Related Experiment Video
Updated: Jul 16, 2025
Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
Published on: July 6, 2014
Pulse-SILAC and Interactomics Reveal Distinct DDB1-CUL4-Associated Factors, Cellular Functions, and Protein
Jennifer Raisch1, Marie-Line Dubois1, Marika Groleau2
1Département d'Immunologie et de Biologie cellulaire, faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada.
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.
Insights
Cullin-RING finger ligases, including the CUL4-DDB1 complex, regulate protein ubiquitination. This study identifies seven high-confidence DDB1/CUL4-associated factors (DCAFs) and characterizes their roles in protein targeting and degradation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Cullin-RING finger ligases are crucial for protein ubiquitination, targeting approximately 20% of cellular proteins for proteasomal degradation.
- The cullin 4 (CUL4)-DNA damage-binding protein 1 (DDB1) complex, a key ubiquitin ligase, utilizes substrate recognition adaptors called DDB1/CUL4-associated factors (DCAFs).
- DCAFs typically contain a WD40 domain and mediate the specificity of the CUL4-DDB1 complex, with over sixty WD40-containing proteins suggested as potential adaptors.
Purpose of the Study:
- To precisely define the association and classification of DCAFs with the DDB1/CUL4 complex.
- To identify novel DCAF partners and potential protein substrates.
- To elucidate the functional roles of DCAFs in regulating CUL4-DDB1 activity and cellular processes.
Main Methods:
- BioID and affinity purification-mass spectrometry were employed to identify and validate DCAFs interacting with DDB1.
- Pulse-stable isotope labeling with amino acids in cell culture (pSILAC) was used to measure changes in protein stability and degradation.
- Bioinformatics and structural analyses were utilized to investigate potential DCAF interactions.
Main Results:
- Seven WD40-containing proteins were confidently identified as DCAFs interacting with the DDB1/CUL4 complex.
- Changes in protein degradation were measured upon expression of each DCAF, revealing their roles in substrate targeting.
- New protein partners and potential substrates for the CUL4-DDB1 ligase were identified.
Conclusions:
- This study provides a high-confidence list of DCAFs, enhancing the understanding of the DDB1/CUL4-DCAF interactome.
- The identified DCAFs play significant roles in regulating CUL4-DDB1 complex activity and substrate specificity.
- Characterization of DCAF-mediated protein targeting and degradation offers new insights into cellular process regulation.
More Related Videos
07:03Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions
Published on: December 23, 2022
09:02Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
Published on: April 20, 2018
Related Concept Videos
02:04Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
01:57Protein Complexes with Interchangeable Parts
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order...
01:30Assembly of Signaling Complexes
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
02:24Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...