CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

Abstract

Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.

Regulation of ClC-2 Chloride Channel Proteostasis by Molecular Chaperones: Correction of Leukodystrophy-Associated Defect

“…Hsp90β is known to stabilize numerous types of client proteins, including E3 ubiquitin ligases that promote degradation of misfolded proteins [ 41 , 42 , 43 ]. For example, Hsp90β directly interacts with and is essential for stabilizing CUL4 [ 34 , 44 ], which serves as an essential component of the CUL4-DDB1-CRBN E3 ubiquitin ligase complex mediating ubiquitination and proteasomal degradation of ClC-2 channels [ 20 ]. Depending on the proteostatic role of Hsp90β in ER quality control, pharmacological suppression of Hsp90β activity may either enhance or reduce protein expression of its client proteins [ 34 , 45 , 46 , 47 ].…”

“…As summarized in the schematic model illustrated in Figure 10 , we propose that ClC-2 protein biogenesis at the ER is promoted by the Hsp90β-Hsc70 chaperone system associated with the co-chaperones HOP, Aha1, and FKBP8. In contrast, leukodystrophy-causing mutations tend to disrupt ClC-2 protein folding at the ER, leading to enhanced ClC-2 polyubiquitination by the CUL4-DDB1-CRBN E3 ubiquitin ligase complex and the ensuing proteasomal degradation [ 20 ].…”

“…To date, more than 10 disease-related CLCN2 mutations have been identified, including missense, insertion, and deletion mutations. Functional and biochemical analyses show that, in addition to altered voltage-dependent gating property and reduced Cl − current amplitude, leukodystrophy-causing ClC-2 mutant channels are associated with defective protein stability and impaired membrane trafficking [ 11 , 13 , 20 ], consistent with the presence of disease-related anomalous ClC-2 protein homeostasis (proteostasis). Proteostasis is largely maintained by multiple translational and post-translational mechanisms, thereby ensuring appropriate conformation, stability, and subcellular localization of proteins [ 21 , 22 ].…”

“…Elucidation of the interplay between ClC-2-related ER protein quality control systems and proteasomal degradation pathways is therefore essential for addressing the molecular pathophysiology of leukodystrophy. We demonstrated recently that the cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase complex promotes ubiquitination and ER-associated degradation of wild-type (WT) and disease-related mutant ClC-2 channels [ 20 ]. The molecular nature of the ER quality control system for ClC-2 protein, however, is still unclear.…”

NEDD4L Regulates the Proliferation and Metastasis of Non-small-cell Lung Cancer by Mediating CPNE1 Ubiquitination

“…Generally, there are three types of ubiquitination modi cation of substrate proteins: a single ubiquitin molecule labelling a single lysine site (single monoubiquitination), multiple ubiquitin molecules labelling a single lysine site (poly monoubiquitination), and ubiquitin chains labelling multiple different lysine residues (polyubiquitination) [31]. If a particular protein has been extensively polyubiquitinated before it is recognized by the proteasome, then the application of lysine-less ubiquitin (UB-K0) will reduce proteolysis [32]. As shown in Fig.…”