A mental retardation-linked nonsense mutation in cereblon is rescued by proteasome inhibition

Abstract

A nonsense mutation in cereblon (CRBN) causes autosomal recessive nonsyndromic mental retardation. Cereblon is a substrate receptor for the Cullin-RING E3 ligase complex and couples the ubiquitin ligase to specific ubiquitination targets. The CRBN nonsense mutation (R419X) results in a protein lacking 24 amino acids at its C terminus. Although this mutation has been linked to mild mental retardation, the mechanism by which the mutation affects CRBN function is unknown. Here, we used biochemical and mass spectrometric approaches to explore the function of this mutant. We show that the protein retains its ability to assemble into a Cullin-RING E3 ligase complex and catalyzes the ubiquitination of CRBN-target proteins. However, we find that this mutant exhibits markedly increased levels of autoubiquitination and is more readily degraded by the proteasome than the wild type protein. We also show that the level of the mutant protein can be restored by a treatment of cells with a clinically utilized proteasome inhibitor, suggesting that this agent may be useful for the treatment of mental retardation associated with the CRBN R419X mutation. These data demonstrate that enhanced autoubiquitination and degradation account for the defect in CRBN activity that leads to mental retardation.

Keywords: Cereblon; E3 Ubiquitin Ligase; Genetic Diseases; Mental Retardation; Mutant; Nonsense Mutation; Proteasome Inhibition; Protein Degradation; Ubiquitin; Ubiquitination.

FIGURE 1.

cereblon (CRBN) R419X transcript is not predicted to undergo nonsense-mediated mRNA decay. The exon-intron structure of the cereblon gene is indicated. The R419X nonsense mutation is located in the final exon. In the wild type (WT) sequence (middle), the normal C nucleotide is underlined. In the mutant cereblon gene, a mutation of C to T results in the introduction of a stop codon (bottom). Because the mutation introduces a nonsense mutation in the final exon, the encoded transcript is not predicted to undergo nonsense-mediated decay. The encoded protein is predicted to be missing the last 24 amino acids. Asterisk represents the stop codon.

FIGURE 2.

CRBN R419X can assemble into a CRL4 E3 ligase complex. A, schematic illustration of the ubiquitin E3 ligase complex, CRL4-CRBN, composed of damage-specific DNA-binding protein 1 (DDB1), CUL4A, X-box protein (ROC1), and CRBN. CRBN is the substrate receptor that targets proteins for ubiquitination. The CRL4 complex (DDB1, CUL4A, and ROC1) can interact with diverse substrate receptors. B, CRBN R419X binds the CRL4 E3 ligase. To test whether the CRBN mutant forms an E3 ligase as is seen with WT CRBN, HEK293T cells were transfected with a plasmid expressing an N-terminal FLAG-StrepII-tagged full-length human CRBN (FS-CRBN) or the nonsense mutant, FS-CRBN R419X. The tagged CRBN was affinity-purified with Strep-Tactin beads that specifically interact with the StrepII tag under native conditions. The purified protein complex was separated on an SDS-PAGE, and the binding of endogenous CRL4 was measured by immunoblotting with anti-DDB1, anti-CUL4A, and anti-ROC1 antibodies. Both WT CRBN and CRBN R419X assemble into CRL4 complexes. WB, Western blot.

FIGURE 3.

CRBN R419X forms an active CRL4-CRBN E3 ligase. A, CRL4-CRBN ubiquitinates CRBN-interacting proteins, BK_Ca and AMPKα1. To test whether the CRBN R419X mutant can ubiquitinate CRL4-CRBN substrates, we sought to identify targets of the CRL4-CRBN E3 ligase complex. To do so, we asked whether potential targets exhibit increased ubiquitination upon expression of CRBN. HEK293T cells were transfected with plasmids expressing BK_Ca-3×FLAG-2×HA-His₆ (BK_Ca-tag) and AMPKα1–3×FLAG-2×HA-His₆ (AMPKα1-tag) in the absence or in the presence of StrepII-CRBN WT and R419X. The tagged BK_Ca and AMPKα1 were purified on TALON resin under denaturing conditions, and the ubiquitination level was determined by anti-ubiquitin (P4D1) immunoblotting. Upon cotransfection with WT and R419X CRBN, both BK_Ca and AMPKα1 exhibited a significant increase in their ubiquitination. This suggests that BK_Ca and AMPKα1 are substrates of the CRL4-CRBN E3 ligase and the premature termination mutation does not significantly affect the E3 ligase activity. B, quantification of the relative ubiquitination level of BK_Ca and AMPKα1 in the presence of WT or R419X CRBN. As with WT CRBN, CRBN R419X induces the ubiquitination of both BK_Ca and AMPKα1, indicating that it can form a functional CRL4-CRBN R419X E3 ligase complex. WB, Western blot.

FIGURE 4.

BK_Ca and AMPKα1 are ubiquitinated by the CRL4 E3 ligase. A, BK_Ca and AMPKα1 are ubiquitinated by CRL4 E3 ligases. BK_Ca-3×FLAG-2×HA-His₆ and AMPKα1–3×FLAG-2×HA-His₆ were coexpressed with WT CRBN in the absence or presence of a dominant negative CUL4A, CUL4A(Δ), in HEK293T, cells and the cells were treated with MG132 (10 μm, 16 h). The ubiquitinated BK_Ca and AMPKα1 were purified with TALON resin under denaturing conditions and blotted with an anti-ubiquitin antibody. FLAG Western blotting is used as a loading control. The results showed that their ubiquitination level is increased upon the expression of CRBN, and this increase is diminished when CUL4A(Δ) is expressed. B, quantification of the relative ubiquitination level of BK_Ca and AMPKα1 in the presence of WT CRBN or WT CRBN and CUL4A(Δ). These data indicate that the ubiquitination of BK_Ca and AMPKα1 by CRBN is due to its binding to the CRL4 E3 ligase. WB, Western blot.

FIGURE 5.

CRBN R419X is more susceptible to ubiquitination and degradation. A, CRBN R419X is more susceptible to ubiquitination. To test whether CRBN R419X exhibits altered levels of ubiquitination, HEK293T cells were transfected with FS-CRBN WT or FS-CRBN R419X and treated with a proteasome inhibitor, MG132 (10 μm, 3 h). The amount of ubiquitinated CRBN was detected by Strep-Tactin pulldown followed by anti-ubiquitin immunoblotting. The Western blot signal indicates that CRBN R419X exhibits higher levels of ubiquitination than WT CRBN. Anti-FLAG immunoblotting is shown as a loading control for the CRBN level. B, CRBN R419X protein levels significantly increase upon proteasome inhibition. To further test the idea that CRBN R419X is targeted for ubiquitination to a larger degree than WT CRBN, we asked if CRBN R419X protein levels exhibit increases upon proteasome inhibition. HEK293T cells were transfected with FS-CRBN WT or FS-CRBN R419X and then treated with a proteasome inhibitor, MG132 (10 μm, 40 h). Cells lysate was blotted with an anti-FLAG antibody. WT CRBN levels are slightly increased after proteasome inhibition, whereas CRBN R419X levels exhibit marked increases upon proteasome inhibition and can rise to a similar level as the WT counterpart. C, CRBN R419X is more labile in cells. The increased ubiquitination of CRBN R419X suggests that it might be more efficiently targeted for proteasomal degradation. To test this, HEK293T cells expressing WT and R419X CRBN were grown for 2 days and then treated with 100 μm cycloheximide to block protein synthesis. Cell lysates were harvested at the indicated time points, and the remaining amount of CRBN was determined by quantitative anti-FLAG Western blotting (WB). The results shown are an average from experiments repeated three times. R419X CRBN exhibits a significantly shorter half-life than the WT protein. D, CRBN R419X mutant is ubiquitinated much faster than the WT protein. Our data suggest that CRBN R419X is more rapidly or efficiently ubiquitinated by CRL4. We examined this by measuring ubiquitin levels in WT and R419X CRBN. The FS-tagged WT and R419X CRBN were expressed in HEK293T cells by calcium phosphate transfection. Cells were treated with MG132 (10 μm) for the indicated time and lysed in denaturing conditions. The tagged proteins were purified by Strep-Tactin beads and immunoblotted with an anti-ubiquitin antibody. As can be seen, R419X exhibits higher levels of ubiquitination at earlier time points than WT.

