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. 2018 Aug 23;13(8):e0202408.
doi: 10.1371/journal.pone.0202408. eCollection 2018.

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

Natália G Sampaio  1   2 , Martina Kocan  3 , Louis Schofield  1   4 , Kevin D G Pfleger  5   6 , Emily M Eriksson  1   2
Affiliations

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

Natália G Sampaio et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

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.

Figures

Fig 1
Fig 1. BRET constructs are functional and appear to be appropriately localized when transfected into cells.
A-D) HEK293FT cells were transfected with 0, 30 or 300 ng of TLR1, TLR2-Rluc8, TIRAP-Venus or MyD88-Venus constructs, alone or in combination, and analysed for protein expression by Western immunoblot (IB) as indicated, along with lysate from untransfected THP1 cells. HEK293FT cells were transfected with E) TIRAP-Venus or F) MyD88-Venus constructs, DAPI stained, and imaged by confocal microscopy. Yellow = Venus, blue = nucleus; representative images of two independent experiments. G) HEK293FT cells were transfected with 100 ng TLR1/2-Rluc8 construct only (negative control), 100 ng TLR1/2-Rluc8 + 100 ng Kras-Venus constructs, and 100 ng V2R-Rluc8 + 100 ng Kras-Venus constructs (positive control) and BRET ratio was measured. Error bars represent SD of three independent experiments. One-way ANOVA; **** = P<0.0001, ** = P<0.01.
Fig 2
Fig 2. TIRAP directly interacts with TLR1/2, but does not show ligand-induced BRET increase.
A) HEK293FT cells were transfected with 100 ng TLR1 and 100 ng TLR2-Rluc8 constructs, and additionally with 300 ng MyD88-Venus, or 300 ng TIRAP-Venus constructs, and BRET measured over time. Graph shown is representative of three independent experiments; error bars represent SD of triplicate wells. B) Transfections as in (A), with additional cells transfected with 300 ng MyD88-Venus + 300 ng TIRAP constructs, or 300 ng MyD88 + 300 ng TIRAP-Venus constructs. Data shown as % of max BRET, where max is TLR1/2-Rluc8 + TIRAP-Venus; n = 4 independent experiments. One-way ANOVA; **** = P<0.0001. HEK293FT cells transfected with 300 ng TLR1 + 300 ng TLR2-Rluc8 constructs and additionally with either C) 300 ng TIRAP-Venus or D) 100 ng MyD88-Venus + 300 ng of TIRAP constructs. Cells were stimulated with 10 μg/ml Pam3CSK4 or vehicle control and BRET measured over time. Data shown are donor-only-subtracted BRET ratio; graphs are representative of three independent experiments. E) Untransfected THP1 cells, or HEK293FT cells transfected with 0 or 300 ng of TLR1/2, MyD88 and TIRAP constructs (TLR pathway), were treated with increasing concentrations of Pam3CSK4, as indicated, and TNF was measured in culture medium after 24 hr. Dose-response curve was fitted using Graphpad Prism non-linear fit with variable slope (EC50 = 14.4 for THP1 cells).
Fig 3
Fig 3. When overexpressed, TIRAP constitutively interacts with TLR1/2 but MyD88 interaction only occurs in the presence of TIRAP.
A) HEK293FT cells were transfected with constant (100 ng) TLR1/2-Rluc8 and increasing (0–1000 ng) TIRAP-Venus (blue circles; combined n = 7), MyD88-Venus (red triangles; combined n = 3), MyD88-Venus + TIRAP (black squares, combined n = 2), or V2R-Venus constructs (green inverted triangles, combined n = 3). B) HEK293 cells were transfected with constant (50 ng) TLR1/2-Rluc8 and increasing (0–1000 ng) MyD88-Venus + TIRAP constructs at the concentrations indicated (combined n = 2). Saturation curves were fitted using ‘one site—specific binding’ function on Prism software. Data shown are combined from independent experiments, as indicated. C) HEK293FT cells were transfected with Venus-tagged and untagged MyD88 and TIRAP constructs as indicated, DAPI stained, and imaged by confocal microscopy. Yellow = Venus, blue = nucleus; representative images of two independent experiments.
Fig 4
Fig 4. Modified BRET using Nluc allowed reduction in protein expression levels, but did not enable observation of ligand-induced BRET ratio.
A) HEK293FT cells were transfected with 100 ng of TLR1/2, MyD88 and TIRAP constructs (TLR pathway) and 0, 100, 200 or 400 ng of NF-κB luciferase constructs, and treated with 1 μg/ml Pam3CSK4. NF-κB luciferase levels measured after 24 hr; representative experiment of n = 3. B and C) HEK293FT cells were transfected with 200 ng of NF-κB-Fluc constructs and 0, 25, 50 or 100 ng of TLR1/2, MyD88 and TIRAP constructs (TLR pathway), and treated with 1 μg/ml Pam3CSK4. NF-κB reporter Fluc luciferase (B) and TLR-Rluc8 luciferase (C) levels measured after 24 hr; representative experiment of n = 2. HEK293FT cells transfected with D) 50 ng TLR1 + 50 ng TLR2-Nluc + 50 ng MyD88-Venus + 100 ng TIRAP constructs, or E) 5 ng TLR1 + 5 ng TLR2-Nluc + 5 ng MyD88-Venus + 10 ng TIRAP constructs. Cells were stimulated with 1 μg/ml Pam3CSK4 (red) or vehicle control (blue) and BRET measured over time. Data shown are donor-only-subtracted BRET ratio; graphs are representative of three independent experiments. F) HEK293FT cells were transfected with 100 ng TLR1 + 100 ng TLR2 + 10 ng MyD88-Nluc and 10 ng (circle) or 50 ng (triangle) of TIRAP-Venus constructs. Cells were stimulated with 1 μg/ml Pam3CSK4 (red and purple) or vehicle control (blue and green) and BRET measured over time. Data shown are donor-only-subtracted BRET ratio; graphs are representative of two independent experiments. G) HEK293FT cells were transfected with constant (100 ng) TLR1/2 and MyD88-Nluc constructs (20 ng, blue; or 10 ng, green), and increasing (0–600 ng) TIRAP-Venus construct. Saturation curves were fitted using ‘one site—specific binding’ function on Prism software.
Fig 5
Fig 5. Immunoprecipitation does not show constitutive interactions between overexpressed TLR1/2, TIRAP and MyD88.
A) HEK293FT cells were transfected as indicated, and cell lysates were analysed by Western blotting. High and low exposure images show both Venus-tagged (arrows) and endogenous (triangles) MyD88 and Venus in the same blot. B and C) HEK293FT cells were transfected with 100 ng of DNA constructs as indicated. Immunoprecipitation of Venus-tagged protein was performed, and analysed by Western blot, along with input samples.
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Grants and funding

This work was supported by Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council (NHMRC) Independent Research Institute Infrastructure Support Scheme; NHMRC Dora Lush Scholarship, Grant/Award Number: APP1038030 (NGS); NHMRC Grant/Award Numbers: APP106722 and APP1126395 (EME); NHMRC RD Wright Biomedical Research Fellowship Grant/Award Number 1085842 (KDGP). BRET work in the laboratory of KDGP is funded in part by Australian Research Council Linkage Grant LP160100857, with Promega, BMG Labtech and Dimerix as industry partners. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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