Abstract
Cullin-RING finger ligases represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ∼20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven cullins are described in vertebrates. Among them, cullin 4 (CUL4) associates with DNA damage-binding protein 1 (DDB1) to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4-associated factors (DCAFs) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and affinity purification-mass spectrometry approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-stable isotope labeling with amino acids in cell culture to identify changes in protein degradation, following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved.