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  • br Materials and methods br Results br Discussion Mistry et

    2019-07-08


    Materials and methods
    Results
    Discussion Mistry et al. have provided convincing data showing that targeting the BB loop pocket is an effective approach for the identification of TLR2 signaling inhibitors [35]. In our study, the combination of blind docking and subsequent MD simulations indicated that site 2 is the most likely to which DMPO would be bound in TLR2-TIR domain. This site where DMPO fits into the TIR domain has been previously found to be important in downstream signaling [17]. Particularly, residue P681 has a critical role in TLR2 triggered signaling as shown by site-directed mutagenesis technique [17,35]. Moreover, a naturally occurring allele of mouse TLR4 has been reported to be unresponsive to LPS due to a point mutation on residue P712, a structural analog of human TLR2 P681 residue [16,17]. Protein-protein interactions between the TLR2-TIR-BB-Loop domain with the BB-loop within MyD88 are important to ensure functional downstream signaling. Thus, according to our in silico data, we tested whether by non-covalently binding to those specific residues at the BB-loop domain DMPO may reduce the effective downstream signaling. To test this possibility we measured the functional effects of DMPO on zymosan-triggered TLR2 signaling and found that DMPO inhibits downstream signaling. It is important to highlight that the HEK293 cells do not express TLRs [36], hence our findings on TLR2 signaling are only linked to this specific receptor. These results support ours in silico simulations where DMPO was found to directly bind to a key region responsible for signal transduction, i.e., the BB-loop within the TLR2-TIR domain. DMPO interferes the signaling triggered by zymosan-induced protein-protein interaction between TLR2-TIR-BB-loop with its adaptor protein MyD88. Because DMPO inhibits the function of the TLR2-TIR-BB-loop/MyD88 protein-protein interaction, it may be because DMPO binds to a critical site needed for this interaction. In other words, by binding to one or more specific amino AngiotensinI residues within the TLR2-TIR-BB-loop region DMPO may block the binding of TLR2-TIR with MyD88. Thus we measured the effect of DMPO on the binding of TLR2 with MyD88 upon zymosan activation in THP-1-derived macrophages. Our co-immunoprecipitation data suggest that DMPO does not inhibit TLR2-MyD88 protein-protein interaction. However, this observation does not necessarily mean that DMPO binding to the BB-loop inhibits, in term of downstream signaling, the functionality of the TLR2-MyD88 protein-protein interaction. The spin trap could be disrupting the proper interaction of TLR2 with the adaptor protein without completely inhibiting the protein-protein interaction. These findings are consistent with our previously published data and propose an explanation for the observed phenomenon of multiple TLRs inhibition by DMPO, as we have previously reported [11].
    Transparency Document
    Conflict of interest
    Acknowledgments Authors are thankful to Dr. Paula Di Sciullo for her excellent technical assistance. This research was supported by the following research awards: FONCYT-Argentina (PICT-2014-3369, to DCR), CONICET-Argentina (PIP-916, to DCR and SEGM), and National University of San Luis-Argentina (PROICO 02-3418 to DCR and PROICO 10-0218, to SEGM). DCR and SEGM want to dedicate this article to their former mentors Dr Ronald P. Mason, Dr Robert A. Floyd and Dr Maria S. Gimenez for seminal scientific contributions to the data published here.
    Introduction Accumulating evidence suggests a role of mast cells in cardiovascular diseases (CVD) [1]. Distinct from most other inflammatory cells, mast cells contain a set of signature serine proteases, including chymase, tryptase, and carboxypeptidase A [2,3]. These enzymes hydrolyze extracellular matrix (ECM) proteins and cell surface proteins, activate pro-enzymes [2], and generate angiotensin-II (Ang-II) [4]. Chymase and tryptase can directly degrade collagens and interrupt the structural integrity of the arterial wall. Chymase also cleaves apoA-I to destroy its activity in suppressing NF-κB-dependent coronary artery endothelial cell (EC) expression of VCAM-1 (vascular cell adhesion molecule-1) and monocyte adhesion [5]. Chymase-mediated production of Ang-II in cardiovascular tissues [6,7] activates macrophage expression of MCP-1 (monocyte chemoattractant protein-1), a chemokine implicated in monocyte recruitment [8]. Chymase and tryptase also activate matrix metalloproteinases (MMPs) MMP-1, MMP-3 and MMP-9 [9,10] and TGF-β [11,12] that contribute to arterial wall ECM degradation or synthesis.