Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression

Sampaio, Natalia G., Kocan, Martina, Schofield, Louis, Pfleger, Kevin D.G., and Eriksson, Emily M. (2018) Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression. PLoS ONE, 13 (8). e0202408.

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Toll like receptors (TLRs) are important pattern recognition receptors that can detect pathogen and danger associated molecular patterns to initiate an innate immune response. TLR1 and 2 heterodimerize at the plasma membrane upon binding to triacylated lipopeptides from bacterial cell walls, or to the synthetic ligand Pam3CSK4. TLR1/2 dimers interact with adaptor molecules TIRAP and MyD88 to initiate a signalling cascade that leads to activation of key transcription factors, including NF-kB. Despite TLRs being extensively studied over the last two decades, the real-time kinetics of ligand binding and receptor activation remains largely unexplored. We aimed to study the kinetics of TLR activation and recruitment of adaptors, using TLR1/2 dimer interactions with adaptors MyD88 and TIRAP. Bioluminescence resonance energy transfer (BRET) allows detection of real-time protein-protein interactions in living cells, and was applied to study adaptor recruitment to TLRs. Energy transfer showed interactions between TLR2 and TI RAP, and between TLR2 and MyD88 only in the presence of TI RAP. Quantitative BRET and confocal microscopy confirmed that TIRAP is necessary for MyD88 interaction with TLR2. Furthermore, constitutive proximity between the proteins in the absence of Pam3CSK4 stimulation was observed with BRET, and was not abrogated with lowered protein expression, changes in protein tagging strategies, or use of the brighter NanoLuc luciferase. However, co-immunoprecipitation studies did not demonstrate constitutive interaction between these proteins, suggesting that the interaction observed with BRET likely represents artefacts of protein overexpression. Thus, caution should be taken when utilizing protein overexpression in BRET studies and in investigations of the TLR pathway.

Item ID: 55711
Item Type: Article (Research - C1)
ISSN: 1932-6203
Copyright Information: © 2018 Sampaio et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funders: Victorian State Government Operational Infrastructure Support, National Health and Medical Research Council of Australia (NHMRC), Independent Research Institute Infrastructure Support Scheme, Australian Research Council (ARC)
Projects and Grants: NHMRC Dora Lush Standard Scholarship, NHMRC APP1038030, NHMRC APP106722, NHMRC APP1126935, NHMRC 108542, ARC Linkage Grant LP160100857
Date Deposited: 26 Sep 2018 09:20
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3204 Immunology > 320407 Innate immunity @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 100%
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