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Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway

Published

Author(s)

Mark A. Nakasone, Timothy A. Lewis, Olivier Walker, Anita Thakur, Wissam Mansour, Carlos A. Castaneda, Jennifer L. Goeckeler-Fried, Frank Parlati, Tsui-Fen Chou, Ortal Hayat, Daoning Zhang, Christina M. Camara, Steven M. Bonn, Urszula K. Nowicka, Susan T. Krueger, Michael H. Glickman, Jeffrey L. Brodsky, Raymond J. Deshaies, David Fushman

Abstract

The success of proteasome inhibitors has led to intense efforts to discover points of therapeutic intervention for nearly every component of the ubiquitin-proteasome system. The discovery of ubistatins, small molecules that inhibit proteasomal degradation of substrates by directly binding to polyubiquitin, intriguingly suggests that ubiquitin itself is a promising target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural and biophysical details of ubiquitin-ubistatin interactions has impeded their continued development. Here, we synthesized a panel of ubistatin derivatives, screened their ability to inhibit substrate ubiquitination and deubiquitination, and assessed ubiquitin binding. Through a series of solution NMR experiments complemented with small-angle neutron scattering measurements we determined that ubistatin B contains two ubiquitin-binding entities that enable the formation of a ternary (1:2 ubistatin:ubiquitin) complex. Conversely a compound that represents one half of ubistatin B forms a 1:1 complex with ubiquitin albeit with reduced affinity. The structures of ubiquitin in complexes with half-ubistatin and full ubistatin B revealed direct interactions involving ubiquitins' hydrophobic surface patch and the basic/polar residues surrounding it. The critical role of these electrostatic interactions in binding was confirmed by site-directed mutagenesis. We also discovered that ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin partner and shows strong preference for K48-linked diubiquitin compared to K11- and K63-linked diubiquitins. In addition, we found that ubistatin B shields polyubiquitin from disassembly by a range of deubiquitinases as well as by the 26S proteasome. Finally, we show that ubistatin B penetrates cancer cells, exhibits localization with ubiquitin, and imp acts the ubiquitin profile. Overall these findings highlight the versatile properties of ubistatin and have implications for their future development and use in targeting ubiquitin-signaling pathways.
Citation
Structure
Volume
25
Issue
12

Keywords

ubiquitin, ubistatin, proteasome inhibitor, signaling, NMR, small-angle neutron scattering

Citation

Nakasone, M. , Lewis, T. , Walker, O. , Thakur, A. , Mansour, W. , Castaneda, C. , Goeckeler-Fried, J. , Parlati, F. , Chou, T. , Hayat, O. , Zhang, D. , Camara, C. , Bonn, S. , Nowicka, U. , Krueger, S. , Glickman, M. , Brodsky, J. , Deshaies, R. and Fushman, D. (2017), Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway, Structure, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=919071 (Accessed June 10, 2023)
Created December 4, 2017, Updated October 12, 2021