Limits...
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.

Show MeSH

Related in: MedlinePlus

Generation of sequential mutants of 14.7K.The top row gives the names of the generated mutants. The bottom row displays the amino acid sequence of 14.7K replaced by a five amino acid Flag-tag (DYKDE). The arrow points to the C119S substitution, which was designated as 14.7K PM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366936&req=5

pone-0038348-g004: Generation of sequential mutants of 14.7K.The top row gives the names of the generated mutants. The bottom row displays the amino acid sequence of 14.7K replaced by a five amino acid Flag-tag (DYKDE). The arrow points to the C119S substitution, which was designated as 14.7K PM.

Mentions: The failure of 14.7K PM to protect against TNF could be attributed to mutation-related loss of function of 14.7K PM or absence of its interaction partner optineurin. In consequence, individual characterization of the contribution of optineurin and 14.7K to TNF-resistance required generation of suitable 14.7K mutants, ideally with no intrinsic protection against TNF but conserved optineurin binding. Accordingly, we performed a systematic mutagenesis and sequentially replaced five amino acids by a Flag-tag sequence (DYKDE), covering the entire 14.7K gene (Figure 4). All generated 14.7K mutants were characterized regarding their capability to bind optineurin using a mammalian-two-hybrid screen (Figure 5A). With one exception, mutations in the C-terminus resulted in loss of optineurin binding, thereby confirming earlier studies stating that the C-terminal region was essential for protein-protein interactions [14], [19]. Consequently, to maximize the probability to obtain a structural integer 14.7K mutant without TNF-resistance, we choose two N-terminal mutants, designated as “14.7K mut 1” and “14.7K mut 3”. Optineurin-14.7K interaction of the selected mutants was additionally validated in coimmunoprecipitation experiments (Figure 5B).


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)

Generation of sequential mutants of 14.7K.The top row gives the names of the generated mutants. The bottom row displays the amino acid sequence of 14.7K replaced by a five amino acid Flag-tag (DYKDE). The arrow points to the C119S substitution, which was designated as 14.7K PM.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038348-g004: Generation of sequential mutants of 14.7K.The top row gives the names of the generated mutants. The bottom row displays the amino acid sequence of 14.7K replaced by a five amino acid Flag-tag (DYKDE). The arrow points to the C119S substitution, which was designated as 14.7K PM.
Mentions: The failure of 14.7K PM to protect against TNF could be attributed to mutation-related loss of function of 14.7K PM or absence of its interaction partner optineurin. In consequence, individual characterization of the contribution of optineurin and 14.7K to TNF-resistance required generation of suitable 14.7K mutants, ideally with no intrinsic protection against TNF but conserved optineurin binding. Accordingly, we performed a systematic mutagenesis and sequentially replaced five amino acids by a Flag-tag sequence (DYKDE), covering the entire 14.7K gene (Figure 4). All generated 14.7K mutants were characterized regarding their capability to bind optineurin using a mammalian-two-hybrid screen (Figure 5A). With one exception, mutations in the C-terminus resulted in loss of optineurin binding, thereby confirming earlier studies stating that the C-terminal region was essential for protein-protein interactions [14], [19]. Consequently, to maximize the probability to obtain a structural integer 14.7K mutant without TNF-resistance, we choose two N-terminal mutants, designated as “14.7K mut 1” and “14.7K mut 3”. Optineurin-14.7K interaction of the selected mutants was additionally validated in coimmunoprecipitation experiments (Figure 5B).

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