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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1865; no. 1; p. 129754
• Main Authors: Spagnol, Valentine, Oliveira, Caio A.B., Randle, Suzanne J., Passos, Patrícia M.S., Correia, Camila R.S.T.B., Simaroli, Natália B., Oliveira, Joice S., Mevissen, Tycho E.T., Medeiros, Ana Carla, Gomes, Marcelo D., Komander, David, Laman, Heike, Teixeira, Felipe Roberti
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2021; Elsevier
• Subjects: alternative splicing; androgens; apoptosis; Cell Cycle Proteins - metabolism; Cell Line, Tumor; cycloheximide; E3 ubiquitin ligase; estrogens; F-box proteins; F-Box Proteins - metabolism; HEK293 Cells; Humans; microscopy; Molecular Chaperones - metabolism; NF-kappa B (NF-κB); NF-kappa B - metabolism; precipitin tests; Proteasome Endopeptidase Complex - metabolism; Protein Isoforms - metabolism; Proteolysis; SCF(Fbxo7); Signal Transduction; SKP Cullin F-Box Protein Ligases - metabolism; transcription (genetics); ubiquitin; ubiquitin-protein ligase; Ubiquitination; Ubiquitylation (ubiquitination); UXT-V1; UXT-V2; UXT-V2; E3 ubiquitin ligase; SCF(Fbxo7); UXT-V1; Ubiquitylation (ubiquitination); NF-kappa B (NF-κB)
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129754

Ubiquitously eXpressed Transcript isoform 2 (UXTV2) 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. 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. The Ubl domain of Fbxo7 contributes to interaction with UXTV2. 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 UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, 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. Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway. Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson. •UXT-V2 is a canonical substrate of SCF(Fbxo7) E3 ubiquitin ligase.•Fbxo7 interacts with both UXT-V1 and UXTV2.•UXT-V2 recruits Fbxo7 to the cell nuclei.•Fbxo7 inhibit NF-kB pathway through degradation of UXT-V2.