H486R optineurin is defective in interaction with CYLD.

<p>Yeast strain PJ69-4A was co-transformed with wild-type optineurin or its mutants and CYLD. Transformants were grown on selection media lacking adenine to assay interaction. GST-optineurin, GST-H486R or GST alone bound to glutathione agarose beads were incubated with lysates of HeLa cells transfected with HA-CYLD. The bound proteins were eluted and immunoblotted with anti-HA antibodies. Western blot was done with optineurin antibody (lower panel) to confirm the identity of the GST fusion proteins. HeLa cells were co-transfected with HA-CYLD and Myc-OPTN or Myc-H486R. Lysates were immunoprecipitated with anti-HA antibody and subjected to western blotting. WCL, whole cell lysate.</p

C-terminal region of optineurin interacts with CYLD.

<p>Interaction of optineurin and its deletion constructs with CYLD was analysed using yeast two-hybrid method. Interaction is denoted by ‘+’. CC, coiled coil; UBD, ubiquitin binding domain; ZF, zinc finger. Yeast strain PJ69-4A was co-transformed with optineurin and CYLD or its deletion constructs. Transformants were grown on selection media lacking Ade to assay interaction. Growth on Ade⁻ plate indicates interaction. CAP, CAP-Gly domain; USP, ubiquitin specific protease. HeLa cells were transfected with HA-CYLD and CYLD was immunoprecipitated using HA tag antbody. Immunoprecipitates were analyzed by western blotting with HA and optineurin antibodies. OPTN, optineurin; WCL, whole cell lysate. HeLa cells were co-transfected with Myc-OPTN and HA-CYLD. CYLD was immunoprecipitated with HA antibody and immunoprecipitates were analyzed by western blotting with HA and Myc antibodies.</p

Optineurin is required for interaction of CYLD with RIP.

<p>HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 h of infection, cells were transfected with HA-tagged mutant (H871N) CYLD, treated with TNFα for 5 min after 30 h of transfection and subjected to immunoprecipitation with HA or control antibodies. Immunoprecipitates were then subjected to western blotting. A schematic representing the regulation of TNFα-induced NF-κB signalling by optineurin. Binding of TNFα to its receptor leads to assembly of a multimolecular complex on TNF receptor in which ubiquitination of RIP takes place. Then NEMO is recruited to ubiquitinated RIP. This leads to activation of IKK (left panel). Optineurin binds to ubiquitinated RIP possibly by displacing NEMO (middle panel) as suggested by Zhu et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017477#pone.0017477-Zhu1" target="_blank">[16]</a> and then recruits CYLD to the molecular complex thus facilitating deubiquitination of RIP by CYLD (right panel). In the absence of optineurin, CYLD is not recruited to ubiquitinated RIP resulting in accumulation of ubiquitinated RIP.</p

Effect of H486R optineurin on CYLD-dependent inhibition of TNFα-induced NF-κB activity.

<p>HeLa cells were transfected with optineurin or its mutants (100 ng) along with or without CYLD (100 ng, left panel and 50 ng, right panel). After 22 h of transfection, the cells were treated with TNFα for 4 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot showing the expression of optineurin and its mutants along with CYLD using HA tag antibody. Yeast strain PJ694A was co-transformed with optineurin or its H486R and D474N mutants and CYLD. Transformants were grown on selection media lacking Ade to assay interaction. Growth on Ade⁻ plate indicates interaction. GST-ubiquitin or GST alone bound to glutathione agarose beads were incubated with lysates of HEK293T cells transfected with wild type optineurin or its mutants. The bound proteins were eluted and immunoblotted with anti-HA antibodies. WCL, whole cell lysates. HeLa cells were infected with adenoviruses expressing HA-tagged wild-type or H486R mutant optineurin. After 30 hrs of infection, the cells were treated with TNFα for 5 min and immunoprecipitations were carried out with HA antibody and analyzed by Western blotting with RIP and HA antibodies.</p

H486R mutant does not inhibit TNFα- induced NF-κB activation.

<p>HeLa cells were transfected with optineurin or its H486R mutant (25, 50 or 100 ng) along with NF-κB reporter plasmid and, after 22 h, treated with TNFα for 4 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows expression of optineurin and its H486R mutant using HA tag antibody. HeLa cells were infected with adenoviruses for expressing HA tagged optineurin (Optn-AdV) or H486R (H486R-AdV) mutant or control virus (AdC). After 36 hours of infection, the cells were treated with TNFα for 5 min or left untreated. Cell lysates were then prepared for western blotting with antibodies for IκBα, HA tag and actin (loading control). GFP expression was used to monitor infection by adenoviruses. RGC-5 cells were transfected with optineurin or its H486R mutant (100 ng) along with NF-κB reporter plasmid and, after 22 h of transfection, treated with TNFα for 3 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows expression of optineurin and its H486R mutant using HA tag antibody.</p

Optineurin is required for CYLD mediated inhibition of NF-κB activation.

