Ubiquitin-specific protease 7 is a regulator of ubiquitin-conjugating enzyme UbE2E1

Abstract

Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme found in all eukaryotes that catalyzes the removal of ubiquitin from specific target proteins. Here, we report that UbE2E1, an E2 ubiquitin conjugation enzyme with a unique N-terminal extension, is a novel USP7-interacting protein. USP7 forms a complex with UbE2E1 in vitro and in vivo through the ASTS USP7 binding motif within its N-terminal extension in an identical manner with other known USP7 binding proteins. We show that USP7 attenuates UbE2E1-mediated ubiquitination, an effect that requires the N-terminal ASTS sequence of UbE2E1 as well as the catalytic activity of USP7. Additionally, USP7 is critical in maintaining the steady state levels of UbE2E1 in cells. This study reveals a new cellular mechanism that couples the opposing activities of the ubiquitination machinery and a deubiquitinating enzyme to maintain and modulate the dynamic balance of the ubiquitin-proteasome system.

Keywords: Deubiquitination; E3 Ubiquitin Ligase; Protein Structure; Ubiquitin-conjugating Enzyme (Ubc); Ubiquitination.

FIGURE 1.

Analysis of the in vitro interaction between USP7-NTD and UbE2E1. A, schematics of UbE2E1 and USP7 indicating domain organization of the two proteins and the putative USP7 binding motif within the N terminus of UbE2E1. CAT, catalytic domain. B, comparison of the substrate peptide sequences recognized by USP7-NTD, featuring the (P/A)XXS motif, which is also found in the N terminus of UbE2E1. C, GST pulldowns were performed using GST-USP7-NTD and UbE2E1. GST-USP7-NTD fusion protein was incubated with UbE2E1, loaded onto glutathione resin (load (L)), washed with wash buffer (wash (W)), and eluted with Laemmli SDS-PAGE loading dye (eluate (E)). A GST pulldown of UbE2E1 protein with GST alone served as a negative control. GST pulldown experiments also tested the interaction of GST-USP7-NTD and the N-terminal UbE2E1 deletion mutant (ΔN-UbE2E1) and between the GST-USP7-NTD double mutant USP7-NTD^DWand UbE2E1.

FIGURE 2.

Molecular analysis of the USP7-NTD-UbE2E1 interaction A, dissociation constants between USP7-NTD and UbE2E1 peptides were measured by intrinsic tryptophan fluorescence assays. B, the crystal structure of the USP7-NTD·UbE2E1^ASTS complex. A ribbon representation of the structure of USP7-NTD (green) with UbE2E1^ASTS in stick form (yellow) is shown. The 2F_o − F_c electron density map showing the UbE2E1^ASTS peptide is contoured at 1σ. C, the molecular details of the interaction between USP7-NTD and UbE2E1^ASTS are shown in stick format with the same color scheme as in B. Hydrogen bonds are indicated by black dashed lines. D, superimposition of UbE2E1^ASTS (yellow) with the peptide from viral interferon regulatory factor protein 4, vIRF4^ASTS (salmon), showing the similarity in mode of binding within the USP7 peptide binding region.

FIGURE 3.

USP7 and UbE2E1 localization and interaction in vivo. A, immunofluorescence images of U2OS cells are shown after staining for endogenous UbE2E1 (Cy-3) and USP7 (Alexa Fluor 488) proteins. The merged image indicates a partially overlapping localization of USP7 and UbE2E1 in the nucleus. The nuclei were counterstained with DAPI. B, 293T cells transfected with FLAG-UbE2E1 were subject to immunoprecipitation (IP) using mouse IgG (negative control) and anti-FLAG followed by immunoblotting (IB) using antibodies against USP7 and UbE2E1. C, 293T cells transfected with Myc-USP7 were subject to immunoprecipitation using rabbit IgG and anti-Myc followed by immunoblotting against USP7 and UbE2E1 D, endogenous UbE2E1 from 293T cells was immunoprecipitated with a monoclonal antibody against UbE2E1 or mouse IgG followed by immunoblotting with polyclonal rabbit antibodies against USP7 or UbE2E1.

FIGURE 4.

USP7 attenuates UbE2E1-mediated ubiquitination. A, in vitro ubiquitination assays were performed using UbE2E1, UbE2D2, or ΔN-UbE2E1 (as the E2) in the presence of ATP, E1, Ub, the catalytic domain of NEDD4 (as the E3 ligase), and increasing amounts of USP7 (0–2 μg). Total ubiquitinated products (Ub(n))were detected by immunoblotting (IB) using a specific antibody against ubiquitin. Polyubiquitinated products were highlighted in boxes. B, ubiquitin loading assays were carried out in a reaction containing E1, His₆-Ub, and ATP with increasing amounts of USP7 (0–2 μg) in the presence of 1 μg of full-length UbE2E1 or ΔN-UbE2E1. The UbE2E2-S∼Ub and ΔN-UbE2E2-S∼Ub intermediates were visualized by immunoblotting, using anti-UbE2E1 (left panel) or anti-His₆ epitope tag (right panel). C, in vitro ubiquitination assays were performed as in A with the addition of active USP7 (lanes 3–5) or USP7 pretreated with ubiquitin-aldehyde (lanes 7–9), which blocks the catalytic activity of USP7. NC (negative control) represents a standard ubiquitination reaction in the absence of ATP.

FIGURE 5.

USP7 deubiquitinates UbE2E1. A, in vitro ubiquitination assays were performed as in Fig. 4A with increasing amounts of USP7. Total ubiquitination (Ub(n)) was detected using anti-Ub (left panel) and ubiquitinated UbE2E1 was detected using anti-UbE2E1 (right panel). NC (negative control) represents a standard ubiquitination reaction in the absence of ATP. IB, immunoblot. B, UbE2E1 is subject to ubiquitination and accumulated in the presence of MG132 in U2OS cells. Ubiquitinated UbE2E1 was immunoprecipitated (IP) and visualized by immunoblot using anti-ubiquitin. C, ubiquitinated UbE2E1 was subject to USP7 deubiquitination using USP7 or USP7-CS and visualized by immunoblot using anti-ubiquitin.

FIGURE 6.

USP7 stabilizes UbE2E1 in vivo. A, U2OS cells were transfected with nonspecific control siRNA (siControl) or siRNA targeting USP7 (siUSP7) and treated with 10 μg/ml cycloheximide (CHX) for the indicated number of hours or left untreated (lane 0). Cell lysates were subjected to SDS-PAGE and Western blot analysis using antibodies indicated. B, WT HCT116 cells or HCT116 USP7^−/− were treated with cycloheximide and harvested for Western blotting as in A. The levels of UbE2E1 were normalized using actin as a loading control and presented as line graphs in the cycloheximide chase experiments. The asterisk represents the statistical analysis comparing levels of UbE2E1 after siRNA treatment with p < 0.01. Error bars indicate S.D. C, WT USP7 and the catalytically inactive mutant C223S, USP7-CS, were transfected into HCT116 USP7^−/− cells. Cells were lysed and blotted for UbE2E1 48 h after transfection. The levels of UbE2E1 were normalized using actin as a loading control and presented in a bar graph (bottom). The asterisk represents the statistical analysis comparing levels of UbE2E1 after USP7 rescue with p < 0.01. Error bars indicate S.D.

FIGURE 7.

USP7 regulates the stability of UbE2E1. USP7 attenuates UbE2E1-mediated total ubiquitination and stabilizes UbE2E1 through an interaction between USP7-NTD and the N-terminal extension of UbE2E1. Inactivation or disruption of the interaction between USP7 and UbE2E1 leads to UbE2E1 destabilization.