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The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway.
BACKGROUND: 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. 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 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. 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.Biotechnology and Biological Science Research Council [BB/J007846/1]
Functional characterization of the interaction between E3 ubiquitin ligase SCF(Fbxo7) and UXT-V1 and UXT-V2 proteins
Fbxo7 é uma das 69 proteínas do tipo F-box em humanos que interage com SKP1, Culina1 e RBX1 para formar o complexo enzimático E3 ubiquitina ligase SCF(Fbxo7), que promove a ubiquitinação de seus substratos. A proteína Fbxo7 é superexpressa em uma variedade de linhagens celulares tumorais humanas, mas não nesses tecidos normais, e é descrita como um regulador negativo da via de sinalização de NF-κB por mediar a ubiquitinação de cIAP-1 e TRAF2. Em um estudo de identificação de substratos em larga escala, foi identificada a proteína UXT (Ubiquitously Expressed Transcript) como um potencial substrato de SCF(Fbxo7). Esta proteína pertence à família das prefoldinas (PFD) e é amplamente expressa em diversos tecidos humanos. Duas isoformas de UXT, UXT-V1 e UXT-V2, foram descritas com funções distintas, sendo a UXT-V2 um cofator transcricional que ativa a transcrição de NF-κB no núcleo e a UXT-V1 modulando a apoptose induzida por TNF no citoplasma. Combinamos as técnicas de co-imunoprecipitação, ubiquitinação in vitro e em células, análise de restrição de cadeia de ubiquitina, ensaio de estabilidade com cicloheximida, fracionamento celular e microscopia confocal para investigar a interação entre Fbxo7 e ambas as isoformas de UXT. Validamos a UXT-V1 e a UXT-V2 como substratos da E3 ubiquitina ligase SCF(Fbxo7) e mostramos que SCF(Fbxo7) realiza uma modificação pós-traducional para regulação de função na UXT-V1 e para degradação do substrato via proteassoma na UXT-V2. Demostramos que a superexpressão de UXT-V2, mas não de UXT-V1, recruta Fbxo7 do citosol para o núcleo das células para mediar a interação entre elas e observamos que as isoformas diferem em suas localizações subcelulares. Também mostramos que o knockdown de FBXO7 ou a superexpressão do mutante Fbxo7-ΔF-box protege a UXT-V2 da degradação proteassomal mediada por Fbxo7, aumentando a resposta dos repórteres da via do NF-κB. Neste trabalho, demonstramos que SCF(Fbxo7) promove a degradação proteassomal da UXT-V2, tendo por consequência funcional a inibição da via de sinalização do NF-κB e caracterizando um novo ponto de regulação da via. Por fim, resultados preliminares demonstraram que a UXT-V2 promove um aumento na ubiquitinação do Estrogen Receptor (ER) e, ao contrário, promove uma redução na ubiquitinação do Androgen Receptor (AR). A caracterização da interação entre Fbxo7/UXT com os receptores AR e ER será avaliada em ensaios posteriores.Fbxo7 is one of 69 F-box-like proteins in humans that interact with SKP1, Cullin1 and RBX1 to form the E3 ubiquitin ligase SCF(Fbxo7) enzyme complex, which promotes the ubiquitination of its substrates. The Fbxo7 protein is overexpressed in a variety of human tumor cell lines, but not in these normal tissues, and is described as a negative regulator of the NF-κB signaling pathway by mediating the ubiquitination of cIAP-1 and TRAF2. In a large-scale substrate identification study, the UXT (Ubiquitously Expressed Transcript) protein was identified as a potential substrate of SCF(Fbxo7). This protein belongs to the prefoldin family (PFD) and is widely expressed in many human tissues. Two isoforms of UXT, UXT-V1 and UXT-V2, have been described with distinct functions, UXT-V2 being a transcriptional cofactor that activates NF-κB transcription in the nucleus and UXT-V1 modulating TNF-induced apoptosis in the cytoplasm. We combined the techniques of co-immunoprecipitation, in vitro and in-cell ubiquitination, ubiquitin chain restriction analysis, cycloheximide stability assay, cell fractionation and confocal microscopy to investigate the interaction between Fbxo7 and both isoforms of UXT. We validated UXT-V1 and UXT-V2 as substrates of E3 ubiquitin ligase SCF(Fbxo7) and showed that SCF(Fbxo7) performs a functional regulation of UXT-V1 and proteasomal degradation of UXT-V2. We demonstrated that overexpression of UXT-V2, but not UXT-V1, recruits Fbxo7 from the cytosol to the cell nucleus to mediate the interaction between them, and we observed that the isoforms differ in their subcellular locations. We also showed that knockdown of FBXO7 or overexpression of the Fbxo7-ΔF-box mutant protects UXT-V2 from Fbxo7-mediated proteasomal degradation, increasing the responsiveness of NF-κB reporter genes. In this work, we demonstrate that SCF(Fbxo7) promotes the proteasomal degradation of UXT-V2, contributing to the inhibition of the NF-κB signaling pathway and characterizing a new point of regulation of the pathway. Finally, preliminary results showed that UXT-V2 promotes an increase in Estrogen Receptor (ER) ubiquitination and, on the contrary, reduces Androgen Receptor (AR) ubiquitination. The characterization of the interaction between Fbxo7/UXT with AR and ER receptors will be evaluated in further assays