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A Mental Retardation-linked Nonsense Mutation in Cereblon Is Rescued by Proteasome Inhibition*

  • Guoqiang Xu
    Correspondence
    To whom correspondence may be addressed: Dept. of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China. Tel.: 0086-512-6588-2723;
    Affiliations
    From the Department of Pharmacology, College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Translational Research for Neuro-Psycho-Diseases, Soochow University, Suzhou, Jiangsu 215123, China and

    the Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10065
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  • Xiaogang Jiang
    Affiliations
    From the Department of Pharmacology, College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Translational Research for Neuro-Psycho-Diseases, Soochow University, Suzhou, Jiangsu 215123, China and
    Search for articles by this author
  • Samie R. Jaffrey
    Correspondence
    To whom correspondence may be addressed: Dept. of Pharmacology, Weill Medical College of Cornell University, 1300 York Ave., Rm. LC-523, Box 70, New York, NY 10065. Tel.: 212-746-6243; Fax: 212-746-6241;
    Affiliations
    the Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10065
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants MH086128 from NIMH (to S. R. J.) and T32CA062948 from NCI (to G. X.). This work was also supported by the National High-tech Research and Development Program of China 973-projects (2012CB947602), National Natural Science Foundation of China Grant 31270874, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (to G. X.).
Open AccessPublished:August 27, 2013DOI:https://doi.org/10.1074/jbc.M113.472092
      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.
      Background: A nonsense mutation in cereblon, which results in the loss of the last 24 amino acids in the protein, causes mental retardation.
      Results: This mutant form of cereblon undergoes autoubiquitination by a CRL4 E3 ligase complex, leading to enhanced proteasomal degradation.
      Conclusion: The protein level of the mutant cereblon can be rescued by proteasome inhibition.
      Significance: These findings provide an approach for the treatment of mental retardation associated with this cereblon mutation.

