Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

Journal of Cellular and Molecular Medicine

Cristinel Sandu¹  Nagaranjan Chandramouli⁴  Joseph Fraser Glickman²  Henrik Molina⁴  Chueh-Ling Kuo³  Nikolay Kukushkin³  Alfred L. Goldberg³ 

[1] Howard Hughes Medical Institute, Strang Laboratory of Apoptosis and Cancer Biology, The Rockefeller University, New York, NY, USA;High Throughput Screening Resource Center, The Rockefeller University, New York, NY, USA;Department of Cell Biology, Harvard Medical School, Boston, MA, USA;Proteomics Resource Center, The Rockefeller University, New York, NY, USA

关键词: thiol;    protein degradation;    protein chemical modification;    proteasome;    ubiquitin;   

DOI  :  10.1111/jcmm.12602

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

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