Heterologous Expression and Assembly of Human TLR Signaling Components in Saccharomyces cerevisiae

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.

Human gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signalling in budding yeast

“…Similarly, we assessed changes in autophagy signalling by measuring Atg13 phosphorylation. TORC1 inhibition leads to its dephosphorylation, triggering autophagy [ 52 ]. Equivalently to figure 7 b , we co-transformed the plasmids carrying GSDMD and MLKL constructs with plasmids expressing either Maf1-HA or HA-Atg13.…”

“…Once activated, GSDMD and MLKL are known to insert into cellular membranes through positively charged patches on their surface that allow them to interact with negatively charged lipids, particularly cardiolipin, phosphatidylserine and phosphoinositides [4][5][6]10]. Previously, we reproduced recognition of the plasma membrane by positively charged human proteins involved in innate immune signalling in yeast, like Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP) [52]. Thus, we expected yeast growth inhibition by GSDMD and MLKL to be linked to their localization at the plasma membrane, leading to its disruption.…”

Human Gasdermin D and MLKL disrupt mitochondria, endocytic traffic and TORC1 signaling in budding yeast

“…Once activated, GSDMD and MLKL are known to insert into cellular membranes through positively charged patches on their surface that allow them to interact with negatively charged lipids, particularly cardiolipin, phosphatidylserine, and phosphoinositides [4–6, 10]. Previously, we reproduced recognition of the plasma membrane by positively charged human proteins involved in innate immune signaling in yeast, like Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP) [45]. Thus, we expected yeast growth inhibition by GSDMD and MLKL to be linked to their localization at the plasma membrane, leading to its disruption.…”

Estrategia de enseñanza de Biología Molecular para la edición genética In Silico: Una experiencia disruptiva

“…Los seis grupos de trabajo lograron obtener resultados, concretar los objetivos y crear un plásmido recombinante que cumplía con las características buscadas, sin embargo, durante el desarrollo del proyecto y sobre todo durante la sustentación de los mismos se logró evidenciar algunas falencias. Una de las limitaciones del estudio fue que un grupo (G-1) no pudo obtener un plásmido vector para trabajar en S. cerevisiae, no obstante, el grupo pudo solucionar esta falencia al trabajar con un plásmido sugerido por literatura, que permitía la edición génica en levaduras (Coronas-Serna et al, 2021).…”