Flow cytometric reporter assays provide robust functional analysis of signaling complexes

Muusse, Timothy W; Lee, Morris Y L; Kim, Hyo‐Young; Parat, Marie-Odile; Nanson, J.D.; Kobe, Boštjan; Vajjhala, Parimala R.; Stacey, Katryn J.

doi:10.1016/j.jbc.2022.102666

Cited by 1 publication

(2 citation statements)

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“…Unlike mouse, we have found that human TLR2 signalling is entirely dependent upon MAL (P. Mudai, unpublished) and so we cannot assess a role for MAL in the same way as in mouse. We examined TLR2 and TLR4 ligand-dependent signalling, as well as spontaneous signalling in response to over-expressed MAL, using HEK293 NF-κB-reporter cell lines expressing all relevant TLR signalling proteins 31 . The spontaneous MAL signalling was inhibited by o-vanillin only at the highest concentration and this was not due to effects on MAL expression ( Figure 9D and Supplemental Figure S8 ).…”

Section: Resultsmentioning

confidence: 99%

“…Human TLR signalling assays were performed as described 31 in transiently transfected fluorescent HEK293 reporter cell lines. Analysis of TLR4 and MAL signalling used the HEK-TLR4-mScarlet cell line which expresses TLR4, MD-2, and CD14 and has an integrated mScarlet-i reporter gene under the control of an NF-κB-driven promoter 31 . For analysis of spontaneous MAL signalling, these cells were plated at 40,000 cells/well in 100 μl of complete DMEM without antibiotics and cultured overnight.…”

Section: Methodsmentioning

confidence: 99%

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o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2

Rahaman,

Thygesen,

Maxwell

et al. 2024

Journal of Enzyme Inhibition and Medicinal Chemistry

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Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MAL TIR ). We show that o-vanillin binds to MAL TIR and inhibits its higher-order assembly in vitro . Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%