      Introduction

      Mental retardation occurs in 1–3% of the population in the United States (
      • Centers for Disease Control and Prevention (CDC)
      State-specific rates of mental retardation–United States, 1993.
      ) and is currently untreatable. In most cases, the genetic causes of mental retardation are highly heterogeneous (
      • Chelly J.
      • Khelfaoui M.
      • Francis F.
      • Chérif B.
      • Bienvenu T.
      Genetics and pathophysiology of mental retardation.
      ); single gene mutations have been identified only in a few instances. Understanding how genetic lesions affect the function of the encoded proteins can suggest the potential therapeutic approaches.
      Recently, a new genetic cause for a mild form of mental retardation was discovered in five nuclear families (
      • Higgins J.J.
      • Pucilowska J.
      • Lombardi R.Q.
      • Rooney J.P.
      A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation.
      ). This form of mental retardation is otherwise nonsyndromic, exhibits an autosomal recessive pattern of inheritance, and is associated with an IQ between 50 and 70 (
      • Higgins J.J.
      • Rosen D.R.
      • Loveless J.M.
      • Clyman J.C.
      • Grau M.J.
      A gene for nonsyndromic mental retardation maps to chromosome 3p25-pter.
      ). Single strand polymorphism analysis and DNA sequencing showed that this form of mental retardation is caused by a single nucleotide mutation (C→T mutation) in the cereblon (CRBN) gene (
      • Higgins J.J.
      • Pucilowska J.
      • Lombardi R.Q.
      • Rooney J.P.
      A mutation in a novel ATP-dependent Lon protease gene in a kindred with mild mental retardation.
      ). This mutation introduces a premature stop codon, which terminates protein translation at arginine 419. The corresponding protein, CRBN R419X, lacks the last 24 amino acids compared with the full-length protein that contains 442 amino acids. Because the mutation exhibits an autosomal recessive pattern of inheritance, it is likely to inactivate the protein. However, it is unknown how this mutation affects the function of CRBN.
      CRBN is thought to function as a substrate receptor for the Cullin 4-RING ubiquitin ligase (CRL4) complex. CRL4 is a complex comprising CUL4A or CUL4B, DDB1 (damage-specific DNA-binding protein 1), and the RING finger protein ROC1 (
      • Jackson S.
      • Xiong Y.
      CRL4s: the CUL4-RING E3 ubiquitin ligases.
      ). DDB1 can interact with a variety of substrate receptors, including damage-specific DNA-binding protein 2 (DDB2) and VS5 V (
      • Angers S.
      • Li T.
      • Yi X.
      • MacCoss M.J.
      • Moon R.T.
      • Zheng N.
      Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.
      ,
      • Lin G.Y.
      • Paterson R.G.
      • Richardson C.D.
      • Lamb R.A.
      The V protein of the paramyxovirus SV5 interacts with damage-specific DNA-binding protein.
      ). Each of these substrate receptors is thought to couple the CRL4 complex to different target proteins. Ubiquitin is then transferred from an E2 ubiquitin-conjugating enzyme to target proteins. The crystal structures and biochemical experiments of two of these E3 ligases, CRL4-DDB2 (
      • Fischer E.S.
      • Scrima A.
      • Böhm K.
      • Matsumoto S.
      • Lingaraju G.M.
      • Faty M.
      • Yasuda T.
      • Cavadini S.
      • Wakasugi M.
      • Hanaoka F.
      • Iwai S.
      • Gut H.
      • Sugasawa K.
      • Thomä N.H.
      The molecular basis of CRL4DDB2/CSA ubiquitin ligase architecture, targeting, and activation.
      ) and CRL4-VS5 V (
      • Angers S.
      • Li T.
      • Yi X.
      • MacCoss M.J.
      • Moon R.T.
      • Zheng N.
      Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.
      ), demonstrated that DDB2 and VS5 V are the substrate receptors, which recognize a set of target proteins for ubiquitination. A structural analysis showed CRBN is one of the substrate receptors (
      • Angers S.
      • Li T.
      • Yi X.
      • MacCoss M.J.
      • Moon R.T.
      • Zheng N.
      Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery.
      ), which contains a DDB1 binding domain similar to those in DDB2 and VS5 V. Ito et al. (
      • Ito T.
      • Ando H.
      • Suzuki T.
      • Ogura T.
      • Hotta K.
      • Imamura Y.
      • Yamaguchi Y.
      • Handa H.
      Identification of a primary target of thalidomide teratogenicity.
      ) further confirmed that CRBN directly interacts with DDB1 and competes with DDB2 for binding with DDB1. Their data and our data in this work also showed that CRBN indeed forms a complex with ROC1, CUL4A, and DDB1. These results support the idea that CRBN is a substrate receptor of the CRL4 E3 ligase.
      It has been reported that CRBN can directly interact with several proteins, such as AMP-activated protein kinase (
      • Lee K.M.
      • Jo S.
      • Kim H.
      • Lee J.
      • Park C.S.
      Functional modulation of AMP-activated protein kinase by cereblon.
      ), the large conductance calcium-activated potassium channel (
      • Jo S.
      • Lee K.H.
      • Song S.
      • Jung Y.K.
      • Park C.S.
      Identification and functional characterization of cereblon as a binding protein for large-conductance calcium-activated potassium channel in rat brain.
      ), voltage-gated chloride channels (
      • Hohberger B.
      • Enz R.
      Cereblon is expressed in the retina and binds to voltage-gated chloride channels.
      ), and the 20 S core proteasome subunit β type 4 (
      • Lee K.M.
      • Lee J.
      • Park C.S.
      Cereblon inhibits proteasome activity by binding to the 20 S core proteasome subunit β type 4.
      ). None of these proteins has yet been shown to be targeted by CRL4 as a result of their binding to CRBN.
      Here, we explore the mechanism by which the premature stop codon in CRBN affects its function. The stop codon is not predicted to destabilize the CRBN mRNA by making it a target for nonsense-mediated decay. Instead, we show that CRBN R419X is expressed in cells and binds the CRL4 complex to mediate the ubiquitination of target proteins. However, we find that CRBN R419X exhibits markedly increased autoubiquitination and degrades faster than the wild type (WT) protein. Furthermore, we show that widely used proteasome inhibitors can restore levels of CRBN R419X in cells, suggesting a potential therapeutic strategy for this form of mental retardation.