FIGURE 6.

Degradation and ubiquitination of CRBN R419X is mediated by CRL4. A, CRBN R419X expression level is regulated by cullin-RING E3 ligases. To test whether the CRL4 complex regulates CRBN itself, we monitored CRBN R419X levels upon activation or inhibition of CRL4. In these experiments, CRBN R419X was coexpressed with the full-length (FL) and dominant negative CUL4A, CUL4A(Δ), and CRBN R419X levels were determined by anti-FLAG immunoblotting. Expression of full-length CUL4A results in a near total loss of CRBN R419X. However, inhibition of the CRL4 complex with CUL4A(Δ) results in an increase in CRBN R419X levels. β-Actin levels are shown as a loading control, and anti-CUL4A immunoblotting is used to show the expression of the CUL4A constructs. Note: the weak signal at ∼75 kDa in the 1st and 3rd lanes of the middle image is from the endogenous CUL4A. B, ubiquitination of CRBN R419X is mediated by CRL4. To further test whether the ubiquitination of CRBNR419X is mediated by CRL4s, we monitored CRBN R419X ubiquitination following activation and inhibition of CRL4 using full-length and dominant negative CUL4A. In these experiments, cells were treated for 3 h with 10 μm MG132 to allow accumulation of ubiquitinated CRBN R419X. CRBN R419X was purified by Strep-Tactin and blotted with anti-ubiquitin and anti-FLAG antibodies. The ubiquitination level of CRBN R419X is increased upon expression of full-length CUL4A, although it is reduced when coexpressed with the dominant negative CUL4A. These results suggest that CRBN R419X ubiquitination and degradation is mediated by CRL4s, presumably when CRBN R419X is bound in a CRL4-CRBN R419X complex. C, CRBN R419X is ubiquitinated with Lys-48-linkage specific polyubiquitin chains. The FS-CRBN R419X was expressed in HEK293T cells. Cells were treated with either DMSO or MG132 and lysed in denaturing conditions. The tagged proteins were purified by Strep-Tactin beads and immunoblotted with a ubiquitin antibody recognizing Lys-48 polyubiquitin chain (clone, Apu2). Upon MG132 treatment, the Lys-48-linked polyubiquitin chain on CRBN is significantly increased. D, CRBN R419X ubiquitination is reduced upon coexpressing a K48R-ubiquitin mutant. FLAG-StrepII-tagged CRBN R419X was coexpressed with a control plasmid, WT-Ub, K48R-Ub, or K63R-Ub, and purified with Strep-Tactin beads. The ubiquitinated proteins were blotted with an anti-ubiquitin antibody. FLAG blotting was used as a loading control. Upon coexpression of K48R-Ub, CRBN R419X exhibited slightly reduced ubiquitination, although its ubiquitination level is increased when the WT and K63R-Ub are expressed. Note: the ubiquitination of the 1st lane, with a control plasmid, and the residual ubiquitination in the 3rd lane are from the endogenous ubiquitin. These data support the idea that the ubiquitination in CRBN R419X is indeed with the Lys-48-linked polyubiquitin chains and leads to its degradation. E, quantification of CRBN R419X ubiquitination in D. F, localization of R419X CRBN is not significantly altered upon proteasome inhibition. To test whether the ubiquitination causes the change of subcellular localization of CRBN R419X, we used HEK293 cells expressing WT and R419X CRBN for immunostaining with an anti-FLAG antibody after DMSO or MG132 treatment (10 μm, 4 h). The subcellular localization of the WT and R419X CRBN is not changed upon the MG132 treatment in HEK293T cells. Red, FLAG (CRBN WT or R419X mutant); blue, DAPI for nuclei; scale bar, 10 μm. WB, Western blot.

FIGURE 7.

