Spagnol V, Oliveira CAB, Randle SJ, Passos PMS, Correia CRSTB, Simaroli NB, Oliveira JS, Mevissen TET, Medeiros AC, Gomes MD, Komander D, Laman H, Teixeira FR

Ubiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-?B signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-?B transcriptional enhanceosome, and its knockdown inhibits TNF-? -induced NF-?B activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 ...

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proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-?B 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-?F-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-?B 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-?B signaling pathway.Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.

Date: Dec. 01, 2020

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