Fig 2: OPTN-UBAN domain is critical for linear ubiquitin binding.(a) The E478G mutation abrogated linear ubiquitin binding. In vitro MBP pull-down experiments using linear (M1)-, K48- or K63-linked tetraubiquitins and MBP-fused lacZ, OPTN-WT, E478G, Q398X and NEMO-WT were performed, and the bound ubiquitin chain was detected by immunoblotting. (b) Kinetic analyses of OPTN and linear ubiquitin. Linear tetraubiquitin was immobilized and various concentrations of OPTN-WT or E478G were tested. Grey lines, a global fit to a 1:1 interaction model. (c) Crystal structure of OPTN-UBAN in complex with linear diubiquitin (crystallized in the presence of linear tetraubiquitin). Each subunit of OPTN is coloured green and pink, respectively. The distal and proximal ubiquitin moieties are coloured purple and cyan, respectively. Phosphorylation sites, such as Ser65 in ubiquitin and Ser473 in OPTN, are coloured orange. (d) Superimposition of the structures of OPTN·linear diubiquitin and NEMO·linear diubiquitin (grey) (PDB 2ZVN). (e) Superimposition of the structures of OPTN·linear diubiquitin and NEMO·K63 diubiquitin (grey) (PDB 3JSV).

Fig 3: OPTN regulates TNFR signalling complex I.(a) Parental and OPTN-KO HeLa cells were stimulated with FLAG-TNF-α for the indicated times and then TNFR complex I was immunoprecipitated using an anti-FLAG antibody, followed by immunoblotting using the indicated antibodies. (b) Association of endogenous OPTN with free and/or conjugated linear ubiquitin chain on TNF-α stimulation. HEK293T cells were treated with FLAG-TNF-α and anti-OPTN immunoprecipitates were blotted with an anti-linear ubiquitin antibody. (c,d) Association of OPTN with RIP1 on TNF-α stimulation, examined at overexpressed (c) and endogenous (d) levels.

Fig 4: Enhanced NF-κB activation in OPTN-KO cells.(a) TNF-α- and IL-1β-induced NF-κB luciferase activities in OPTN-KO HeLa cells and their parental cells. (b) Enhanced NF-κB activation in TNF-α-treated OPTN-KO HeLa cells. Cells were treated with TNF-α for the indicated times and immunoblotting was performed using the indicated antibodies. (c) Accelerated TNF-α-induced nuclear translocation of p65 in OPTN-KO HeLa cells. (d) Quantitative PCR (qPCR) analyses of NF-κB targets in TNF-α-treated parental and OPTN-KO HeLa cells. Relative expression of target mRNAs was analysed using GAPDH as an internal standard. (e) Quantification of secreted IL-6 from TNF-α-treated parental and OPTN-KO HeLa cells. (f) qPCR analyses of NF-κB targets in CD40L-treated parental and OPTN-KO BJAB cells. (a,d,e,f) Mean±s.e.m. (n=3). Statistical significance was assessed using one-way analysis of variance. *P<0.001, **P<0.01 and ***P<0.05.

Fig 5: ALS-associated OPTN mutants fail to suppress NF-κB activity.(a) Domain structure of OPTN and disease-associated mutations. CC, coiled-coil; LZ, leucine zipper; LIR, LC3-interacting region; NZF, Npl4-type zinc finger; UBAN, ubiquitin binding in A20-binding IκB (ABIN) and NEMO proteins. Blue, ALS-associated mutations; pink, POAG-associated mutations. (b) Effects of WT and mutants of OPTN on LUBAC- and TNF-α-induced NF-κB activation were examined by luciferase assays in HEK293T cells. Expression of WT and mutants of FLAG-OPTNs is shown by immunoblotting. (c) Effects of WT and E478G mutant of OPTN on linear diubiquitin-conjugated NEMO were examined as in b. (b,c) Induction folds of NF-κB activity by luciferase assay are shown as mean±s.e.m. (n=3). Statistical significance was assessed using one-way analysis of variance. *P<0.001 and **P<0.01, NS, not significant. (d) WT-OPTN, but not the E478G mutant, binds to linear di-ubiquitinated NEMO. Immunoprecipitation and immunoblotting were performed as indicated.