X-linked Intellectual Disability Gene CUL4B Targets Jab1/CSN5for Degradation

Zou Yongxin et. al. used screening of candidate genes, identified CUL4B gene caused XLID (X-linked intellectual disability), then analyzed the localization and expression pattern of CUL4B and its role on the cell proliferation and cell cycle regulation. CUL4B encodes a scaffold protein that assembles a Cullin-RING (really interesting new gene) ubiquitin ligase (E3) complex in ubiquitylation. CUL4B E3complexes contain ROC1(RING-box protein1) and the adaptor protein DDB1(DNA damage binding1).Identifying the specific protein substrates for CUL4B by comparative proteomics, Xi Li et. al found differentially expressed proteins (Peroxiredoxin Ⅲ、 Triosephosphate isomerase1、Annexin A5、Jab1/CSN5) between the miNeg HEK293(Human Embryonic Kidney293cells) cells and miCUL4B HEK293cells. The study found that CUL4B ubiquitin ligase complexes targets PrxⅢ that as a ROS scavenger for degradation. There is a significant decrease in cellular reactive oxygen species (ROS) production in CUL4B-silenced cells. CUL4B-silenced cause PrxⅢ accumulation, and reduce a significant decrease in cellular ROS production. But proliferative, self-renewing multipotent neural progenitors maintain a high ROS status and are highly responsive to ROS stimulation. WDR5, another substrate of CUL4B E3ubiquitin ligase, may regulate neuronal gene expression and suggest epigenetic change.The specificity substrates of CUL4B E3ubiquitin ligase may play an important role in the development of the nervous system. Thus, investigation of additional target substrates of CUL4B E3ubiquitin ligase and their functional roles will be necessary to delineate the full range of physiological functions of CUL4B.Part I:CUL4B E3ligase targets Jabl/CSN5for polyubiquitination and degradationWhile there is only a single Cul4gene in lower organisms, two closely related paralogs, CUL4A and CUL4B, carry out the CUL4function in mammalian cells. CUL4is composed of C-terminal domain that binds the RING finger protein ROC1(also known as Rbx1) and the N-terminal domain allows for association with DDB1. CUL4B has a unique N-terminus that is149amino acids longer than CUL4A, suggesting that CUL4B may selectively ubiquitinate specifically recruited substrates. In order to study specifically substrates of CUL4B ubiquitin ligase and identify its association with XLID, we screen the substrates of CUL4B E3ubiquitin ligase in HEK293after RNAi-mediated CUL4B depletion. We found that Jabl/CSN5is a specifically substrate of CUL4B ubiquitin ligase.1) In HEK293cells, there is a significant increase of Jab1in CUL4B RNAi cells compared with that of control cells. At the same time, CUL4B-silencing resulted in a significant increase in Jabl half-life in treated cells compared with that observed in control cells. These results suggest that Jab1protein levels may be regulated by CUL4B E3ligase.2) In HEK293cells, treatment with an inhibitor of the26S proteasome, resulted in increased accumulation of Jab1, suggesting that the enhanced accumulation of Jab1results from inhibition of proteasomal degradation.3) In HEK293cells, Jabl could not interact directly with CUL4B, but endogenous Jabl was apparently present in anti-CUL4B immunoprecipitates.4) In HEK293cells, silencing DDB1or ROC1produced a significant increase in Jab1levels; in the presence of CHX resulted in a significant increase in the half-life of Jabl. However, Jabl protein levels and half-life were not affected by silencing of CUL4A. Integrity of DDB1-CUL4B-ROC1was further demonstrated to be indispensable for the degradation of Jab1.5) In vitro and vivo ubiquitination assay have verified that the Jabl/CSN5protein could be polyubiquitination by the CUL4B E3ubiquitin ligase. These assays can prove that the CUL4B E3ubiquitin ligase target Jabl for polyubiquitination and mediate the Jabl degradation.Collectively, the above results suggest that the integrity of DDB1-CUL4B-ROC1E3ligase is critical for Jabl degradation. CUL4A, however, does not appear to be involved in the degradation of Jabl. These data establish Jabl as an ubiquitination target for the CUL4B ubiquitin ligase complex.Part Ⅱ:CUL4B silencing enhances BMP signallingSmad7negatively regulate BMP signaling by interfering with the phosphorylation of R-Smads. Jabl/CSN5has been shown to bind to Smad7, and promote Smad7degradation, and enhance TGF-(3and BMP signaling. The impaired CUL4B-mediated degradation of Jab1enhances BMP signaling.1) In HEK293cells, CUL4B silencing was associated with increased Jabl, decreased Smad7and increased pSmad1/5/8levels. Importantly, the changes in Smad7and pSmad1/5/8concentrations caused by siCUL4B were offset in si(CUL4B+Jabl) cells, demonstrating that the reduction in Smad7of siCUL4B cells was indeed mediated by the up-regulation of Jab1.2) In HEK293cells, silencing of CUL4B significantly increased BMP-specific Idl luciferase reporter activities cells. And the upregulation of BMP signaling in response to CUL4B silencing could be partially rescued by co-depletion of Jab1, thus restoring BMP signaling to a level not significantly different from that observed in the control. These assays demonstrat that CUL4B silencing could enhance the BMP signaling. 3) In the Embryonic fibroblasts were derived from the Cul4b-deficient mice, the Jabl levels was increased, and the Smad7was decreased than those observed in normal cells. At the same time, we found that the level of pSmad1/5/8was increased. Idl and Msx-2mRNA levels in Cul4b-deficient MEFs were increased significantly.Collectively, the changes in BMP signalling concentrations caused by silence of CUL4B were offset by Jab1silencing. The expression of direct BMP-target gene was enhanced in Cul4b-deficient MEFs.All the Part Ⅰ and Part Ⅱ findings collectively demonstrate that Jabl is a novel proteolytic target for the CUL4B ubiquitin ligase complex, and the impaired CUL4B mediated abnormal upregulation of BMP signaling. This observation may provide novel insights into the molecular mechanisms underlying CUL4B-associated XLID pathogenesis.