The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway

https://doi.org/10.1016/j.bbagen.2020.129754Get rights and content
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Highlights

  • UXT-V2 is a canonical substrate of SCF(Fbxo7) E3 ubiquitin ligase.

  • Fbxo7 interacts with both UXT-V1 and UXTsingle bondV2.

  • UXT-V2 recruits Fbxo7 to the cell nuclei.

  • Fbxo7 inhibit NF-kB pathway through degradation of UXT-V2.

Abstract

Background

Ubiquitously eXpressed Transcript isoform 2 (UXTsingle bondV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination.

Methods

We combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA.

Results

The Ubl domain of Fbxo7 contributes to interaction with UXTsingle bondV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTsingle bondV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTsingle bondV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus.

Conclusions

Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway.

General significance

Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.

Keywords

E3 ubiquitin ligase
NF-kappa B (NF-κB)
SCF(Fbxo7)
UXT-V2
UXT-V1
Ubiquitylation (ubiquitination)

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