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. 2009;4(5):e5571.
doi: 10.1371/journal.pone.0005571. Epub 2009 May 15.

The UBA-UIM domains of the USP25 regulate the enzyme ubiquitination state and modulate substrate recognition

Amanda Denuc  1 , Anna Bosch-Comas, Roser Gonzàlez-Duarte, Gemma Marfany
Affiliations

The UBA-UIM domains of the USP25 regulate the enzyme ubiquitination state and modulate substrate recognition

Amanda Denuc et al. PLoS One. 2009.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. USP25 domain dissection and their contribution to the catalytic activity.
A. Sequence homologies revealed five highly conserved USP motifs (I to V) in two domains (USP1 and USP2) that catalogue USP25m as a deubiquitinating enzyme. Cys-178 is the putative active site of the enzyme, since it is conserved in all analyzed members of the family. B. Deubiquitinating activity assays in E.coli cells co-transformed with the recombinant substrate Ub-βgalactosidase and either wild type (WT) USP25m or the C178S mutant confirmed that Cys 178 is the active site of USP25m. βgal immunodetectection shows a lower band using WT USP25m, indicating hydrolysis of Ub from βgal, while the mutated form is catalytically inactive and displays the band of the uncleaved fusion Ub-βgal. Note that the endogenous β-galactosidase is of lower molecular weight.
Figure 2
Figure 2. Localization of the USP25m UBDs and analysis of their contribution to the deubiquitinating activity.
A. USP25m contains one UBA and two UIM (USP25_1, USP25_2) domains, as shown by alignments with other UBAs or UIMs. B. Schematic representation of the USP25m C-terminal and UBD deletion mutants: ΔUBA (Δ19-58 aa, inclusive), ΔUIM1 (Δ96-115 aa, inclusive), ΔUIM2 (Δ121-141 aa, inclusive), ΔUBA-UIM1 (Δ19-115 aa, inclusive), ΔUBA-UIM1-UIM2 (Δ19-141 aa, inclusive), ΔUIM1-UIM2 (Δ96-141 aa, inclusive). The constructs bearing serial deletions of USP25m at the C-terminus are also shown (E679X, E769X, Q863X, E1020X). C. Deubiquitinating activity assays indicated that UBDs were not required to cleave off ubiquitin (left upper panel). The mutant USP25mE679X was unable to hydrolyze Ub from the Ub-βgal substrate, indicating that the region between the amino acids 679 and 769 was required for enzymatic activity (right upper panel). The empty GST vector and the full length USP25m were respectively used as negative and positive controls. The expression level of each USP25m mutant was comparable (lower panels).
Figure 3
Figure 3. Dimerization/oligomerization of USP25m.
A. Coimmunoprecipitation assays after co-expressing two differently tagged forms (cMyc- or GFP-) of either the wild-type USP25m or the C178S mutant, showed that USP25 dimerized in vivo (upper panel). The catalytic Cys was not required for interaction. The empty GFP vector was used as a negative control. B. The same co-immunoprecipitation experiment co-expressing the cMyc-USP25m with each of the UBD deletion mutants fused to GFP showed that none of the UBDs was critical for dimerization or formation of the complex. Last lanes of the panels correspond to the co-immunoprecitation of the two mutants bearing the deletion of the 3 UBD domains (Δ19-141, inclusive). Single transfection with the GFP-USP25m construct was used as a negative control. C. The same assays using the constructs with serial deletions of the C-terminal region of the enzyme showed that the C-terminus was not required for dimerization of USP25m. The last lane of the panels at the left corresponds to the cotransfection with two differently tagged E679X mutants. Single transfection with the GFP-USP25m construct was used as a negative control (first lane). The separated panels at the right correspond to the co-immunoprecipitation of the double mutant USP25m bearing the deletion of the first 153 amino acids and truncated at residue 679 (USP25mΔ153-E679X) with the wild-type USP25m, and their positive control.
Figure 4
Figure 4. USP25m is ubiquitinated and autodeubiquitinated.
A. Immunodetection of cell lysates expressing Myc-tagged USP25m showed one additional high molecular-weight band. This band was stronger when co-expressing His(6x)-Ub, suggesting that it corresponded to mono-ubiquitinated USP25m. The high molecular weight bands were stronger when co-expressing His(6x)-Ub and the catalytically inactive mutant USP25mC178S. The lower histogram shows the percentage of non-modified versus mono-Ub-conjugated USP25m. B. The same experiment was performed co-expressing His(6x)-Ub with all the UBD USP25m deletion mutants, in combination or not with the C178S mutation. Again, the ubiquitinated band was much visible in the C178S version of the mutants. C. Ni2+ pull-down assays to purify His(6x)Ub-conjugated proteins confirmed that USP25m was ubiquitinated. All the mutant constructs were tested, confirming that monoubiquitination (and multi- or poly-ubiquitination) did not depend on UBDs, neither on the presence of the C-terminus. The ratio output/input is 4. (Output samples were eluted at pH 4.5, which could account for the slight variation in the apparent protein molecular weight compared to inputs). D. Protein stability of the USP25m full-length and mutant constructs. Cells were grown in standard conditions (−), or treated with MG132 (+). Immunodetection of α-tubulin was used as a loading control.
Figure 5
Figure 5. UBDs modulate substrate recognition by USP25m and K99 is the key regulatory residue.
A. MyBPC1 is differentially rescued from proteasome degradation depending on the presence of the distinct UBDs. Transfection of MyBPC1 with the empty GFP vector was used as the negative control, and addition of MG132 was used as a positive control. B. Relative quantification of the MyBPC1 rescue by different USP25m mutants. α-tubulin was used for normalization of protein concentration (data not shown) and USP25m expression levels were used to normalize for transfection efficiency. The rescue achieved by the wild-type USP25m was considered as the reference (value of one). At least three different replicates were used for quantification. Asterisks indicate statistical significance (p<0.05, Mann-Whitney test). C. The catalytically inactive C178S and the K99R mutants behaved similarly and are unable to rescue MyBPC1 from proteasome degradation in a time-course experiment when new protein synthesis is inhibited. The rescue achieved by expression of the wild-type USP25m was used as a control. D. The MyBPC1 levels (normalized by α-tubulin expression) were quantified and expressed relatively to those observed at time 0 h (30 h post-transfection, before cycloheximide treatment), which were considered 100%. The values corresponded to a minimum of three different replicates in several independent experiments. Asterisks indicate statistical significance (p<0.05, Mann-Whitney test). CHX- cicloheximide.
Figure 6
Figure 6. Model of the USP25m activity regulation upon specific substrates.
A. Post-translational modifications of USP25m concerning monoubiquitination in Lys99 (K99) and sumoylation in K99 and K141. U: Ubiquitin; S: SUMO; UBA: Ubiquitin associated domain; SIM: SUMO interacting motif; UIM: Ubiquitin interacting motif; USP: Ubiquitin specific protease catalytic domains; CC: coiled coil domain; 19a19b: peptide encoded by the muscle-specific exons. B. Model on the regulation of USP25m activity through alternative and mutually exclusive conjugation of SUMO (inhibiting) and ubiquitin (activating) on the same lysine residue (K99) (see discussion). C. The dimerization of USP25m might be relevant to the regulation of the enzymatic activity as autodeubiquitination could occur either intra- or inter-molecular in the dimer.
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