Inhibition of the inflammatory response to stress by targeting interaction between PKR and its cellular activator PACT

Abstract

PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication

“…After having assessed that IBV prevents SGs formation not only in chicken cells but also in mammalian cells, due to the availability of antibodies directed against mammalian proteins and their lack of cross-reactivity to the chicken proteins of interest, we next proceeded with Vero and H1299 cells. Poly I:C transfection or sodium arsenite treatment were included as positive control to stimulate phosphorylation of PKR and eIF2α [ 57 ]. As shown in Fig 2A and 2B , compared to mock-infected cells, the level of phospho-PKR was gradually increased along with the infection time course, peaked at 20 and 24 h.p.i., which was lower than those in poly I:C-transfected or sodium arsenite-treated cells.…”

A tale of two proteins: PACT and PKR and their roles in inflammation

“…This article is protected by copyright. All rights reserved microglial cells, although it did lead to increased PKR-mediated inflammasome activation [238]. Various PKR regulators can limit or promote inflammasome activation; for example, amyloid β 1-42 can induce activation of the NLRP3 inflammasome dependent on PKR [13], and the inhibitor protein P58 IPK can bind to PKR to inhibit NF-κB and JNKmediated proinflammatory signaling and activation of the NLRP3 inflammasome [239].…”

Antiviral activity of iridoid glycosides extracted from Fructus Gardeniae against influenza A virus by PACT-dependent suppression of viral RNA replication

“…The interactions between influenza polymerase and host cell factors were identified by diverse methods, such as genome-wide RNA interference (RNAi) screens 21 , proteomic approaches 22 , and systematic immunoprecipitation analysis 23 . Host factor PACT is a double-stranded RNA-binding protein that is also known to be an activator of the cellular dsRNA-dependent protein kinase, PKR 24 . PACT is involved in the interaction between influenza A virus (IAV) and host cells, and it can substantially enhance RIG-I activation and phosphorylate eukaryotic translation initiation factor 2 alpha (eIF2α) to induce an IFN type I response, which is suppressed by the nonstructural influenza virus protein NS1 25,26 .…”