Date of Award
Open Access Dissertation
Rekha C. Patel
An integral aspect of innate immune response to viral infections is the ability to detect non-self molecules to initiate antiviral signaling via pattern recognition receptors (PRRs). One subset of these receptors are cytoplasmic receptors that contain double stranded (dsRNA) binding domains, which allow them to identify non-self dsRNA produced during a viral infection and mount a protective cellular response. PKR is a dsRNA-activated eIF2α kinase that is a key regulator of cellular antiviral and stress response pathways. Activation of PKR’s catalytic activity requires binding to one of its activators, viral dsRNAs or the cellular protein PACT (PKR activator). Although PACT also binds to dsRNA, in uninfected cells PACT activates PKR by a direct interaction in the absence of dsRNA in response to oxidative stress, ER stress, and serum starvation. Prolonged PKR activation and downstream eIF2α phosphorylation and inhibition leads to cell death by apoptosis. A third dsRNA-binding protein TRBP (TAR-RNA-binding protein), which is homologous to PACT, inhibits PKR by a direct interaction as well as by sequestration of dsRNA and PACT. Recently, an inherited, early-onset form of the neuromuscular disorder dystonia has been identified to be associated with multiple missense mutations in the coding region of PACT. We investigated alterations in PACT activity caused by DYT16 mutations by examining changes in interactions with known protein partners and ability to activate PKR during cellular stress. Our results establish that each point mutation may alter PACT’s functions differently. Patient lymphoblasts containing homozygous mutant P222L activate PKR with slower kinetics, albeit more robustly and for longer duration, as compared to wt lymphoblasts. In addition, the affinity of PACT-PACT and consequently PACT-PKR interaction is enhanced in vivo in these P222L dystonia lymphoblast lines, thereby leading to intensified PKR activation and consequently enhanced cellular death. Our results elucidate new mechanistic details of PKR regulation by PACT and shed new light on its impact on stress induced cellular apoptosis. Another cytoplasmic dsRNA receptor similar to PKR is the RNA helicase RIG-I, which has the ability to detect and be activated by 5’triphosphate uncapped dsRNA as well as the viral mimic dsRNA polyI:C. Once activated, RIG-I’s CARD domains oligomerize and initiate downstream mitochondrial anti-viral signaling (MAVS) to induce interferon (IFN) production. PACT stimulates RIG-I signaling in response to polyI:C treatment, in part, by stimulating RIG-I’s helicase activity and resulting in an enhanced induction of IFN. Despite the domain homology and similar structure of PACT and TRBP, the role of TRBP is unknown in RIG-I like receptor (RLR) signaling. We investigated the role of TRBP in RIG-I signaling and IFN production. Our results establish an inhibitory role of TRBP on RIG-I signaling, opposing PACT’s activating role. This inhibitory effect is also seen in the absence of PACT and PKR, indicating a direct role in RIG-I inhibition. The effect of DYT16 causing mutations in PACT on RIG-I signaling in response to viral stress mimics was also investigated and appears to be unaffected by the DYT16 mutations.
Vaughn, L. S.(2015). Regulation of Stress Response and Innate Immunity by DSRNA-Binding Proteins PACT and TRBP. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3219