EhrlichiaWnt short linear motif ligand mimetic deactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis

Abstract

Ehrlichia chaffeensisTRP120 effector has evolved short linear motif (SLiM) ligand mimicry to repurpose multiple evolutionarily conserved cellular signaling pathways including Wnt, Notch and Hedgehog. In this investigation, we demonstrate thatE. chaffeensisand recombinant TRP120 deactivate Hippo signaling resulting in activation of Hippo transcription coactivator Yap and target gene expression. Moreover, a homologous 6 amino acid (QDVASH) SLiM shared by TRP120 and Wnt3a/5a ligands phenocopied Yap and β-catenin activation induced byE. chaffeensis, rTRP120 and Wnt5a. Similar Hippo gene expression profiles were also stimulated byE. chaffeensis, rTRP120, SLiM and Wnt5a. Single siRNA knockdown of Hippo transcription co-activator/factors (Yap and TEAD) significantly decreasedE. chaffeensisinfection. Yap activation was abolished in THP-1 Wnt Frizzled-5 (Fzd5) receptor knockout cells (KO), demonstrating Fzd5 receptor dependence. In addition, TRP120 Wnt-SLiM antibody blocked Hippo deactivation (Yap activation). Expression of anti-apoptotic Hippo target geneSLC2A1(encodes glucose transporter 1; GLUT1) was upregulated byE. chaffeensisand corresponded to increased levels of GLUT1. Conversely, siRNA knockdown ofSLC2A1significantly inhibited infection. Higher GLUT1 levels correlated with increased BCL-xL and decreased Bax levels. Moreover, blocking Yap activation with the inhibitor Verteporfin induced apoptosis that corresponded to significant reductions in levels of GLUT1 and BCL-xL, and activation of Bax and Caspase-3 and -9. This study identifies a novel shared Wnt/Hippo SLiM ligand mimetic and demonstrates thatE. chaffeensisdeactivates the Hippo pathway to engage the anti-apoptotic Yap-GLUT1-BCL-xL axis.

Ehrlichia chaffeensis TRP120 ubiquitinates tumor suppressor APC to modulate Hippo and Wnt signaling

“…We previously demonstrated the activation of Yap and β-catenin via TRP120 Wnt ligand mimicry ( Rogan et al, 2021 ; Byerly et al, 2023b ). However, multiple host cellular proteins are involved in Yap and β-catenin negative regulation, including APC.…”

“…APC knockdown stabilizes active Yap and β-catenin levels in the nucleus during E. chaffeensis infection. E. chaffeensis is a known Hippo and Wnt modulator that activates Yap and β-catenin transcriptional regulators for intracellular survival ( Rogan et al, 2021 ; Byerly et al, 2023b ). Since APC negatively regulates active Yap and β-catenin by shuttling them out of the nucleus, we examined the effect of APC on E. chaffeensis infection using RNAi to knockdown APC in THP-1 cells.…”

“…Notably, the 120 kDa tandem repeat protein (TRP120) has emerged as a model moonlighting effector that functions as a cell signaling pathway ligand mimic, ubiquitin ligase and nucleomodulin. More recently, TRP120 has been shown to activate evolutionarily conserved signaling pathways such as Hippo, Wnt, Notch, and Hedgehog through short linear motif (SLiM) ligand mimicry ( Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023b ; Rogan et al, 2021 ; Byerly et al, 2022 ; Patterson et al, 2022 ; Byerly et al, 2023a ).…”

“…Considering E . chaffeensis TRP120 is known to exploit Hippo and Wnt signaling ( Rogan et al, 2021 ; Byerly et al, 2023b ), we examined the relationship between TRP120 HECT E3 ligase activity and the tumor suppressor APC in modulating Hippo and Wnt transcriptional activators, Yap and β-catenin. We determined that E. chaffeensis TRP120 promotes intracellular infection by ubiquitinating APC for degradation to maintain nuclear Yap and β-catenin levels.…”