Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

Abstract

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

“…Indeed, it is recently being increasingly appreciated that Ub chain length in addition to linkage type is a determinant of DUB activity (Hermanns et al, 2018; Kwasna et al, 2018; Mevissen et al, 2013). Moreover, the recent identification of UCHL5 as an enzyme that debranches Ub chains at the proteasome suggests that DUBs may also prefer bifurcated polyUb architectures (Deol et al, 2020; Song et al, 2021).…”

Assembly and function of branched ubiquitin chains

“…This includes UCH37, which is activated by binding to the proteasomal lid subunit RPN13 [90,91] (Figure 3A). UCH37 removes K48 linkages from branched ubiquitin molecules, while leaving the variable second linkage intact [92,93] (Figure 3B). Although it is not fully understood how UCH37 gains its specificity for branched chains, it does recognize both moieties that are attached to the shared ubiquitin molecule [93] (Figure 3C).…”

“…UCH37 removes K48 linkages from branched ubiquitin molecules, while leaving the variable second linkage intact [92,93] (Figure 3B). Although it is not fully understood how UCH37 gains its specificity for branched chains, it does recognize both moieties that are attached to the shared ubiquitin molecule [93] (Figure 3C). Debranching by UCH37 is required for continued proteasomal activity, potentially by keeping ubiquitin receptors accessible for further rounds of substrate engagement [92,93].…”

“…As the proteasome contains three ubiquitin receptors with slightly distinct linkage preferences [103][104][105], simultaneous recognition of multiple linkages in a branched chain, as well as the high local ubiquitin concentration at the substrate, could also increase the affinity of a modified protein for the proteasome. Because multivalent interactions often delay complex dissociation, the synergistic recognition of branched chains might explain the need to trim such conjugates at the proteasome to preserve the degradation capacity of this essential machine [92,93].…”

Intracellular localization of the proteasome in response to stress conditions

“…Proteasome foci associated with active site mutation of UCH37 retained polyubiquitinated proteins and RAD23B. Again, this defect of substrate processing resulted in the immobility and irreversibility of proteasome foci ( 83 ). Here, it would be interesting to investigate whether nuclear foci either surrounded by or containing proteasomes share similar qualities or are even identical structures independently of the inducing stress factor, that is, osmotic stress or amino acid deprivation.…”