Chapter title |
Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER)
|
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Chapter number | 8 |
Book title |
Innate Immune Activation
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7519-8_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7518-1, 978-1-4939-7519-8
|
Authors |
Olaf-Oliver Wolz, Manfred Koegl, Alexander N. R. Weber |
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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