The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

Abstract

Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B

“…CRL4B was shown to promote the ubiquitination of the reactive oxygen species scavenging protein peroxiredoxin III (Li et al, 2011b). Recently, Chen et al reported that interference with CRL4 CRBN expression via RNAi or dominant-negative constructs stabilizes the voltage-gated chloride channel protein CLC-1, which is expressed in skeletal muscle cells and is sometimes mutated in patients with myotonia congenita (Chen et al, 2015). Moreover, CUL4B has been found to assemble a noncanonical CRL4 complex independent of any DCAF substrate receptor.…”

CLCN1 Myotonia congenita mutation with a variable pattern of inheritance suggests a novel mechanism of dominant myotonia

“…In other studies with the mutation R894X, the mRNA allelic expression ratio found in heterozygous members of a dominant pedigree was identical to that found in members of recessive pedigrees . We speculate that it is possible that mRNA levels do not correlate with the levels of the expressed protein, which may be controlled by different regulatory mechanisms, such as the recently identified ubiquitin ligases or molecular chaperones …”

“…6 We speculate that it is possible that mRNA levels do not correlate with the levels of the expressed protein, which may be controlled by different regulatory mechanisms, such as the recently identified ubiquitin ligases or molecular chaperones. 13,14 Thus, taking into account these results, we suggest that strategies aimed at increasing expression of the WT allele or reducing expression of the mutant allele may be beneficial for these patients.…”

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

“…Additionally, yeast two-hybrid screening has identified CRBN as a potential binding protein of the ClC channel, a voltagegated chloride channel that mediates degradation via the ubiquitin-proteasome dependent pathway [7,8]. Recently, we reported that CRBN is recruited to aggresomes and functions in cytoprotection against the ubiquitin-proteasome system-impaired condition [5].…”