Abstract
Recent structural, biochemical, and functional studies have led to the notion that many of the post-receptor signaling complexes in innate immunity have a multimeric, multi-protein architecture whose hierarchical assembly is vital for function. The Myddosome is a post-receptor complex in the cytoplasmic signaling of Toll-like receptors (TLR) and the Interleukin-1 receptor (IL-1R), involving the proteins MyD88, IL-1R-associated kinase 4 (IRAK4), and IRAK2. Its importance is strikingly illustrated by the fact that rare germline mutations in MYD88 causing high susceptibility to infections are characterized by failure to assemble Myddosomes; conversely, gain-of-function MYD88 mutations leading to oncogenic hyperactivation of NF-κB show increased Myddosome formation. Reliable methods to probe Myddosome formation experimentally are therefore vital to further study the properties of this important post-receptor complex and its role in innate immunity, such as its regulation by posttranslational modification. Compared to structural and biochemical analyses, luminescence-based mammalian interactome mapping (LUMIER) is a straightforward, automatable, quantifiable, and versatile technique to study protein-protein interactions in a physiologically relevant context. We adapted LUMIER for Myddosome analysis and provide here a basic background of this technique, suitable experimental protocols, and its potential for medium-throughput screening. The principles presented herein can be adapted to other signaling pathways.
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Acknowledgment
We thank Dr. Julie George and Dr. Hui Wang for contributing to the setup of LUMIER in our laboratory.
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Wolz, OO., Koegl, M., Weber, A.N.R. (2018). Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER). In: De Nardo, D., De Nardo, C. (eds) Innate Immune Activation. Methods in Molecular Biology, vol 1714. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7519-8_8
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DOI: https://doi.org/10.1007/978-1-4939-7519-8_8
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