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E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin.

Klingseisen L, Ehrenschwender M, Heigl U, Wajant H, Hehlgans T, Schütze S, Schneider-Brachert W - PLoS ONE (2012)

Bottom Line: In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF.Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation.Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany.

ABSTRACT
Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

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Optineurin binding to 14.7K is not sufficient for protection against TNF.(A) H1299 cells expressing indicated 14.7K variants were seeded in triplicates in 96-well plates and treated with 12.5 µg/mL CHX and increasing amounts of TNF (0.001–10 ng/mL) the next day. Dead cells were removed by washing with PBS followed by staining of viable cells with crystal violet. (B) H1299 cells expressing either 14.7K, 14.7K mut 3 or untransfected cells were treated (right panel) or not (left panel) with CHX (12.5 µg/mL) followed by stimulation with 20 ng/mL TNF for 5 h. Lysates were analyzed by Western blotting with caspase-8 and caspase-3 antibodies. Tubulin was used as loading control.
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pone-0038348-g006: Optineurin binding to 14.7K is not sufficient for protection against TNF.(A) H1299 cells expressing indicated 14.7K variants were seeded in triplicates in 96-well plates and treated with 12.5 µg/mL CHX and increasing amounts of TNF (0.001–10 ng/mL) the next day. Dead cells were removed by washing with PBS followed by staining of viable cells with crystal violet. (B) H1299 cells expressing either 14.7K, 14.7K mut 3 or untransfected cells were treated (right panel) or not (left panel) with CHX (12.5 µg/mL) followed by stimulation with 20 ng/mL TNF for 5 h. Lysates were analyzed by Western blotting with caspase-8 and caspase-3 antibodies. Tubulin was used as loading control.

Mentions: Selected 14.7K mutants with retained optineurin binding capacity were stably expressed in H1299 cells and assessed for TNF-protection (Figure 6A). 14.7K mut 1 exhibited a comparable degree of protection as 14.7K wild-type protein. This made the construct unfeasible for tracing back TNF-resistance to either 14.7K or 14.7K-optineurin complex and it was therefore excluded. However, 14.7K mut 3 failed to rescue cells after challenge with TNF although optineurin binding was retained, making it a suitable candidate for further investigations.


E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin.

Klingseisen L, Ehrenschwender M, Heigl U, Wajant H, Hehlgans T, Schütze S, Schneider-Brachert W - PLoS ONE (2012)

Optineurin binding to 14.7K is not sufficient for protection against TNF.(A) H1299 cells expressing indicated 14.7K variants were seeded in triplicates in 96-well plates and treated with 12.5 µg/mL CHX and increasing amounts of TNF (0.001–10 ng/mL) the next day. Dead cells were removed by washing with PBS followed by staining of viable cells with crystal violet. (B) H1299 cells expressing either 14.7K, 14.7K mut 3 or untransfected cells were treated (right panel) or not (left panel) with CHX (12.5 µg/mL) followed by stimulation with 20 ng/mL TNF for 5 h. Lysates were analyzed by Western blotting with caspase-8 and caspase-3 antibodies. Tubulin was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366936&req=5

pone-0038348-g006: Optineurin binding to 14.7K is not sufficient for protection against TNF.(A) H1299 cells expressing indicated 14.7K variants were seeded in triplicates in 96-well plates and treated with 12.5 µg/mL CHX and increasing amounts of TNF (0.001–10 ng/mL) the next day. Dead cells were removed by washing with PBS followed by staining of viable cells with crystal violet. (B) H1299 cells expressing either 14.7K, 14.7K mut 3 or untransfected cells were treated (right panel) or not (left panel) with CHX (12.5 µg/mL) followed by stimulation with 20 ng/mL TNF for 5 h. Lysates were analyzed by Western blotting with caspase-8 and caspase-3 antibodies. Tubulin was used as loading control.
Mentions: Selected 14.7K mutants with retained optineurin binding capacity were stably expressed in H1299 cells and assessed for TNF-protection (Figure 6A). 14.7K mut 1 exhibited a comparable degree of protection as 14.7K wild-type protein. This made the construct unfeasible for tracing back TNF-resistance to either 14.7K or 14.7K-optineurin complex and it was therefore excluded. However, 14.7K mut 3 failed to rescue cells after challenge with TNF although optineurin binding was retained, making it a suitable candidate for further investigations.

Bottom Line: In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF.Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation.Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology and Hygiene, University of Regensburg, Regensburg, Germany.

ABSTRACT
Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

Show MeSH
Related in: MedlinePlus