The UBA-UIM Domains of the USP25 Regulate the Enzyme Ubiquitination State and Modulate Substrate Recognition

Abstract

USP25m is the muscle isoform of the deubiquitinating (DUB) enzyme USP25. Similarly to most DUBs, data on USP25 regulation and substrate recognition is scarce. In silico analysis predicted three ubiquitin binding domains (UBDs) at the N-terminus: one ubiquitin-associated domain (UBA) and two ubiquitin-interacting motifs (UIMs), whereas no clear structural homology at the extended C-terminal region outside the catalytic domains were detected. In order to asses the contribution of the UBDs and the C-terminus to the regulation of USP25m catalytic activity, ubiquitination state and substrate interaction, serial and combinatorial deletions were generated. Our results showed that USP25m catalytic activity did not strictly depend on the UBDs, but required a coiled-coil stretch between amino acids 679 to 769. USP25 oligomerized but this interaction did not require either the UBDs or the C-terminus. Besides, USP25 was monoubiquitinated and able to autodeubiquitinate in a possible loop of autoregulation. UBDs favored the monoubiquitination of USP25m at the preferential site lysine 99 (K99). This residue had been previously shown to be a target for SUMO and this modification inhibited USP25 activity. We showed that mutation of K99 clearly diminished USP25-dependent rescue of the specific substrate MyBPC1 from proteasome degradation, thereby supporting a new mechanistic model, in which USP25m is regulated through alternative conjugation of ubiquitin (activating) or SUMO (inhibiting) to the same lysine residue (K99), which may promote the interaction with distinct intramolecular regulatory domains.

Regulation of USP28 Deubiquitinating Activity by SUMO Conjugation

“…3b). As has been described for USP25 (35), an extension of the C-terminal catalytic domain to Tyr 757 yielded a recombinant protein with deubiquitinating activities comparable with the full-length USP28 and USP25 (Fig. 3, b and c).…”

“…2a) (Table 2). Remarkably, Lys 99 was also found to correspond to the major site for SUMOylation and monoubiquitination of USP25 in previous works (35,36).…”

“…1, a and b). In a recent report on USP25, in silico comparative searches predicted three different ubiquitin binding motifs in the N-terminal region: one UBA and two UIMs (35). The N-terminal regions of USP28 and USP25 are highly homologous, particularly in these ubiquitin-binding motifs (Fig.…”

“…Finally, the DUB protease USP25 has been shown to be either SUMOylated or monoubiquitinated on Lys 99 . The ubiquitin modification of Lys 99 enhanced USP25 deubiquitinating activity on the model substrate MyBPC1 (myosine-binding protein C), whereas SUMO modification had an inhibitory effect on its activity (35,36). Interestingly, the ubiquitin-binding domains in the N-terminal region of USP25 seem to play a role in the regulation of its protease activity (35,36).…”

Activity and Cellular Functions of the Deubiquitinating Enzyme and Polyglutamine Disease Protein Ataxin-3 Are Regulated by Ubiquitination at Lysine 117

“…Doing so is important because several DUbs are reportedly ubiquitinated, including JosD1, which belongs to the same DUb family as AT3 (8,(25)(26)(27)29); 4 consequently, direct regulation of DUb activity through ubiquitination may apply to DUbs other than AT3. Also, greater insight into the AT3 structurefunction relationship and the mechanisms by which it is regulated will be important to fully understand the normal functions of AT3 and its dysfunction when the poly(Q) is expanded in the neurodegenerative disease, SCA3.…”