| Several classes of mammalian innate immune receptors alert the host cell to the presence of pathogens. A subset of the receptors, including RIG-I-like receptors, NOD-like receptors, and DNA-sensing receptors, detect the presence of pathogen-associated molecular patterns in the cytoplasm. Many of the molecular details of the signaling events leading to type I interferon production and NF-kappaB activation have yet to be elucidated. We used two approaches to identify novel pathway member candidates: the yeast-two hybrid method, and affinity-based purification of protein complexes coupled with mass spectrometry.;Two candidates were selected for follow-up studies: optineurin, a candidate identified as a TBK-1 interactor using a yeast two-hybrid assay, and Smac, a candidate identified as an IPS-1 interactor by a yeast two-hybrid assay. Since optineurin is highly homologous to NEMO, the regulatory subunit of the IKK complex in NE-kappaB signaling, we hypothesized that optineurin may play a role in innate immune signaling. We stimulated murine bone marrow-derived macrophages that were pretreated with OPTN siRNA using LPS, Listeria monocytogenes, or PBS as a mock treatment. Using microarrays, we found that the global transcriptional profile of innate-immune-regulated genes was unaffected by reduced protein levels of optineurin.;Smac is a proapoptotic protein that is localized to the mitochondria. IPS-1, the adaptor for RLR signaling also localizes to the mitochondria, and its localization is important for its function. Since Smac and IPS-1 localize to the mitochondria, and both Smac and IPS-1 have been previously shown to bind TRAF2/3, we selected the interaction for further study. Here we present evidence that the precursor form of Smac interacts with IPS-1 in a HEK 293T cells. This interaction could possibly be involved in the integration of apoptotic and innate immune signaling responses. |
