Pulse-SILAC and Interactomics Reveal Distinct DDB1-CUL4–Associated Factors, Cellular Functions, and Protein Substrates

• Published in: Molecular & cellular proteomics Vol. 22; no. 10; p. 100644
• Main Authors: Raisch, Jennifer, Dubois, Marie-Line, Groleau, Marika, Lévesque, Dominique, Burger, Thomas, Jurkovic, Carla-Marie, Brailly, Romain, Marbach, Gwendoline, McKenna, Alyson, Barrette, Catherine, Jacques, Pierre-Étienne, Boisvert, François-Michel
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.10.2023; American Society for Biochemistry and Molecular Biology
• Subjects: BioID; cullin4; DCAFs; DDB1; interactome; Life Sciences; pulse-SILAC; ubiquitination; BF; CUL4; MS; cullin4; MV; MEC; SILAC; ACN; BioID; DDB1; DCAFs; ubiquitination; POV; cDNA; interactome; DCAF; FA; pulse-SILAC; CRL; AP-MS; FC
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1016/j.mcpro.2023.100644

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. [Display omitted] •Analyzing DDB1 and DCAFs interactomes to define true DDB1/Cul4-associated factors.•Proteomic investigation of DCAFs degradome to identify protein substrates of CRL4.•DCAFbase allows easy visualization and browsing of each DCAFs results. Previous analyses revealed that DDB1 interacts with up to sixty DCAFs shown to serve as substrate receptors for the CRL4 complex. Several DCAFs have yet to be confirmed and their targets remain to be identified. We identified partners and substrates for each DCAFs by interactomics and pulse-SILAC labeling. We created an interface allowing visualization, where each result is accessible for all the DCAFs. The characterization of 58 DCAFs gives us a new insight into targets of the DDB1–CRL4 complex.