Figure 1.
Identification of interacting partners for DC-UbP protein from HEK 293T cell lysates.
A, SDS-PAGE analysis of the proteins pulled down by GST-DC-UbP. There exist two protein bands around 250 kDa from the protein species pulled down by GST-fused DC-UbP. By using LC-MS/MS assay, UbE1, USP5 and Ub have been identified from each band. GST was set as a control. The samples were eluted by Buffer A containing 350 mM NaCl. *, this band around 100 kDa was identified as P97/VCP. B, Immunoblotting detection of the identified proteins with antibodies against UbE1, USP5 or Ub. #, this high-molecular-weight species could also be detected by all the three antibodies applied. C, Dissociation of the high-molecular-weight species in the presence of 8 M urea. The 250-kDa bands were disappeared in the silver-staining gel. After TCA precipitation, the pellet was re-suspended with 8 M urea. D, Western blotting detection of the individual components in 8 M urea dissociated from the high-molecular-weight species. The antibodies against UbE1, USP5 and Ub were applied. The 120- and 95-kDa proteins were UbE1 and USP5, respectively. The smear bands for Ub derivatives could not be detected due to very small amounts in the gel.
Figure 2.
Confirmation of the interaction between DC-UbP and USP5 or UbE1.
A, DC-UbP contains two separate domains, the N-terminal UBD and the C-terminal UbL. The N-terminal construct (UbP_N) includes residues 14–141, while the C-terminal (UbP_C) contains residues 129–234. B, Co-IP experiment testing the interactions between DC-UbP and USP5 or UbE1. FLAG-tagged DC-UbP, UbP_N or UbP_C was co-transfected with Myc-USP5 into HEK 293T cells. The mouse anti-FLAG M2-agarose was used for the IP experiment. Endogenous UbE1 was detected with an antibody against UbE1. Ctrl, protein-A/G beads without anti-FLAG antibody. C, NMR titration of DC-UbP with USP5. The relative peak intensities for both N- and C-terminal domains of DC-UbP were displayed upon addition of molar ratios of USP5 into the 15N-labeled DC-UbP protein. For DC-UbP alone, the peak intensities (heights) were normalized as 1 for all the peaks of DC-UbP except those for prolines and unassigned residues. The lines indicate the mean peak intensities for each domain of DC-UbP at a molar ratio of 1∶1, which are 0.56 for the UBD domain and 0.27 for the UbL domain, respectively. D, As in (C), NMR titration of DC-UbP with UbE1. The mean peak intensities are 0.54 and 0.39 for UBD and UbL, respectively, at a molar ratio of 1∶1.
Figure 3.
Characterization of the DC-UbP-binding domain in USP5.
A, Domain architecture of USP5. NT, N-terminal domain; ZnF, zinc-finger domain; UBA1 and UBA2, tandem UBA domains (UBA12); C-box and H-box, two boxes of the USP domain. The constructs of USP5_ZnF, USP5_UBA12 and USP5_UBA1 include residues 169–289, 625–749 and 631–692, respectively. B, Overlay of the HSQC spectra of 15N-labeled UbP_C (200 µM) and addition of UBA12 at different molar ratios. C, As in (B), overlay of the HSQC spectra of 15N-labeled UbP_C (200 µM) and addition of ZnF at different molar ratios. D, Titration of UbP_C with UBA12 for measuring the binding affinity. The concentration of 15N-labeled UbP_C was 150 µM. E, Diagram of the chemical-shift changes of UbP_C upon titration with UBA12 versus residue number. The molar ratio of UBA12 over UbP_C was 1∶1. The solid and dashed lines indicate the threshold values of Mean and Mean+SD for the chemical shift changes. The residues with chemical shift changes above the Mean+SD value (dashed line) are considered involved in substantial contact with UBA12. F, Mapping of the UBA12-binding surface (blue) on the UbL domain. This binding-site surface is similar to that of the UBA binding on Ub molecule. The structural model was generated based on chemical-shift perturbation data and HADDOCK analysis and displayed with PyMOL.
Figure 4.
NMR titration of USP5_UBA1 with UbP_C or its mutants.
A, Wild-type UbP_C. B, F195A. C, R199A. D, Q219A/I221A. The concentration of 15N-labeled UBA1 was 100 µM. The chemical-shift changes of residues Gln639, Leu640 and Glu642 on UBA1 were selected to plot with the increasing concentration of UbP_C. The KD value for R199A was not obtained due to neglected chemical-shift changes. n.d., not detectable.
Figure 5.
Characterization of the DC-UbP-binding domain in UbE1.
A, Domain architecture of human UbE1. A, adenylation domain, CC, catalytic cysteine domain; FCCH, first CC half domain; SCCH, second CC half domain; UFD, Ub-fold domain. B, NMR titration of DC-UbP with GST-UFD. The relative peak intensities for both N- and C-terminal domains of DC-UbP were displayed upon addition of GST-UFD at the molar ratios of 0∶1 (blue) and 5∶1 (pink) into the 15N-labeled DC-UbP protein. The lines indicate the mean peak intensities for each domain of DC-UbP at 5∶1, which are 0.69 for the UBD domain (residues 27–126) and 0.44 for the UbL domain (152–225), respectively. The bars indicate significantly perturbed regions. C, GST pull-down for testing the interaction between UFD and DC-UbP or its C-terminal part. GST-UFD and His-tagged DC-UbP or UbP_C were used for the experiments, and the anti-His antibody for Western blotting. The arrows indicate the bands for pull-down proteins. D, Mapping of the UFD-binding surface (blue) on the UbL domain. This binding-site surface is resided on the positively-charged surface just opposite to that of the UBA binding (see Fig. 3F). The structural model was displayed with PyMOL.
Figure 6.
Effects of DC-UbP binding on the catalytic activities of UbE1 and USP5.
A, Fluorescence assay for deubiquitinating activity of the components from GST-DC-UbP pull-down. The GSH conjugated beads were incubated with purified GST-DC-UbP and HEK 293T cell lysates and then eluted with GSH. The components that may contain the USP5 enzyme were subjected to activity assay with Ub-AMC (250 nM) as a substrate. The cell lysates was set as a positive control; and the GST pull-down components was a negative control. B, Fluorescence assay for deubiquitinating activities of purified USP5 in the presence of DC-UbP or UbP_C. USP5 (10 nM) was pre-incubated with 10 mM DTT for 15 min, then DC-UbP or UbP_C was added with 10-fold excess in amounts. C, In vitro ubiquitin conjugation assay for the components from GST-DC-UbP pull-down. The pull-down components were subjected to assaying the activity of UbE1 enzyme (lanes 7 and 8). Purified UbE1 and cell lysates were set as positive controls, and the GST pull-down components were a negative control. In this reaction, the UbcH5C-Ub (E2-Ub) conjugates were generated, while dissociated in the presence of DTT. D, Effect of purified DC-UbP on the diUb synthesis in vitro. The concentrations of UbE1 and E2-25K were 0.25 µM and 2 µM, respectively, while those of His6-DC-UbP and BSA were 80 µM. The reaction system (100 µL) included 0.25 mg Ub, 0.25 mg Ub-K48R and 2 mM ATP in a buffer of 50 mM Tris, 10 mM MgCl2, pH 8.0.
Figure 7.
Effects of DC-UbP overexpression on the association of UbE1 and USP5.
A, Co-IP for detecting the association of UbE1, USP5 and DC-UbP. Myc-USP5 was co-transfected with FLAG-DC-UbP or mock vector into HEK 293T cells and then precipitated with anti-Myc antibody. The endogenous UbE1 was monitored by anti-UbE1 antibody. B, As in (A), detected with Ub antibody. After IP, the Ub conjugate level and especially the 250-kDa species were increased when co-transfected with DC-UbP. *, IgG heavy chain. C, Effect of the F195A/R199A mutant of DC-UbP on the association of USP5 and UbE1. Due to partial removal of the FLAG tag during co-IP experiment, two bands for DC-UbP by an antibody against DC-UbP were observed. D, As in (C), effect of the DC-UbP mutants on the association of USP5 and UbE1. Myc-USP5 was co-transfected with FLAG-tagged WT or its mutants (F195A, R199A, and F195A/R199A) and analyzed by Western blotting. The gels were blotted with anti-Myc, anti-DC-UbP and anti-UbE1 antibodies, respectively. Due to proteolytic degradation, two bands were detected in the gel by anti-DC-UbP antibody.
Figure 8.
Overexpression of DC-UbP alters the total level of Ub conjugates in HEK 293T cells.
A, Effect of DC-UbP overexpression on the total level of Ub conjugates. The transfected plasmid for DC-UbP was 0.8 µg for one well of a 6-well plate. The USP5 and UbE1 levels remained unchanged. GAPDH expression worked as a reference. B, Dose-dependent assay for the level of the Ub conjugates with the increase of DC-UbP plasmid (0, 0.5, 1.0 and 2.0 µg DNA). HA-Ub was co-transfected with DC-UbP and blotted with anti-HA antibody. C, DC-UbP mutation caused a reduced effect on the level of Ub conjugates. The amount of plasmid for DC-UbP or its mutants was ∼0.6 µg. F/R, F195A/R199A. D, Dose-dependent assay for the Ub conjugation level with the increase of F195A/R199A.
Figure 9.
Effect of DC-UbP knockdown on the cellular ubiquitination level.
A, Knockdown of DC-UbP by siRNAs in HEK 293T cells. B, Detection of UbE1 after knockdown of DC-UbP. The Western blotting for the cell lysates was performed with an anti-UbE1 antibody. C, As in (B), detection of USP5 after knockdown of DC-UbP with an anti-USP5 antibody. D, As in (B), detection of the Ub conjugates after knockdown of DC-UbP with an anti-Ub antibody. The Ub conjugate level was reduced when DC-UbP was silent. GAPDH was applied for a loading control.
Figure 10.
Schematic representation for DC-UbP coupled ubiquitination and deubiquitination in cell.
In this cartoon, DC-UbP mediates USP5 and UbE1 to form a population of complex through transient interactions. This dynamic complex can integrate the functions of USP5 and UbE1. USP5 processes the unanchored polyUb chains or Ub precursors to constitute the free Ub pool, while UbE1 activates the Ub molecules from the pool for further conjugating to the substrates. Thus, DC-UbP reconciles the two opposite processes, ubiquitination and deubiquitination, through linking the UbE1 and USP5 enzymes.