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

Author(s):Show less -

Spagnol, Valentine ^[1] ; Oliveira, Caio A. B. ^[1] ; Randle, Suzanne J. ^[2] ; Passos, Patricia M. S. ^[1] ; Correia, Camila R. S. T. B. ^[1] ; Simaroli, Natalia B. ^[1] ; Oliveira, Joice S. ^[1] ; Mevissen, Tycho E. T. ^{[3, 4]} ; Medeiros, Ana Carla ^[5] ; Gomes, Marcelo D. ^[5] ; Komander, David ^{[3, 6]} ; Laman, Heike ^[2] ; Teixeira, Felipe Roberti ^[1]

Affiliation

^[1] Univ Fed Sao Carlos, Dept Genet & Evolut, Rod Washington Luis, Km 235, BR-13565905 Sao Carlos - Brazil

^[2] Univ Cambridge, Dept Pathol, Tennis Court Rd, Cambridge CB2 1QP - England

^[3] MRC Lab Mol Biol, Francis Crick Ave, Cambridge Biomed Campus, Cambridge CB2 0QH - England

^[4] Harvard Med Sch, Dept Biol Chem & Mol Pharmacol, 250 Longwood Ave, Boston, MA 02115 - USA

^[5] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Sao Paulo - Brazil

^[6] Walter & Eliza Hall Inst Med Res, Ubiquitin Signalling Div, 1G Royal Parade, Parkville, Vic 3052 - Australia

• Document type: Journal article
• Source: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS; v. 1865, n. 1 JAN 2021.
• Web of Science Citations: 0

Abstract

Background: Ubiquitously eXpressed Transcript isoform 2 (UXT-V2) is a prefoldin-like protein involved in NF-kappa B signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-kappa B transcriptional enhanceosome, and its knockdown inhibits TNF-alpha -induced NF-kappa B 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-kappa B 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 Ub1 domain of Fbxo7 contributes to interaction with UXT-V2. 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 UXT-V1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXT-V2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-Delta F-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-kappa B 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-kappa B 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. (AU)

• FAPESP's process: 16/25798-3 - Functional consequences of rearrangements to the FBXL17 gene detected in patient samples and cancer cell lines, on the E3 ubiquitin-ligase SCF(Fbxl17) activity
• Grantee: Felipe Roberti Teixeira
• Support type: Regular Research Grants
• FAPESP's process: 16/21310-6 - Regulations mechanisms of the Wnt signalling pathway by E3 ubiquitin ligase SCF(Fbxo7)
• Grantee: Caio Almeida Batista de Oliveira
• Support type: Scholarships in Brazil - Master
• FAPESP's process: 16/00792-2 - Functional characterization of the E3 ubiquitin-ligase SCF(Fbxo7/PARK15) in the oncogenic Wnt pathway and mitochondrial homeostasis
• Grantee: Felipe Roberti Teixeira
• Support type: Regular Research Grants
• FAPESP's process: 17/22153-4 - Identification of the ubiquitination sites in mitochondrial protein TOMM20 modified by the E3 ubiquitin-ligase SCF(Fbxo7)
• Grantee: Natália Borges Simaroli
• Support type: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 17/07879-9 - Does the protein UXT, which is a required for the NF-kB pathway, a substrate of the E3 ubiquitin-ligase SCF(Fbxo7)?
• Grantee: Valentine Spagnol
• Support type: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 18/09204-1 - Functional consequences of truncations in the Fbxl17 gene on the stability and activity of E3 ubiquitin ligase SCF (Fbx17)
• Grantee: Joice Silva de Oliveira
• Support type: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 19/03943-0 - Cloning of Fbxl17 domains in bacterial expression vectors
• Grantee: Camila Rolemberg Santana Travaglini Berti de Correia
• Support type: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 18/01308-2 - Genes Differentially Expressed During Reversion Skeletal Muscle Atrophy
• Grantee: Marcelo Damário Gomes
• Support type: Regular Research Grants