UBQLN2 Promotes the Production of Type I Interferon via the TBK1-IRF3 Pathway

Abstract

Mutations of Ubiquilin 2 (UBQLN2) or TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). However, the mechanisms whereby UBQLN2 or TBK1 mutations lead to ALS and FTD remain unclear. Here, we explored the effect of UBQLN2 on TBK1 in HEK-293T cells or in CRISPR–Cas9-mediated IRF3 and IRF7 knockout (KO) cells. We found an interaction between TBK1 and UBQLN2, which was affected by ALS/FTD-linked mutations in TBK1 or UBQLN2. Co-expression of UBQLN2 with TBK1 elevated the protein level of TBK1 as well as the phosphorylation of TBK1 and IRF3 in a UBQLN2 dose-dependent manner, and this phosphorylation was reduced by mutant UBQLN2. In addition, the cellular production of IFN1 and related pro-inflammatory cytokines was substantially elevated when UBQLN2 and TBK1 were co-expressed, which was also decreased by mutant UBQLN2. Functional assay revealed that mutant UBQLN2 significantly reduced the binding affinity of TBK1 for its partners, including IRF3, (SQSTM1)/p62 and optineurin (OPTN). Moreover, complete loss of IRF3 abolished the induction of IFN1 and related pro-inflammatory cytokines enhanced by UBQLN2 in HEK-293T cells, whereas no significant change in IRF7 knockout cells was observed. Thus, our findings suggest that UBQLN2 promotes IRF3 phosphorylation via TBK1, leading to enhanced IFN1 induction, and also imply that the dysregulated TBK1-IRF3 pathway may play a role in UBQLN2-related neurodegeneration.

Impaired 26S Proteasome Assembly Precedes Neuronal Loss in Mutant UBQLN2 Rats

“…All rats were maintained on Sprague-Dawley background. As characterized previously [ 6 ], CaMKα2-tTA transgenic rats [ 31 ] were crossed with TRE-UBQLN2 P497H transgenic rats to generate bigenic rats. Breeding rats were fed doxycycline (DOX) (50 μg/mL) (Sigma, St Louis, MI, USA) in drinking water during embryonic development.…”

“…Plasmids were constructed as previously described [ 31 ], and a 3× FLAG tag was fused to the C-terminal of human UBQLN2. HEK-293T cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (ampicillin and streptomycin).…”

UBQLN proteins in health and disease with a focus on UBQLN2 in ALS/FTD

“…Previous studies had shown that upregulation of UBQLN2 regulates Nuclear Factor kappa-B signaling in inflammation and pathogenicity [175]. More recently Chen et al found UBQLN2 binds directly with TBK1, promoting phosphorylation and activation of the kinase and interferon signaling [174].…”

Regulation of antiviral innate immune signaling and viral evasion following viral genome sensing

“… Signaling molecule Classification Regulator Function Ref. TBK1 PTMs Positive MIB K63-linked ubiquitination 161 TBK1 K63-linked ubiquitination 154 Nrdp1 K63-linked ubiquitination 163 RNF128 K63-linked ubiquitination 162 USP1–UAF1 complex Deubiquitination 164 UBQLN2 Phosphorylation 159 Src Autophosphorylation 160 TBK1 Autophosphorylation 155 RKIP Autophosphorylation 158 GSK3β Self-association and autophosphorylation 157 Dnmt3a Recruitment of HDAC9 for deacetylation 165 HDAC3 Deacetylation 166 TRIM9 Recruitment of GSK3β for activation 157 Negative ASB8 K48-linked ubiquitination 172 USP38 K48-linked ubiquitination 176 DYRK2 K48-linked ubiquitination 174 THOC7 K48-linked ubiquitination 175 TRIP K48-linked ubiquitination 173 Siglec1 Recru...…”

“…TRIM9 short isoform (TRIM9s) facilitates the recruitment of GSK3β to TBK1 upon viral infection 157 , and Raf kinase inhibitory protein serves as a positive regulator 158 ; both of these proteins promote autophosphorylation of TBK1. Moreover, ubiquilin 2 (UBQLN2) promotes the stability and facilitates the phosphorylation of TBK1 159 , and Tyr179 (Y179) phosphorylation (catalyzed by the tyrosine kinase Src) is essential for the initiation of TBK1 autophosphorylation 160 . Ubiquitination also plays a critical role in the activation of TBK1.…”