title: Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates.
abstract: Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.
authors.affiliation: Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Cristinel
authors.initials: C
authors.lastname: Sandu
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, NY, USA.
authors.forename: Nagaranjan
authors.initials: N
authors.lastname: Chandramouli
authors.affiliation: High Throughput Screening Resource Center, The Rockefeller University, New York, NY, USA.
authors.forename: Joseph Fraser
authors.initials: JF
authors.lastname: Glickman
authors.affiliation: Proteomics Resource Center, The Rockefeller University, New York, NY, USA.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Chueh-Ling
authors.initials: CL
authors.lastname: Kuo
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Kukushkin
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
authors.affiliation: Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY, USA.
authors.forename: Hermann
authors.initials: H
authors.lastname: Steller
mesh_terms: D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism*; D059748:Proteolysis / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013883:Thiostrepton / Q000494:pharmacology*
chemical_list: D061988:Proteasome Inhibitors; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D013883:Thiostrepton
keywords: proteasome; protein chemical modification; protein degradation; thiol; ubiquitin