      DISCUSSION

      Although the premature termination codon in CRBN has been linked to mild mental retardation, the mechanism by which this mutation affects CRBN has, until now, not been explored. In many cases, premature stop codons lead to reduced mRNA stability due to NMD. However, the R419X mutation is present in the final exon of CRBN and is therefore not expected to trigger NMD of the mutant transcript. Thus, the truncated protein is expected to be translated in cells. We show that this mutant protein is capable of forming a CRL4-CRBN E3 ligase and promoting the ubiquitination of different CRL4-CRBN targets. Our data indicate that the mutation affects CRBN function by promoting its autoubiquitination and degradation. As a result, the R419X mutation leads to CRBN deficiency in cells.
      Our data suggest that the R419X mutation interferes with the normal regulation of CRBN autoubiquitination. Autoubiquitination of E3 ligase components has important roles for the activity of these complexes (
      • Bosu D.R.
      • Kipreos E.T.
      Cullin-RING ubiquitin ligases: global regulation and activation cycles.
      ). Autoubiquitination of a substrate receptor and its subsequent degradation may be important to enable the replacement of one adaptor for another, enabling CRL4 to form other active E3 ligases (
      • Silva A.J.
      • Ehninger D.
      Adult reversal of cognitive phenotypes in neurodevelopmental disorders.
      ,
      • Zeier Z.
      • Kumar A.
      • Bodhinathan K.
      • Feller J.A.
      • Foster T.C.
      • Bloom D.C.
      Fragile X mental retardation protein replacement restores hippocampal synaptic function in a mouse model of fragile X syndrome.
      ,
      • Ehninger D.
      • Matynia A.
      • Silva A.J.
      Trafficking in emotions.
      ). The C terminus of CRBN seems to have an important role as an autoinhibitory domain that normally limits the ubiquitination rate of CRBN. The loss of the C-terminal 24 amino acids in CRBN R419X results in unrestrained ubiquitination. Because this ubiquitination constitutes Lys-48-linked polyubiquitin chains, this results in CRBN degradation. Thus, the C terminus comprises a ubiquitin autoinhibitory domain that is removed as a result of the R419X mutation. Further truncation of the mutant significantly reduces the ubiquitination level of the protein, which suggests the presence of a ubiquitination domain between amino acids 339 and 418. These results suggest that there are two functional domains, an autoinhibitory domain and a ubiquitination domain, at the C terminus of CRBN.
      It is important to note that it is still currently unclear how CRBN deficiency leads to mental retardation. Because of the subtle nature of the human phenotype, it is likely that it will be difficult to model this disease in cultured cells. Nevertheless, the disease is likely due to CRBN deficiency that is caused by enhanced CRL4-mediated autoubiquitination. The reduction in CRBN levels could affect neuronal function by either reducing the levels of CRL4-CRBN activity or by reducing the levels of CRL4-independent CRBN functions. Indeed, there is evidence that CRBN can influence the proteasome activity by binding one of its subunits (
      • Lee K.M.
      • Lee J.
      • Park C.S.
      Cereblon inhibits proteasome activity by binding to the 20 S core proteasome subunit β type 4.
      ), which may not be involved in CRL4. It will be important to determine the specific functional roles of CRBN to gain insight into how this protein influences neuronal function.
      Although our data suggest a potential route to increase CRBN levels, at present it is not known if restoration of CRBN levels in patients will result in improved cognitive performance. It is possible that CRBN deficiency during embryonic development results in neurodevelopmental abnormalities that cannot be restored by augmenting CRBN levels later in development. However, recent studies focusing on Fragile X mental retardation and tuberous sclerosis have suggested that postnatal restoration of protein function can restore some cognitive phenotypes (
      • Soucy T.A.
      • Smith P.G.
      • Milhollen M.A.
      • Berger A.J.
      • Gavin J.M.
      • Adhikari S.
      • Brownell J.E.
      • Burke K.E.
      • Cardin D.P.
      • Critchley S.
      • Cullis C.A.
      • Doucette A.
      • Garnsey J.J.
      • Gaulin J.L.
      • Gershman R.E.
      • Lublinsky A.R.
      • McDonald A.
      • Mizutani H.
      • Narayanan U.
      • Olhava E.J.
      • Peluso S.
      • Rezaei M.
      • Sintchak M.D.
      • Talreja T.
      • Thomas M.P.
      • Traore T.
      • Vyskocil S.
      • Weatherhead G.S.
      • Yu J.
      • Zhang J.
      • Dick L.R.
      • Claiborne C.F.
      • Rolfe M.
      • Bolen J.B.
      • Langston S.P.
      An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.
      ). Thus, it will be important to determine whether augmentation of CRBN levels can also improve cognitive performance in patients harboring CRBN mutations.
      Our results indicate that therapeutic strategies that prevent the autoubiquitination or degradation of CRBN can restore the levels of CRBN R419X. Our experiments focused on proteasome inhibitors, one of which is currently in use for treatment of multiple myeloma. However, more selective inhibitors are likely to have fewer side effects. Thalidomide has previously been shown to prevent the ubiquitination of CRBN. Because this drug binds to the C terminus of CRBN, which is affected in CRBN R419X, this drug likely is incapable of binding CRBN R419X and as a result cannot prevent its ubiquitination. Another approach could be to use selective inhibitors that target CRL4. However, currently there are no drugs that selectively target this complex. Nevertheless, a family of inhibitors that target other cullin-RING E3 ligases have been described (
      • Xu G.
      • Paige J.S.
      • Jaffrey S.R.
      Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling.
      ), suggesting that CRL4 inhibitors may also be possible for increasing CRBN R419X levels.

      Acknowledgments

      The lentiviral system was a generous gift from Livio Pellizzoni (Columbia University). The full-length and dominant negative CUL4As and His6-ubiquitin plasmids were from Pengbo Zhou (Weill Medical College of Cornell University). We are grateful to Michael S. Cohen and Alessia Deglincerti for assistance in molecular biology and plasmid transfection and Steven S. Gross (Weill Medical College) and Yuliang Ma (Weill Medical College) for helpful discussions in MS/MS analysis. The mass spectrometry work was performed at the Weill Medical College of Cornell University mass spectrometry core facility using instrumentation supported by National Institutes of Health Grants RR19355 and RR22615.

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