemistry, University of Nevada, Reno, Nevada, United States of America.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Bell
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.
authors.forename: Victor
authors.initials: V
authors.lastname: Van Puyenbroeck
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Giraut
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.
authors.forename: Sam
authors.initials: S
authors.lastname: Noppen
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.
authors.forename: Sandra
authors.initials: S
authors.lastname: Liekens
authors.affiliation: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Virology and Chemotherapy, Rega Institute for Medical Research, Leuven, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Schols
authors.affiliation: Institute of Biology, CSCM, University of Lübeck, Lübeck, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Hartmann
authors.affiliation: Institute of Biology, CSCM, University of Lübeck, Lübeck, Germany.
authors.forename: Kai-Uwe
authors.initials: KU
authors.lastname: Kalies
authors.affiliation: MRC-Laboratory for Molecular Cell Biology, University College London, London, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Marsh
mesh_terms: D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000737:chemistry / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000187:drug effects*; D011487:Protein Conformation; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology
chemical_list: D015704:CD4 Antigens; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D013449:Sulfonamides