CRBN R419X exhibits increased ubiquitination in its C-terminal domain. A, MS/MS spectra of the ubiquitin remnant-containing peptides derived from CRBN. Two ubiquitination sites, Lys-300 and Lys-413, were identified from ubiquitin-remnant profiling experiments. The b- and y-ions are labeled in the MS/MS spectra and in the peptide sequences. The ubiquitinated lysine residues are indicated. The Spectrum Mill search score, the percentage of the scored peak intensity, measured mass, charge state, and Δmass for the peptide in the top panel are 11.39, 68.9%, 1453.8857 Da, +3, 0.6 ppm; those for the peptide in the bottom panel are 11.28, 66.0%, 1579.7831 Da, +3, 9.2 ppm. B, schematic illustration of the ubiquitination sites in CRBN that were identified by tandem MS from our experiments and previous proteomic experiments. Besides Lys-300 and Lys-413, other ubiquitination sites, such as Lys-43, Lys-166, and Lys-269, were identified. The C-terminal truncation introduced by the R419X mutation in CRBN is indicated with a jagged line, and the thalidomide (Thal)-binding domain (339–442) is shown. C, 339–418 domain is the major ubiquitination domain in CRBN. To understand which portion of CRBN exhibits increased ubiquitination as a result of the R419X mutation, we monitored ubiquitination in CRBN mutants truncated at different positions in the protein. HEK293T cells were transfected with plasmids expressing FS-CRBN WT, FS-CRBN R419X, or FS-CRBN(1–338) and then treated with 10 μm MG132 for 3 h. Cells were lysed, and ubiquitin levels in the CRBN constructs were assessed in the Strep-Tactin pulldown by anti-ubiquitin Western blotting. The R419X protein is readily ubiquitinated, but this is lost in the 1–338 construct that lacks the C-terminal ubiquitination site. These data indicate these lysine residues within residues 339–418 exhibit increased ubiquitination in the R419X mutant. D, CRBN(1–338) mutant can interact with DDB1 to form an E3 ligase complex. C shows that the CRBN(1–338) mutant is not efficiently ubiquitinated. We thought this could be due to its inability to bind CRL4. To rule this out, we tested whether this mutant can still bind its interacting partner DDB1. FS-tagged WT (WT) and CRBN(1–338) were expressed in HEK293T cells. Cells were lysed using native conditions. The tagged proteins were purified by Strep-Tactin beads under native conditions and immunoblotted with DDB1 and FLAG antibodies. The experiment shows that CRBN(1–338) can interact with DDB1 at a similar level as that of the WT CRBN, which suggests that the reduction of ubiquitination of CRBN(1–338) is indeed through the reduction of ubiquitination on the lysine residues but not the inability to form a CRL4 E3 ligase complex. Note: the experiments were done in parallel and imaged simultaneously.

FIGURE 8.

Restoration of CRBN R419X levels by bortezomib. A, thalidomide (Thal) inhibits the ubiquitination of WT CRBN but not CRBN R419X. To test whether thalidomide could block the enhanced autoubiquitination of CRBN R419X, HEK293T cells expressing either WT or R419X CRBN were treated with DMSO, 10 or 100 μm thalidomide for 1 h. Ubiquitinated protein was then allowed to accumulate by treating cells with 10 μm MG132 for 3 h. CRBN was purified with Strep-Tactin beads under denaturing conditions and immunoblotted with anti-ubiquitin or anti-FLAG antibody (loading control). This experiment showed that the ubiquitination level of WT CRBN is reduced by thalidomide treatment, but the ubiquitination level of CRBN R419X was unaffected by thalidomide. B, quantification of the relative ubiquitination level after thalidomide treatment for WT CRBN and R419X mutant. C, clinically utilized proteasome inhibitor, bortezomib, significantly increases the cellular level of CRBN R419X. To test whether bortezomib inhibits CRBN degradation, HEK293T cells were transfected with WT and R419X CRBN and treated with DMSO or bortezomib (Btz, 1 μm) for 24 h. Lysates were separated by SDS-PAGE, and CRBN levels were determined by immunoblotting with an anti-FLAG antibody. The Western blot (WB) showed that bortezomib slightly increases the protein level of WT CRBN, although it significantly increases the level of CRBN R419X. The right panels show the relative quantification of the WT and R419X CRBN after bortezomib treatment. These data suggest that bortezomib may be useful for restoring CRBN R419X levels in patients with this mutation.