<p>HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection cells were transfected with NF-κB-Luc with or without CYLD (100 ng). The cells were treated with TNFα for 4 h after 22 h of transfection. Luciferase activities relative to untreated control are shown (n = 4). Western blot shows the expression of HA-CYLD in control and optineurin knockdown cells. RIP ubiquitination is enhanced in optineurin knockdown cells. HeLa cells were infected with Ad-shOPTN or with control (Cont-AdV) adenoviruses. After 72 h of infection cell lysates were subjected to immunoprecipitation with RIP antibody or control antibody. The immunoprecipitates were analyzed by western blotting. ns, non-specific. * indicates ubiquitinated RIP.</p

Optineurin is required for deubiquitination of RIP by CYLD.

<p>HeLa cells were infected with adenoviruses expressing shRNA against optineurin or with control adenoviruses. After 48 hrs of infection, cells were either left untransfected or transfected with HA- CYLD, treated with TNFα for 10 min after 30 h of transfection and subjected to immunoprecipitation with RIP or control antibodies. Immunoprecipitates were then subjected to western blotting with RIP antibody. The blot was then reprobed with Lys63-linked polyubiquitin (K63-Ub) antibody. HeLa cells were infected with adenoviruses expressing wild-type optineurin or H486R mutant. After 4 h of infection, cells were either left untransfected or transfected with HA- CYLD, treated with TNFα for 10 min after 24 h of transfection and subjected to immunoprecipitation with RIP or control antibodies. Immunoprecipitates were then subjected to western blotting with RIP antibody. The blot was then reprobed with Lys63-linked polyubiquitin (K63-Ub) antibody.</p

Optineurin and E50K mutant inhibit TNFα-induced NF-κB activity.

<p>(A) NF-κB-Luc reporter plasmid (25 ng) was transfected without or with optineurin expression plasmid (100 ng) in HeLa cells. After 22 hours of transfection cells were treated with TNFα for 4 hours. Luciferase activities relative to untreated control are shown (n = 3). (B) Western blot showing expression of optineurin and its mutants using HA tag antibody. Cdk2 was used as control. (C) Optineurin and E50K mutant inhibit TNFα-induced nuclear translocation of NF-κB p65. HeLa cells grown on coverslips were transfected with optineurin expression plasmid. After 24 hours of transfection cells were treated with TNFα for 30 min. The cells were then fixed and stained for optineurin (HA tag, FITC green) and p65 (Cy3, red) and visualized using a fluorescence microscope. (D) HeLa cells were infected with adenoviruses for expressing optineurin (Optn-AdV), its E50K mutant (E50K-AdV) or control virus (AdC). After 36 hours of infection, the cells were treated with TNFα for 6 min or 12 min or left untreated. Cell lysates were then prepared for western blotting with antibodies for IκBα, HA tag and Cdk2.</p

NF-κB mediates optineurin gene expression.

<p>(A) NF-κB p65 activates optineurin promoter through NF-κB site. Optineurin minimal promoter constructs pGL-DP or pGL-mDP (100 ng) were transfected without or with NF-κB p65 expression plasmid (100 ng) in A549 cells. After 24 hours of transfection cell lysates were prepared for reporter assays. Luciferase activities relative to control pGL-DP (taken as 1.0) are shown (n = 3). (B) IκBα inhibits TNFα-induced NF-κB activity. pGL-DP was transfected without or with IκBα super repressor expression plasmid (100 ng). TNFα was added 6 hours after transfection. Luciferase activities relative to control are shown (n = 3). (C) Blocking of NF-κB activation inhibits TNFα-induced optineurin gene expression. A549 cells were preincubated with 25 µM MG132 or solvent DMSO (0.1%) for 30 minutes prior to treatment with TNFα. After 6 hours of treatment with TNFα, RNA was isolated and the level of optineurin mRNA was determined by real time RT-PCR analysis. GAPDH was used as a control. (D) A549 cells were treated with MG132 as in panel C and after 6 hours of treatment with TNFα cell lysates were subjected to Western blotting. (E) A549 cells were treated with 100 µg/ml of SN-50 peptide for 30 minutes prior to treatment with TNFα for 6 hours. The picture shows RT-PCR analysis for optineurin gene expression.</p

Knockdown of optineurin by shRNA enhances TNFα-induced NF-κB activity.

<p>(A) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses and after 72 hours cell lysates were subjected to western blotting using antibodies for optineurin, IκBα and p65 NF-κB. Control virus (AdC sh) expresses shRNA of unrelated sequence of same length. (B) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses. After 48 h of infection these cells were transfected with NF-κB reporter plasmid (25 ng) along with β-galactosidase expression plasmid. After 22 hours of transfection the cells were treated with TNFα (10 ng/ml) for 4 hours. Cell lysates were then made for reporter assays. The data represent luciferase activities relative to untreated control taken as 1.0 (n = 3). (C) HeLa cells were infected with indicated adenoviruses and after 72 hours treated with TNFα for 6 min or left untreated. Cell lysates were subjected to western blot analysis using antibodies for IκB, optineurin and tubulin (loading control). AdC, control virus not expressing any shRNA.</p