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, 18 (12), 1336-44

Bilateral Inhibition of HAUSP Deubiquitinase by a Viral Interferon Regulatory Factor Protein

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Bilateral Inhibition of HAUSP Deubiquitinase by a Viral Interferon Regulatory Factor Protein

Hye-Ra Lee et al. Nat Struct Mol Biol.

Abstract

Herpesvirus-associated ubiquitin-specific protease (HAUSP) regulates the stability of p53 and the p53-binding protein MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated antitumor activity. Here we report the structural analysis of HAUSP with Kaposi's sarcoma-associated herpesvirus viral interferon (IFN) regulatory factor 4 (vIRF4) and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction that results in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, whereas the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell-cycle arrest and apoptosis in culture and to tumor regression in xenograft mouse model. Thus, the virus has developed a unique strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists.

Figures

Figure 1
Figure 1. vIRF4 interacts with HAUSP
(a) Silver-stained purified V5-vIRF4 complexes 48 h post-transfection with V5-vIRF4. Arrow, HAUSP; asterisk, V5-vIRF4. (b) The HAUSP TRAF domain is sufficient to interact with vIRF4 (wt). Schematic representation of HAUSP: TRAF, TRAF-like domain; DUB, deubiquitinase domain. 293T cells were transiently transfected and coimmunoprecipitated (Co-IP) with an anti-V5 antibody and immunoblotted (IB) with an anti-Flag antibody. (c) 293T cells transfected with the indicated constructs were subjected to Co-IP and IB. (d) The central region of vIRF4 is required for HAUSP interaction. Schematic representation of vIRF4: DBD, DNA-binding domain; PRD, proline-rich domain; and TAD, transactivation domain. 293T cells were transfected with the indicated constructs, followed by Co-IP and IB. (e) Co-IP of HAUSP with wt or several vIRF4 mutants. 293T cells transfected with the indicated vIRF4 constructs along with HAUSP subjected to IP and IB as in (b). (f) vIRF4153–256 is sufficient to bind to HAUSP. Cells were transfected with the indicated vIRF4 mutants in combination with empty vector or HAUSP, and subjected to GST pull-down and IB with an anti-Flag antibody.
Figure 2
Figure 2. Structural basis for the interaction between HAUSP and vIRF4
(a) Ribbon representation of the vIRF4-HAUSP TRAF domain complex. Alpha-helix and β-sheets of the TRAF domain are shown in cyan and green, respectively. The β6 and β7 strands are indicated. The viral peptide (Ser202 to Met216) bound to the TRAF domain is represented in magenta. (b) Distinctiveness of HAUSP TRAF-vIRF4 interaction. Cellular substrates and EBV EBNA1 utilize a similar strategy in the interaction with HAUSP TRAF (left) whereas vIRF4 employs an unusual strategy for binding to the TRAF (middle). All target binding peptides are superimposed onto the HAUSP TRAF domain (right). The peptides include vIRF4 (magenta), p53 (green), MDM2 (cyan), MDM2 (gray), MDMX (yellow), and EBNA1 (olive). Residues in vIRF4 are labeled in magenta. The consensus sequence motif for each peptide is shown, and the most conserved residues are circled. (c) Thermodynamic parameters of competitive binding of vIRF4 with the TRAF domain against cellular substrates. Each cellular substrate peptide was first titrated into the TRAF domain, and the competitor vIRF4202–216 was then titrated against each peptide.
Figure 3
Figure 3. Bilateral interaction of vIRF4 with HAUSP
(a) NMR analysis of the interaction between the vIRF4 peptide and HAUSP TRAF-DUB domain. Shown is the backbone amide region of the 2D 1H-15N correlation spectra of vIRF4153–256 in the presence of an equimolar amount of HAUSP62–205 (red) or HAUSP62–560 (blue). The 1H-15N spectrum of free vIRF4153–256 is represented in black. Blue triangles, signal changes of vIRF4153–256 observed upon binding of HAUSP62–205. Orange triangles, additional changes detected upon binding of HAUSP62–560. Magenta arrows, residues close to vIRF4 Trp232. (b) Signal changes of the tryptophan ε-NH protons upon interaction with HAUSP62–205 (red) or HAUSP62–560 (blue). Free vIRF4153–256 is represented in black contours. (See also Supplementary Fig. 3a). (c) The Trp232 backbone assignment. Superposition of the 1H-15N correlation spectra of free vIRF4153–256 (black) and vIRF4 (W232A)153–256 (green). Residues located close to Trp232 were identified by comparing the two spectra (magenta arrows). Blue arrow, the assigned Trp232 backbone. (See also Supplementary Fig. 3b). (d) Proposed molecular interaction scheme between HAUSP and two different vIRF4-derived peptides. This model is based on the vIRF4-TRAF complex structure from the present study and the HAUSP structure containing the TRAF and DUB domains (PDB accession code 2F1Z). The vIRF4202–216 peptide is displayed as a magenta loop, while the vIRF4217–236 peptide is depicted as magenta short-dashed line. Catalytic triad (yellow) is highlighted in the catalytic site. The ubiquitin binding pocket is indicated.
