Tandem UIMs confer Lys48 ubiquitin chain substrate preference to deubiquitinase USP25

Abstract

Ubiquitin-specific protease (USP) 25, belonging to the USP family of deubiquitinases, harbors two tandem ubiquitin-interacting motifs (UIMs), a ~20-amino-acid α-helical stretch that binds to ubiquitin. However, the role of the UIMs in USP25 remains unclear. Here we show that the tandem UIM region binds to Lys48-, but not Lys63-, linked ubiquitin chains, where the two UIMs played a critical and cooperative role. Purified USP25 exhibited higher ubiquitin isopeptidase activity to Lys48-, than to Lys63-, linked ubiquitin chains. Mutations that disrupted the ubiquitin-binding ability of the tandem UIMs resulted in a reduced ubiquitin isopeptidase activity of USP25, suggesting a role for the UIMs in exerting the full catalytic activity of USP25. Moreover, when mutations that convert the binding preference from Lys48- to Lys63-linked ubiquitin chains were introduced into the tandem UIM region, the USP25 mutants acquired elevated and reduced isopeptidase activity toward Lys63- and Lys48-linked ubiquitin chains, respectively. These results suggested that the binding preference of the tandem UIMs toward Lys48-linked ubiquitin chains contributes not only to the full catalytic activity but also to the ubiquitin chain substrate preference of USP25, possibly by selectively holding the Lys48-linked ubiquitin chain substrates in the proximity of the catalytic core.

Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities

“…The Catalytic Domains of USP25 and USP28 Mediate Oligomerization The two closely related DUBs USP25 and USP28 (Figures 1A and S1) (Valero et al, 2001;Zhen et al, 2014) possess an N-terminal domain, harboring a Ub-associated domain (UBA) and two Ub-interacting motifs (UIMs), which have been shown in USP25 to be responsible for Ub linkage-type specificity (Kawaguchi et al, 2017). The two central catalytic USP domains share 57% sequence identity, and both contain an insertion site with a length of $170 amino acids (aa) (Figure 1A) (Ye et al, 2009).…”

Usp25m protease regulates ubiquitin-like processing of TUG proteins to control GLUT4 glucose transporter translocation in adipocytes

“…In general, Usp25 proteins contain three ubiquitin-binding domains near the N terminus, which can bind the small ubiquitin-like modifier (SUMO) as well as ubiquitin (22,61). Recent work shows that two of these, a pair of tandem ubiquitin-interacting motifs, bind Lys-48 -linked ubiquitin chains (62). Possibly, in Usp25m these motifs interact with tandem ubiquitin-like folds in TUGUL (13,30).…”

The ubiquitin interacting motifs of USP37 act on the proximal Ub of a di-Ub chain to enhance catalytic efficiency

“…UIM binding to Ub is routinely disabled by mutation of the consensus alanine position to glycine or the consensus serine position to alanine. In other DUBs, UIMs have been shown to confer cleavage preference for specific Ub chain types, such as K63-linked chains in the case of OTUD1 and Ataxin-3 or K48-linked Ub chains in the case of USP25 21 – 23 . Additionally, the UIMs of USP25 and Ataxin-3 have been shown to increase the ubiquitination state of the DUB itself, although the precise mechanism by which this is achieved remains unknown 24 , 25 .…”