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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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Blockade of 14.7K-mediated TNFR1 internalization persists in optineurin-knockdown cells.Confocal microscopy of H1299 parental or H1299 14.7K expressing cells 48 h after transfection with 100 pmol optineurin-specific or control siRNA. Cells were labeled with biotin-TNF/strepavidin-FITC complexes for one hour on ice and analyzed immediately for TNFR1 internalization (pictures A, C, E, G) or shifted to 37°C for another hour before microscopy (pictures B, D, F, H). Pictures were acquired using a Zeiss LSM 510, magnification 630-fold. A representative experiment of three independent experiments is shown.
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pone-0038348-g008: Blockade of 14.7K-mediated TNFR1 internalization persists in optineurin-knockdown cells.Confocal microscopy of H1299 parental or H1299 14.7K expressing cells 48 h after transfection with 100 pmol optineurin-specific or control siRNA. Cells were labeled with biotin-TNF/strepavidin-FITC complexes for one hour on ice and analyzed immediately for TNFR1 internalization (pictures A, C, E, G) or shifted to 37°C for another hour before microscopy (pictures B, D, F, H). Pictures were acquired using a Zeiss LSM 510, magnification 630-fold. A representative experiment of three independent experiments is shown.

Mentions: The TNF-resistant phenotype of optineurin knockdown cells was also confirmed by the persistent blockade of TNFR1 internalization. Confocal microscopy of H1299 cells stably transfected with 14.7K wild-type protein exhibited blocked TNFR1 internalization, irrespective of the absence or presence of optineurin (Figure 8). Taken together, a significant contribution of optineurin to the 14.7K-mediated protective effect was unlikely.


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)

Blockade of 14.7K-mediated TNFR1 internalization persists in optineurin-knockdown cells.Confocal microscopy of H1299 parental or H1299 14.7K expressing cells 48 h after transfection with 100 pmol optineurin-specific or control siRNA. Cells were labeled with biotin-TNF/strepavidin-FITC complexes for one hour on ice and analyzed immediately for TNFR1 internalization (pictures A, C, E, G) or shifted to 37°C for another hour before microscopy (pictures B, D, F, H). Pictures were acquired using a Zeiss LSM 510, magnification 630-fold. A representative experiment of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038348-g008: Blockade of 14.7K-mediated TNFR1 internalization persists in optineurin-knockdown cells.Confocal microscopy of H1299 parental or H1299 14.7K expressing cells 48 h after transfection with 100 pmol optineurin-specific or control siRNA. Cells were labeled with biotin-TNF/strepavidin-FITC complexes for one hour on ice and analyzed immediately for TNFR1 internalization (pictures A, C, E, G) or shifted to 37°C for another hour before microscopy (pictures B, D, F, H). Pictures were acquired using a Zeiss LSM 510, magnification 630-fold. A representative experiment of three independent experiments is shown.
Mentions: The TNF-resistant phenotype of optineurin knockdown cells was also confirmed by the persistent blockade of TNFR1 internalization. Confocal microscopy of H1299 cells stably transfected with 14.7K wild-type protein exhibited blocked TNFR1 internalization, irrespective of the absence or presence of optineurin (Figure 8). Taken together, a significant contribution of optineurin to the 14.7K-mediated protective effect was unlikely.

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