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

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Lipopeptides/*pharmacology ; Membrane Glycoproteins/*metabolism ; Myeloid Differentiation Factor 88/*metabolism ; Receptors, Interleukin-1/*metabolism ; Signal Transduction/*drug effects ; Toll-Like Receptor 2/*metabolism; Fluorescence Resonance Energy Transfer ; Gene Expression ; HEK293 Cells ; Humans ; Membrane Glycoproteins/genetics ; Microscopy, Confocal ; Myeloid Differentiation Factor 88/genetics ; Receptors, Interleukin-1/genetics ; Signal Transduction/genetics ; Toll-Like Receptor 2/genetics

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 TIRAP, and between TLR2 and MyD88 only in the presence of TIRAP. 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.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: KDGP is Chief Scientific Advisor of Dimerix Limited and has a shareholding in the company. KDGP receives funding from Promega, BMG Labtech and Dimerix as Partner Organisations of Australian Research Council Linkage Grant LP160100857. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. NGS, MK, LS and EME have no conflicts of interest.

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0 (Lipopeptides)
0 (MYD88 protein, human)
0 (Membrane Glycoproteins)
0 (Myeloid Differentiation Factor 88)
0 (Pam(3)CSK(4) peptide)
0 (Receptors, Interleukin-1)
0 (TIRAP protein, human)
0 (TLR2 protein, human)
0 (Toll-Like Receptor 2)

Date Created: 20180824 Date Completed: 20190204 Latest Revision: 20190215

20231215

PMC6107161

10.1371/journal.pone.0202408

30138457