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

Abstract

Summary The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin-derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin-B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface-patch and the basic/polar residues surrounding it. Ubistatin-B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin-receptor and shows strong preference for K48-linkages over K11 and K63. Furthermore, ubistatin-B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S-proteasome. Finally, ubistatin-B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.

Proteasome Inhibitors: Harnessing Proteostasis to Combat Disease

“…Ubistatin directly interacts with a hydrophobic patch and the surrounding basic/polar residues on the ubiquitin surface. Ubistatin shows a strong preference for K48 linkages over K11 and K63 linkages due to differences in the conformation and distribution of the hydrophobic patches on the surfaces [152]. The major challenge to further develop ubistatins as therapeutics is their negative charge, which precludes efficient membrane penetration, thereby limiting their potency in cells.…”

In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides

“…10 An attractive alternative is to target the signal for degradation: find molecules to bind to and interfere with the recognition of Lys48-linked Ub chains. [11][12][13][14][15] However, given the breadth of cellular processes that rely on differently linked Ub chains, specificity is crucial 5 and also a challenge due to the following reasons: Ub chains are assembled from the same monomer, and the differences in structure and dynamics of differently linked-Ub chains can be subtle. 16,17 Moreover, chain length is also important.…”

Rub1/NEDD8, a ubiquitin-like modifier, is also a ubiquitin modifier

“…In this manner, introduction of Rub1 mutated at position 72 is a new tool for specifically affecting the Ub system without greatly affecting the Rub1 signaling system because it is inert to NAE. Thus ubiquitinized Rub1/NEDD8 may be added to other experimental tools for perturbing the ubiquitination landscape such as expression of lysineless non-polymerizable Ub (44), ubistatins (93,94), or the anti-cancer drug bortezomib (95).…”