Contribution of the two dsRBM motifs to the double‐stranded RNA binding and protein interactions of PACT

Abstract

PACT is a stress-modulated activator of protein kinase PKR (protein kinase, RNA activated), which is involved in antiviral innate immune responses and stress-induced apoptosis. Stress-induced phosphorylation of PACT is essential for PACT's increased association with PKR leading to PKR activation, phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. PACT-induced PKR activation is negatively regulated by TRBP (transactivation response element RNA-binding protein), which dissociates from PACT after PACT phosphorylation in response to stress signals. The conserved double-stranded RNA binding motifs (dsRBMs) in PKR, PACT, and TRBP mediate protein-protein interactions, and the stress-dependent phosphorylation of PACT changes the relative strengths of PKR-PACT, PACT-TRBP, and PACT-PACT interactions to bring about a timely and transient PKR activation. This regulates the general kinetics as well as level of eIF2α phosphorylation, thereby influencing the cellular response to stress either as recovery and survival or elimination by apoptosis. In the present study, we evaluated the effect of specific mutations within PACT's two evolutionarily conserved dsRBMs on dsRNA-binding, and protein-protein interactions between PKR, PACT, and TRBP. Our data show that the two motifs contribute to varying extents in dsRNA binding, and protein interactions. These findings indicate that although the dsRBM motifs have high sequence conservation, their functional contribution in the context of the whole proteins needs to be determined by mutational analysis. Furthermore, using a PACT mutant that is deficient in PACT-PACT interaction but competent for PACT-PKR interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.

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

“…Type I interferon (IFN-I) is a key component of the innate antiviral response, and constitutes the first line of host defence against influenza virus infection [41][42][43] . The activation of the IFN-I response requires PACT, which interacts with RNA polymerase, and nonstructural proteins NS1 and NS2 of influenza A virus 44 . Influenza virus replication depends on the interaction of virus-encoded proteins with host factors, which regulate pivotal aspects of cell survival; virus recognition, replication and pathogenicity; and host defence 45,46 .…”

Double-Stranded RNA Sensors and Modulators in Innate Immunity

“…They were shown to form homodimers as well as heterodimers with each other, other dsRBDs (70,(167)(168)(169), and a helicase domain (170) (Figure 4b). To make matters more complex, dsRBD1 and 2 of PACT and TRBP were also proposed to interact with other proteins in dsRNA-dependent and -independent manners (171). As such, despite having a simple repeat of dsRBDs, PACT and TRBP are involved in multiple aspects of dsRNA biology through various protein-protein interactions as well as protein-dsRNA interactions.…”

A truncated PACT protein resulting from a frameshift mutation reported in movement disorder DYT16 triggers caspase activation and apoptosis

“…This results in a frameshift that would produce a 109 amino acid long truncated protein with 1 to 88 amino acids of original PACT protein followed by 21 extraneous amino acids due to the frameshift (Figure A and B). The resulting truncated (mutant FS) protein does not have a complete copy of the evolutionarily conserved dsRNA‐binding motif (dsRBM1) and thus is missing the crucial part of the motif that is essential for binding dsRNA as well as protein‐protein interactions . Therefore, the FS mutant protein is expected to be devoid of dsRNA‐binding ability, as well as interaction with PKR and PACT.…”

“…The mutant FS protein also showed no interaction with PKR, although it showed interaction with PACT. Based on our previous work on PACT‐PKR and PACT‐PACT interactions, it is expected that FS mutant protein may not interact with PKR or PACT as it lacks the alanine residues at positions 91 and 92 . Thus, it was surprising that the FS mutant protein interacted with PACT in vitro and the FS protein aggregates showed colocalization of PKR and PACT in HeLa cells.…”

“…The resulting truncated (mutant FS) protein does not have a complete copy of the evolutionarily conserved dsRNA-binding motif (dsRBM1) and thus is missing the crucial part of the motif that is essential for binding dsRNA as well as protein-protein interactions. 28,30 Therefore, the FS mutant protein is expected to be devoid of dsRNA-binding ability, as well as interaction with PKR and PACT. To determine if the frameshift mutation affects dsRNA binding activity, an in vitro dsRNAbinding assay previously well established for PKR and PACT 1 was performed (Figure 2A and 2B).…”