A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants

RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2∼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2∼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes

Structure of UBE2K-Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension

Ubiquitin (Ub) chain types govern distinct biological processes. K48-linked polyUb chains target substrates for proteasomal degradation, but the mechanism of Ub chain synthesis remains elusive due to the transient nature of Ub-handover. Here, we present the structure of a chemically trapped complex of E2 UBE2K covalently linked to donor Ub and acceptor K48-linked di-Ub, primed for K48-linked Ub chain synthesis by a RING E3. The structure reveals the basis for acceptor Ub recognition by UBE2K active site residues and the C-terminal Ub-associated (UBA) domain to impart K48-linked Ub specificity and catalysis. Furthermore, the structure unveils multiple Ub binding surfaces on the UBA domain that allow distinct binding modes for K48-linked and K63-linked Ub chains. This multivalent Ub binding feature serves to recruit UBE2K to ubiquitinated substrates to overcome weak acceptor Ub affinity and thereby promote chain elongation. These findings elucidate the mechanism of processive K48-linked polyUb chain formation by UBE2K

The rejuvenating power of the Buena Vista Social Club

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes

Extended ubiquitin species are protein-based DUB inhibitors

A frame-shift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin, UBB+1. UBB+1 has been considered to inhibit proteasomes, and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrated that expression of extended ubiquitin variants led to accumulation of heterogeneously-linked polyubiquitin conjugates indicating a pervasive effect on ubiquitin-dependent turnover. 20S proteasomes selectively proteolysed ubiquitin extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of lysine-48 linkages at 26S proteasomes. Processing of lysine-48 and lysine-63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of ubiquitin-dependent degradation by extended ubiquitin variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs

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

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

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-Methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a β-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite

Bivalent binding of p14ARF to MDM2 RING and acidic domains inhibits E3 ligase function

ARF tumor suppressor protein is a key regulator of the MDM2-p53 signaling axis. ARF interferes with MDM2-mediated ubiquitination and degradation of p53 by sequestering MDM2 in the nucleolus and preventing MDM2-p53 interaction and nuclear export of p53. Moreover, ARF also directly inhibits MDM2 ubiquitin ligase (E3) activity, but the mechanism remains elusive. Here, we apply nuclear magnetic resonance and biochemical analyses to uncover the mechanism of ARF-mediated inhibition of MDM2 E3 activity. We show that MDM2 acidic and zinc finger domains (AD-ZnF) form a weak intramolecular interaction with the RING domain, where the binding site overlaps with the E2∼ubiquitin binding surface and thereby partially reduces MDM2 E3 activity. Binding of human N-terminal 32 residues of p14ARF to the acidic domain of MDM2 strengthens the AD-ZnF-RING domain interaction. Furthermore, the N-terminal RxFxV motifs of p14ARF participate directly in the MDM2 RING domain interaction. This bivalent binding mode of p14ARF to MDM2 acidic and RING domains restricts E2∼ubiquitin recruitment and massively hinders MDM2 E3 activity. These findings elucidate the mechanism by which ARF inhibits MDM2 E3 activity

Unique structural, dynamical, and functional properties of k11-linked polyubiquitin chains

SummaryK11-linked polyubiquitin chains play important signaling and regulatory roles in both degradative and nonproteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other polyubiquitins, we determined solution structures of K11-linked diubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and nonproteasomal pathways but with intermediate affinity and different binding modes than either K48-linked or K63-linked diubiquitin. Our data support the hypothesis that polyubiquitin chains of different linkages possess unique conformational and dynamical properties, allowing them to be recognized differently by downstream receptor proteins

Mixed-linkage ubiquitin chains send mixed messages

SummaryResearch on ubiquitin (Ub) signaling has focused primarily on homogeneously linked polyUb. Although polyUb containing different linkages within the same chain exist, their structures and signaling properties are unknown. These mixed-linkage chains could be unbranched (i.e., no more than one lysine or methionine linkage per Ub) or branched. Here, we examined the structure, dynamics, receptor selectivity, and disassembly of branched and unbranched tri-Ub containing both K48 and K63 linkages. Each linkage was virtually indistinguishable from its counterpart in homogeneously linked polyUb. Linkage-selective receptors from hHR23A and Rap80 preferentially bound to the K48 or K63 linkages in the branched trimer. Linkage-selective deubiquitinases specifically cleaved their cognate Ub-Ub linkages in mixed-linkage chains, and the 26S proteasome recognized and processed branched tri-Ub. We conclude that mixed-linkage chains retain the distinctive signaling properties of their K48 and K63 components and that these multiple signals can be recognized by multiple linkage-specific receptors. Finally, we propose a new, comprehensive notation for Ub and Ub-like polymers