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.
Highlights
Mutations in the autophagy receptor Ubiquilin 2 (UBQLN2) have been associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD) [1]
We explored the effect of UBQLN2 on TANK-binding kinase 1 (TBK1), and we found that over-expression of UBQLN2 increased TBK1 protein stability and TBK1 physically interacted with UBQLN2
We found that pathogenic mutations in either TBK1 (R47H substitution) or UBQLN2 (P497H and P506T substitution) enhanced their binding to one another (Figure 1A,B)
Summary
Mutations in the autophagy receptor Ubiquilin 2 (UBQLN2) have been associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD) [1]. The protein has an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), four heat-shock chaperonin-binding STI1 motifs, and a proline-rich disordered domain (PXX) [2]. Both UBL and UBA domains participate in interactions with proteasomes, and in autophagic protein degradation [3,4]. As a ubiquitin-like protein, it is known that most ALS/FTD-causing mutations in UBQLN2 are within the PXX region, leading to the impairment of its proteasomal function [5,6]. Recent reports have revealed that mutant UBQLN2 compromises autophagy in transgenic rodents [7,8,9], and stress granule formation are impaired by ALS/FTD-linked UBQLN2 mutations [10]
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