Description

Suppressor of IKKepsilon (SIKE) is a naturally occurring protein in humans that becomes phosphorylated when double-stranded(ds) RNA is introduced into the body typically as part of a viral infection. The phosphorylation of SIKE occurs due to a signaling pathway induced by Toll-like receptor 3 (TLR3), that activates host defenses and leads to the interruption of the viral replication. Although the functions of SIKE are unknown, it has been shown that SIKE associates with cytoskeletal proteins. To determine the interactions that SIKE has with cytoskeletal proteins, In Vitro Precipitations (IVP) were performed. During the IVP experiments, the 6x His-tag labeled SIKE is first incubated with various cytoskeletal proteins, and then incubated with Ni-NTA resin. By analyzing the samples of the reaction that are bound and not bound to the resin, the proteins that directly interact with SIKE can be identified. When using an N-terminal 6XHis-tagged SIKE, alpha-actinin appeared to prevent SIKE from interacting with the Ni-NTA resin suggesting that alpha-actinin was binding near the N-terminus of SIKE blocking the Ni-NTA-SIKE interaction. To test this hypothesis, a C-terminal 6XHis-tagged SIKE was incubated with alpha-actinin. The C-terminally labeled SIKE retained its interaction with the Ni-NTA resin and alpha-actinin consistent with the SIKE-alpha-actinin interaction occurring at the N-terminus of SIKE. These results confirm a direct interaction between SIKE and alpha-actinin. This work lays the foundation for understanding how this interaction may affect the function of alpha-actinin in the context of cytoskeletal protein rearrangements and function in the host?s response to viral infection.

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SIKE's Direct Protein-Protein Interactions

Suppressor of IKKepsilon (SIKE) is a naturally occurring protein in humans that becomes phosphorylated when double-stranded(ds) RNA is introduced into the body typically as part of a viral infection. The phosphorylation of SIKE occurs due to a signaling pathway induced by Toll-like receptor 3 (TLR3), that activates host defenses and leads to the interruption of the viral replication. Although the functions of SIKE are unknown, it has been shown that SIKE associates with cytoskeletal proteins. To determine the interactions that SIKE has with cytoskeletal proteins, In Vitro Precipitations (IVP) were performed. During the IVP experiments, the 6x His-tag labeled SIKE is first incubated with various cytoskeletal proteins, and then incubated with Ni-NTA resin. By analyzing the samples of the reaction that are bound and not bound to the resin, the proteins that directly interact with SIKE can be identified. When using an N-terminal 6XHis-tagged SIKE, alpha-actinin appeared to prevent SIKE from interacting with the Ni-NTA resin suggesting that alpha-actinin was binding near the N-terminus of SIKE blocking the Ni-NTA-SIKE interaction. To test this hypothesis, a C-terminal 6XHis-tagged SIKE was incubated with alpha-actinin. The C-terminally labeled SIKE retained its interaction with the Ni-NTA resin and alpha-actinin consistent with the SIKE-alpha-actinin interaction occurring at the N-terminus of SIKE. These results confirm a direct interaction between SIKE and alpha-actinin. This work lays the foundation for understanding how this interaction may affect the function of alpha-actinin in the context of cytoskeletal protein rearrangements and function in the host?s response to viral infection.

 

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