Dataset Information

Identification of protein substrates of the Cullin-RING E3 ubiquitin ligase CRL4 through its substrate recognition factors DCAFs

ABSTRACT: Cullin-RING finger Ligases (CRLs) represent the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through the proteasome, catalyzing the transfer of E2-loaded ubiquitin to a substrate. Eight Cullins were described in vertebrates and among them, CUL4 associates with DDB1 acting as an adaptor to form the CUL4-DDB1 ubiquitin ligase complex, involved in protein ubiquitination and regulation of many cellular processes. The specificity of CUL4-DDB1 is mediated by substrate recognition adaptors named DDB1/CUL4 associated factors (DCAFs), which are characterized by the presence of a short structural motif of approximately 40 amino acids terminating in a tryptophan (W)-aspartic acid (D) dipeptide, the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested at least 60 different WD40 containing proteins that could act as adaptors for the DDB1/CUL4 complex. In this study, we aimed to validate the DCAFs based on their interaction with DDB1 and to define new partners and potential substrates of each DCAF using affinity purification followed by mass spectrometry. Using BioID and AP-MS approaches, we confirmed seven WDR40 protein that can be considered as DCAF with a high confidence level. Because identification of protein substrates of E3-ubiquitin ligases is not always guaranteed by identifying protein interactions, changes in protein stability and protein degradation was measured by pulse-SILAC to identify the protein substrates targeted for proteasomal degradation by each DCAFs. In conclusion, this work provides new insights into the roles of DCAFs as substrate adaptors for the DDB1-CUL4 complex, identifies the proteins targeting and the cellular processes that are involved.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Disease Free

SUBMITTER: Dominique Levesque

LAB HEAD: François-Michel Boisvert

PROVIDER: PXD037482 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

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

Raisch Jennifer J Dubois Marie-Line ML Groleau Marika M Lévesque Dominique D Burger Thomas T Jurkovic Carla-Marie CM Brailly Romain R Marbach Gwendoline G McKenna Alyson A Barrette Catherine C Jacques Pierre-Étienne PÉ Boisvert François-Michel FM

Molecular & cellular proteomics : MCP 20230907 10

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 cell ...[more]

PMID: 37689310

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Project description:Adipose tissues play a pivotal role in energy homeostasis. They are existent in two forms: white adipose tissue and brown adipose tissue (BAT). Being the primary source of non-shivering thermogenesis of mammals, BAT plays an irreplaceable role in maintaining body temperature . Damaged DNA binding protein 1 (DDB1) is usually recognized as a component of the CUL4-DDB1 E3 complex. We have previously shown that DDB1 functions independently of CUL4 to promote adipogenesis. In the thesis, we focus on the function of DDB1 in mature adipocytes. We crossed Ddb1f/f mice with Adipoq-Cre or Ucp1-Cre mice to generate adipose tissue or BAT-specific mice, designated Ddb1-AKO and Ddb1-BKO mice respectively. At 8-12 weeks, BAT in these mice was servely whitened with decreased mitochdiral content. The thermogenic genes were also significantly downregulated at both RNA and protein levels. To further characterize the effect of DDB1 on thermogenesis, we first examined the bdy temperature of the pulps on postnatal day 3, and found that depletion of DDB1 caused a significant decrease in surface temperature.When the adult mice were subjected to cold challenge, both Ddb1-AKO and Ddb1-BKO mice showed dramatically decreased body temperature compared with control mice. At the end of the experiment, Ddb1-AKO and Ddb1-BKO mice had higher contents in BAT triglycerides and lower blood glucose level. Furthermore, the oxygen comsumption in Ddb1-AKO and Ddb1-BKO mice failed to respond to epinephrine injection. . All these results above demonstrated that lacking of DDB1 in brown adipocytes leads to a destruction in thermogenesis of BAT. We have also examined the phenotypes of Ddb1-AKO and Ddb1-BKO mice on high-fat-diet feeding. Compared with control mice, these mice showed decreased body weight, but significantly glucose intolerance and increased triglycierde content in liver, indicating that these mice have partial lipodystrophy. To study whether the effect of DDB1 was dependent on CUL4, we generated adipose tissue-specific Cul4a or CUl4b knockout mice, and found that these mice have no visible defect in BAT morphology compared with control mice, indicating a CUL4-independent function of DDB1. RNA-Seq analysis revealed that DDB1 is required for cold-induced expression of around 900 genes. ChIP-Seq analysis revelaed that these genes were subjected to regulation of promoter-proximally paused RNA polymerase II (Pol II), and that DDB1 is required for the release of paused Pol II upon cold stimulation. Our findings have thus revealed that DDB1 plays an important role in maintaining the theremogenic function of BAT by regulating the expression of thermogenic genes upon cold stimulation. Our studies will shed insights into transcriptional regulation of thermogenic gene

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	341_DCAF17_BioID_N1.raw	Raw
	341_IP_WDR59_N2.raw	Raw
	341_TLE3_BioID_N2.raw	Raw
	343_IP_WDR39_N1.raw	Raw
	343_IP_WDR57_N1.raw	Raw