Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.

Susceptibility of pentylenetetrazole-induced seizures in mice with Cereblon gene knockout.

Epilepsy is a neurological disorder characterized by unpredictable seizures, which are bursts of electrical activity that temporarily affect the brain. Cereblon (CRBN), a DCAFs (DDB1 and CUL4-associated factors), is a well-established protein associated with human mental retardation. Being a substrate receptor of the cullin-RING E3 ubiquitin ligase (CRL) 4 complex, CRBN mediates ubiquitination of several substrates and conducts multiple biological processes. In the central nervous system, the largeconductance Ca2+-activated K+ (BKCa) channel, which is the substrate of CRBN, is an important regulator of epilepsy. Despite the functional role and importance of CRBN in the brain, direct injection of pentylenetetrazole (PTZ) to induce seizures in CRBN knock-out mice has not been challenged. In this study, we investigated the effect of PTZ in CRBN knock-out mice. Here, we demonstrate that, compared with WT mice, CRBN knock-out mice do not show the intensification of seizures by PTZ induction. Moreover, electroencephalography recordings were also performed in the brains of both WT and CRBN knockout mice to identify the absence of significant differences in the pattern of seizure activities. Consistently, immunoblot analysis for validating the protein level of the CRL4 complex containing CRBN (CRL4Crbn) in the mouse brain was carried out. Taken together, we found that the deficiency of CRBN does not affect PTZ-induced seizure. [BMB Reports 2020; 53(9): 484-489].

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity.

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn-/- mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn-/- mice. Increased stability of AMPKγ in Crbn-/- MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4CRBN directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4CRBN regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.