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Examining Myddosome Formation by Luminescence-Based Mammalian Interactome Mapping (LUMIER)

  • Olaf-Oliver Wolz
  • Manfred Koegl
  • Alexander N. R. WeberEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

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.

Keywords

Toll-like receptor Myddosome Luminescence-based mammalian interactome mapping Renilla luciferase Protein-protein interactions Signaling complex 

Notes

Acknowledgment

We thank Dr. Julie George and Dr. Hui Wang for contributing to the setup of LUMIER in our laboratory.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Olaf-Oliver Wolz
    • 1
  • Manfred Koegl
    • 2
  • Alexander N. R. Weber
    • 1
    Email author
  1. 1.Department of Immunology, Interfaculty Institute for Cell BiologyUniversity of TübingenTübingenGermany
  2. 2.Boehringer Ingelheim RCV GmbH & Co KGViennaAustria

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