Figure 4
Figure 4. Effect of vIRF4 peptides on HAUSP DUB enzymatic activity
(a) Effect of vif1 and vif2 peptides on HAUSP DUB activity toward ubiquitin chains. Right: time-course measuring the appearance of cleaved mono- and diubiquitin reaction products was determined by semiquantification of IB, shown on the left. (b) The vif1 and vif2 peptides cannot inhibit USP8 deubiquitinase enzymatic activity in vitro. Purified USP8 was premixed with the vif1, vif2, or Amp (nonspecific) peptide for 5 min and then subjected to an in vitro DUB assay with the K48-Ub3–7 chain. (c) Effect of vif1 and vif2 peptides on HAUSP DUB activity toward ubiquitinated MDM2. Human recombinant purified MDM2 was incubated with purified E1, E2, and ubiquitin prior to the DUB assay. HAUSP preincubated with increasing concentrations of each peptide or HAUSP alone was incubated with ubiquitinated MDM2. (d) Ex vivo effect of TAT-vif1 and TAT-vif2 peptides on HAUSP DUB activity. At 24 h posttransfection with vector or Flag-tagged HAUSP, 293T cells were treated with 100 µM of each peptide for an additional 12 h, followed by IP with anti-Flag agarose beads and elution with Flag peptide. Purified HAUSP complexes were incubated with K48-Ub3–7 chains for the indicated intervals. 1% of the IP complex was used as the input.
Figure 5
Figure 5. Cytotoxic effect of TAT-vif1 or TAT-vif2 peptide on PEL cells
(a) Growth inhibition of PEL cells induced by TAT-vif1 and TAT-vif2 peptides. BC3 (p53wt/wt), VG1 (p53wt/wt), BCBL-1 (p53wt/mt), and BJAB (p53mt/mt) cells were treated with 100 µM of individual peptide for the indicated periods of time. *P < 0.01 and **P < 0.001. (b) TAT-vif1 and TAT-vif2 peptides induce cell cycle arrest of PEL cells. Asynchronously growing BC-1 (p53wt/wt) cells were treated with 100 µM of each peptide or 10 µM of Nutlin-3a for 48 h. Cells were pulse-labeled with BrdU and analyzed for DNA content by flow cytometry. BrdU incorporation during the S phase is quantified as the percentage of stained cells. The sub-G1 population in TAT-vif2 peptide–treated BC-1 cells is denoted by an arrow. (c) TAT-vif1- and TAT-vif2 induced cell death of PELs. Apoptosis in BC-1 cells was assessed at 48 h after treatment with 10 µM of Nutlin-3a or 100 µM of each peptide by Annexin V/PI staining. Apoptosis was measured by flow cytometry analysis. Numbers indicate the percentage of cells in each quadrant. (d) Effect of TAT-vif1 and TAT-vif2 peptides on p53 and its transcription target protein levels. VG1 and BJAB cells were treated with the same dose as that used in (a–c) for 6 h. Aliquots of cell lysates containing 10 mg of protein were analyzed by IB with the indicated antibody.
Figure 6
Figure 6. TAT-vif1 and TAT-vif2 induce tumor suppression in vivo
Time course of bioluminescent images of tumors formed from BCBL-1-Luc cells in NOD/SCID in response to intraperitoneal injection with 1 mg of TAT, TAT-vif1, or TAT-vif2 peptide for two weeks (See also Supplementary Fig. 10).
Figure 7
Figure 7. Combination therapeutic effects of low dose of TAT-vif1 and TAT-vif2 peptides
(a) BCBL-1 cells were treated with 25 µM of each peptide, alone or in combination, for the indicated time periods. Cells were examined by trypan blue staining for cell death analysis or cell number counting for cell growth. *P < 0.05 and ** P < 0.01. (b) BCBL-1 cells were incubated for 6 h with 25 µM of TAT-vif1, TAT-vif2, or both. WCL were subjected to SDS-PAGE followed by IB and were analyzed for p53, MDM2, p21, and HAUSP expression. (c) Asynchronously growing BCBL-1 cells were treated with 25 µM of the designated peptide or combined for the indicated time periods. Cells were pulse-labeled with BrdU and analyzed for DNA content by flow cytometry. BrdU incorporation during the S phase is indicated as the percentage of stained cells. (d) Scatter plot of Annexin V-FITC/PI flow cytometry of BCBL-1 cells after exposure to 25 µM peptide treatments for different time periods. Data are representative of three independent experiments. (e) After tumors were established in the NOD/SCID mice, 0.25 mg each of TAT-vif1 and TAT-vif2 peptide were injected together for 2 weeks. The tumors were measured by in vivo bioluminescence imaging.

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