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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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14.7K-mediated protection against TNF cytotoxicity and recruitment to TNFR1 is independent from endogenous optineurin.(A) Knockdown efficacy of optineurin-specific siRNA was confirmed by Western blotting and lasted at least for 6 days (B) H1299 parental or 14.7K expressing cells transfected with 100 pmol optineurin specific or control siRNA were seeded in 96-well plates and after 48 h challenged with increasing amounts of TNF (0.001–1 ng/mL) in the presence of CHX (12.5 µg/mL). Cell viability was assayed by staining with crystal violet. Knockdown of endogenous protein was confirmed by Western blotting. (C) Cells were seeded in a 10 cm culture plate, transfected with 600 pmol optineurin-specific siRNA or control siRNA and treated with 50 ng/mL biotin-labeled TNF 24 hours after transfection. Proteins associated with biotinylated TNF were analyzed for the presence of TNFR1 and 14.7K. Cell lysates confirmed efficient knockdown of optineurin in the corresponding samples.
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pone-0038348-g007: 14.7K-mediated protection against TNF cytotoxicity and recruitment to TNFR1 is independent from endogenous optineurin.(A) Knockdown efficacy of optineurin-specific siRNA was confirmed by Western blotting and lasted at least for 6 days (B) H1299 parental or 14.7K expressing cells transfected with 100 pmol optineurin specific or control siRNA were seeded in 96-well plates and after 48 h challenged with increasing amounts of TNF (0.001–1 ng/mL) in the presence of CHX (12.5 µg/mL). Cell viability was assayed by staining with crystal violet. Knockdown of endogenous protein was confirmed by Western blotting. (C) Cells were seeded in a 10 cm culture plate, transfected with 600 pmol optineurin-specific siRNA or control siRNA and treated with 50 ng/mL biotin-labeled TNF 24 hours after transfection. Proteins associated with biotinylated TNF were analyzed for the presence of TNFR1 and 14.7K. Cell lysates confirmed efficient knockdown of optineurin in the corresponding samples.

Mentions: We therefore used siRNA-mediated knockdown of optineurin to investigate the impact on 14.7K-mediated TNF-resistance. Efficacy and duration of siRNA-mediated optineurin knockdown was measured and lasted at least over a period of 6 days (Figure 7A). H1299 and H1299/14.7K cells treated with optineurin-specific or control siRNA were challenged in a cytotoxicity assay with increasing amounts of TNF in the presence of the sensitizing agent CHX (Figure 7B). Knockdown efficacy of siRNA transfection was ensured by Western blotting. H1299 cells expressing 14.7K were protected against TNF-mediated cytotoxicity in the presence and absence of optineurin, excluding an essential contribution of optineurin to the protective effect. Although knockdown of optineurin led to a slight, statistically not significant (p>0.05, Kruskal-Wallis test) loss of cell viability upon TNF treatment (Figure 7B, approx. 9% in H1299 parental cells and 13% in H1299/14.7K cells), wildtype 14.7K expressing cells were clearly protected against cytotoxic effects of TNF in the absence of optineurin. As TNF cytolysis was evident in H1299 parental cells in absence of optineurin, it was likely that this molecule is not involved in TNFR1-associated apoptotic mechanisms either.


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)

14.7K-mediated protection against TNF cytotoxicity and recruitment to TNFR1 is independent from endogenous optineurin.(A) Knockdown efficacy of optineurin-specific siRNA was confirmed by Western blotting and lasted at least for 6 days (B) H1299 parental or 14.7K expressing cells transfected with 100 pmol optineurin specific or control siRNA were seeded in 96-well plates and after 48 h challenged with increasing amounts of TNF (0.001–1 ng/mL) in the presence of CHX (12.5 µg/mL). Cell viability was assayed by staining with crystal violet. Knockdown of endogenous protein was confirmed by Western blotting. (C) Cells were seeded in a 10 cm culture plate, transfected with 600 pmol optineurin-specific siRNA or control siRNA and treated with 50 ng/mL biotin-labeled TNF 24 hours after transfection. Proteins associated with biotinylated TNF were analyzed for the presence of TNFR1 and 14.7K. Cell lysates confirmed efficient knockdown of optineurin in the corresponding samples.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366936&req=5

pone-0038348-g007: 14.7K-mediated protection against TNF cytotoxicity and recruitment to TNFR1 is independent from endogenous optineurin.(A) Knockdown efficacy of optineurin-specific siRNA was confirmed by Western blotting and lasted at least for 6 days (B) H1299 parental or 14.7K expressing cells transfected with 100 pmol optineurin specific or control siRNA were seeded in 96-well plates and after 48 h challenged with increasing amounts of TNF (0.001–1 ng/mL) in the presence of CHX (12.5 µg/mL). Cell viability was assayed by staining with crystal violet. Knockdown of endogenous protein was confirmed by Western blotting. (C) Cells were seeded in a 10 cm culture plate, transfected with 600 pmol optineurin-specific siRNA or control siRNA and treated with 50 ng/mL biotin-labeled TNF 24 hours after transfection. Proteins associated with biotinylated TNF were analyzed for the presence of TNFR1 and 14.7K. Cell lysates confirmed efficient knockdown of optineurin in the corresponding samples.
Mentions: We therefore used siRNA-mediated knockdown of optineurin to investigate the impact on 14.7K-mediated TNF-resistance. Efficacy and duration of siRNA-mediated optineurin knockdown was measured and lasted at least over a period of 6 days (Figure 7A). H1299 and H1299/14.7K cells treated with optineurin-specific or control siRNA were challenged in a cytotoxicity assay with increasing amounts of TNF in the presence of the sensitizing agent CHX (Figure 7B). Knockdown efficacy of siRNA transfection was ensured by Western blotting. H1299 cells expressing 14.7K were protected against TNF-mediated cytotoxicity in the presence and absence of optineurin, excluding an essential contribution of optineurin to the protective effect. Although knockdown of optineurin led to a slight, statistically not significant (p>0.05, Kruskal-Wallis test) loss of cell viability upon TNF treatment (Figure 7B, approx. 9% in H1299 parental cells and 13% in H1299/14.7K cells), wildtype 14.7K expressing cells were clearly protected against cytotoxic effects of TNF in the absence of optineurin. As TNF cytolysis was evident in H1299 parental cells in absence of optineurin, it was likely that this molecule is not involved in TNFR1-associated apoptotic mechanisms either.

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