Abstract
Toll-like receptor (TLR) signaling is key to detect pathogens and initiating inflammation. Ligand recognition triggers the assembly of supramolecular organizing centers (SMOCs) consisting of large complexes composed of multiple subunits. Building such signaling hubs relies on Toll Interleukin-1 Receptor (TIR) and Death Domain (DD) protein-protein interaction domains. We have expressed TIR domain-containing components of the human myddosome (TIRAP and MyD88) and triffosome (TRAM and TRIF) SMOCs in Saccharomyces cerevisiae, as a platform for their study. Interactions between the TLR4 TIR domain, TIRAP, and MyD88 were recapitulated in yeast. Human TIRAP decorated the yeast plasma membrane (PM), except for the bud neck, whereas MyD88 was found at cytoplasmic spots, which were consistent with endoplasmic reticulum (ER)-mitochondria junctions, as evidenced by co-localization with Mmm1 and Mdm34, components of the ER and Mitochondria Encounter Structures (ERMES). The formation of MyD88-TIRAP foci at the yeast PM was reinforced by co-expression of a membrane-bound TLR4 TIR domain. Mutations in essential residues of their TIR domains aborted MyD88 recruitment by TIRAP, but their respective subcellular localizations were unaltered. TRAM and TRIF, however, did not co-localize in yeast. TRAM assembled long PM-bound filaments that were disrupted by co-expression of the TLR4 TIR domain. Our results evidence that the yeast model can be exploited to study the interactions and subcellular localization of human SMOC components in vivo.
Highlights
Eukaryotic complex organisms have evolved diverse mechanisms to react to the presence of pathogens and discern them from their microbiota
To track in vivo behavior of TIRAP in yeast, we developed a system for the heterologous expression, from GAL1 promoter, of human cDNA encoding
We have recently described that Toll Interleukin-1 Receptor (TIR) domains that bear NAD+ hydrolase activity are severely toxic in yeast [42], so tolerance to high levels of expression is consistent with the fact that such enzymatic activity has not been reported in this particular human innate immune Toll-like receptor (TLR) adaptor
Summary
Eukaryotic complex organisms have evolved diverse mechanisms to react to the presence of pathogens and discern them from their microbiota. Of Gram-negative bacteria is a major pathogen-associated molecular pattern (PAMP). It is recognized by the Toll-Like Receptor 4 (TLR4), leading to the activation of innate immunity signaling. Those downstream PAMP-recognizing receptors (PRR) rarely rely on secondary messengers. Rather, their signaling mechanism depends on the assembly of supramolecular organizing centers (SMOCs) [1]. The signal, conveyed through protein-protein interactions and posttranslational modifications, ends up in nuclear translocation of transcription factors to induce the expression of cytokines, which eventually set off typical innate immunity responses [3,4]
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