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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
Biochimica et Biophysica Acta (BBA) - General Subjects ( IF 3 ) Pub Date : 2020-09-30 , DOI: 10.1016/j.bbagen.2020.129754
Valentine Spagnol , Caio A.B. Oliveira , Suzanne J. Randle , Patrícia M.S. Passos , Camila R.S.T.B. Correia , Natália B. Simaroli , Joice S. Oliveira , Tycho E.T. Mevissen , Ana Carla Medeiros , Marcelo D. Gomes , David Komander , Heike Laman , Felipe Roberti Teixeira

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.



中文翻译:

E3泛素连接酶SCF(Fbxo7)介导UXT亚型2(UXT-V2)的蛋白酶体降解,从而抑制NF-κB信号通路

背景

普遍表达的转录亚型2(UXT 单键V2)是一种前折叠蛋白样蛋白,参与NF-κB信号传导,细胞凋亡以及雄激素和雌激素反应。UXT-V2是NF-κB转录增强体中的辅助因子,其敲低抑制TNF-α诱导的NF-κB活化。Fbxo7是一种F-box蛋白,可与SKP1,Cullin1和RBX1蛋白相互作用,形成SCF(Fbxo7)E3泛素连接酶复合体。Fbxo7通过TRAF2和cIAP1泛素化负调节NF-κB信号传导。

方法

我们结合免疫共沉淀,泛素化在体外体内,放线菌酮大通化验,泛素链限制性分析和显微镜研究Fbxo7和过表达UXT-V2-HA之间的相互作用。

结果

Fbxo7的Ubl域有助于与UXT 单键V2进行交互。该底物在体外体内通过具有K48和K63泛素链连接的SCF(Fbxo7)进行聚泛素化。这种翻译后修饰降低了UXT-V2的稳定性,并促进了其蛋白酶体降解。我们进一步表明,UXT 单键V1是UXT的另一种剪接同工型,与UXT 单键V2相比,它在N端包含12个额外的氨基酸,也与Fbxo7相互作用并被其泛素化。此外,FBXO7敲低会促进UXT-V2积累,而Fbxo7-ΔF-box的过度表达可保护UXT-V2免受蛋白酶体降解,并增强NF-κB报告基因的反应性。我们发现UXT-V2与Fbxo7在细胞核中共定位。

结论

在一起,我们的研究表明SCF(Fbxo7)介导UXT-V2的蛋白酶体降解,导致NF-κB信号通路的抑制。

一般意义

发现E3泛素连接酶SCF(Fbxo7)的新底物有助于了解其在不同疾病(例如癌症和帕金森病)中的功能。

更新日期:2020-10-06
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