Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1.

Yoon Min,Eunyoung Chun,Mi-Jeong Kim,Sena Lee,Ki-Young Lee

doi:10.3389/fimmu.2017.01827

Abstract

Ubiquitin-specific protease 14 (USP14), one of three proteasome-associated deubiquitinating enzymes, has multifunctional roles in cellular context. Here, we report a novel molecular mechanism and function of USP14 in regulating autophagy induction and nuclear factor-kappa B (NF-κB) activation induced by toll-like receptor (TLR) 4 (TLR4). USP14 interacted with tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and interrupted the association of Beclin 1 with TRAF6, leading to inhibition of TRAF6-mediated ubiquitination of Beclin 1. Reduced expression of USP14 in USP14-knockdown (USP14KD) THP-1 cells enhanced autophagy induction upon TLR4 stimulation as shown by the increased conversion of cytosolic LC3-I to membrane-bound LC3-II. Moreover, USP14KD human breast carcinoma MDA-MB-231 cells and USP14KD human hepatic adenocarcinoma SK-HEP-1 cells showed increased cell migration and invasion, indicating that USP14 is negatively implicated in the cancer progression by the inhibition of autophagy induction. Furthermore, we found that USP14 interacted with TAK1-binding protein (TAB) 2 protein and induced deubiquitination of TAB 2, a key factor in the activation of NF-κB. Functionally, overexpression of USP14 suppressed TLR4-induced activation of NF-κB. In contrast, USP14KD THP-1 cells showed enhanced activation of NF-κB, NF-κB-dependent gene expression, and production of pro-inflammatory cytokines such as IL-6, IL-1β, and tumor necrosis factor-α. Taken together, our data demonstrate that USP14 can negatively regulate autophagy induction by inhibiting Beclin 1 ubiquitination, interrupting association between TRAF6 and Beclin 1, and affecting TLR4-induced activation of NF-κB through deubiquitination of TAB 2 protein.

Highlights

Ubiquitin-specific proteases (USPs) are deubiquitinating enzymes (DUBs) that play a special role in rescuing proteins from degra dation by trimming ubiquitin chains from their substrate-distal tips [1,2,3,4,5]
We demonstrated that Ubiquitin-specific protease 14 (USP14) induced deubiquitination of TAK1-binding protein (TAB) 2, a ubiquitination substrate of TRAF6, thereby suppressing the activation of TLR4-mediated signaling molecules such as TAK1 and IKKs, leading to inhibition of NF-κB activation upon TLR4 stimulation
FlagUSP14, Myc-Beclin 1 wild type and Myc-Beclin 1 truncated mutants were transiently transfected into HEK293T cells, and IP assay was performed with anti-Flag antibody

Summary

Introduction

Ubiquitin-specific proteases (USPs) are deubiquitinating enzymes (DUBs) that play a special role in rescuing proteins from degra dation by trimming ubiquitin chains from their substrate-distal tips [1,2,3,4,5]. Ubiquitin-specific protease 14 (USP14) as one of three proteasomeassociated DUBs is a major regulator of proteasomal degradation and implicated in the development and progression of several tumors [3, 8,9,10,11,12]. The downregulation of USP14 significantly inhibited breast cancer cell proliferation and metastasis [12]. A recent report has shown that deubiquitination of disheveled (Dvl) by USP14 is required for Wnt signaling [7]. USP14 regulated the ubiquitination of Dvl and its subsequent phosphorylation, which is essential for the activation of the downstream Wnt signaling [7]. It has been reported that USP14 regulates autophagy by suppressing K63 ubiquitination of Beclin 1 [13]. Activation of USP14 by Aktmediated phosphorylation modulated autophagy through controlling K63 deubiquitination of Beclin 1 [13]. The precise molecular mechanism by which USP14 suppresses Beclin 1 remains unclear

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Results

Conclusion