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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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Related in: MedlinePlus

Optineurin and 14.7K are recruited to TNFR1 complex.Parental KB cells and KB cells stably expressing 14.7K were transfected with 30 µg HA-tagged optineurin. 24 hours after transfection, cells were challenged with 50 ng/mL TNF (A) or biotinylated TNF (B) for 10 min or left untreated. Proteins associated with HA-tagged optineurin (A) or biotinylated TNF (B) were analyzed together with the corresponding lysates for the presence of TNFR1, optineurin, 14.7K and RIP1.
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pone-0038348-g001: Optineurin and 14.7K are recruited to TNFR1 complex.Parental KB cells and KB cells stably expressing 14.7K were transfected with 30 µg HA-tagged optineurin. 24 hours after transfection, cells were challenged with 50 ng/mL TNF (A) or biotinylated TNF (B) for 10 min or left untreated. Proteins associated with HA-tagged optineurin (A) or biotinylated TNF (B) were analyzed together with the corresponding lysates for the presence of TNFR1, optineurin, 14.7K and RIP1.

Mentions: Immunoprecipitation of 14.7K-optineurin complexes were performed in KB cells and stably transduced 14.7K expressing variants thereof (designated KB/14.7K). Before immunoprecipitation, cells were transfected with HA-tagged optineurin and 24 hours after transfection treated with 50 ng/mL TNF (Figure 1A) or biotinylated TNF (Figure 1B) for 10 min or left untreated. Cell lysates were subjected to immunoprecipitation with anti-HA agarose or streptavidin-coupled agarose. To control specificity when precipitating the ligand, 50 ng/mL biotin-TNF was added in excess to cell lysates from unstimulated cells.


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 and 14.7K are recruited to TNFR1 complex.Parental KB cells and KB cells stably expressing 14.7K were transfected with 30 µg HA-tagged optineurin. 24 hours after transfection, cells were challenged with 50 ng/mL TNF (A) or biotinylated TNF (B) for 10 min or left untreated. Proteins associated with HA-tagged optineurin (A) or biotinylated TNF (B) were analyzed together with the corresponding lysates for the presence of TNFR1, optineurin, 14.7K and RIP1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038348-g001: Optineurin and 14.7K are recruited to TNFR1 complex.Parental KB cells and KB cells stably expressing 14.7K were transfected with 30 µg HA-tagged optineurin. 24 hours after transfection, cells were challenged with 50 ng/mL TNF (A) or biotinylated TNF (B) for 10 min or left untreated. Proteins associated with HA-tagged optineurin (A) or biotinylated TNF (B) were analyzed together with the corresponding lysates for the presence of TNFR1, optineurin, 14.7K and RIP1.
Mentions: Immunoprecipitation of 14.7K-optineurin complexes were performed in KB cells and stably transduced 14.7K expressing variants thereof (designated KB/14.7K). Before immunoprecipitation, cells were transfected with HA-tagged optineurin and 24 hours after transfection treated with 50 ng/mL TNF (Figure 1A) or biotinylated TNF (Figure 1B) for 10 min or left untreated. Cell lysates were subjected to immunoprecipitation with anti-HA agarose or streptavidin-coupled agarose. To control specificity when precipitating the ligand, 50 ng/mL biotin-TNF was added in excess to cell lysates from unstimulated cells.

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