Dystonia 16 (DYT16) mutations in PACT cause dysregulated PKR activation and eIF2α signaling leading to a compromised stress response.

Dystonia 16 (DYT16) is caused by mutations in PACT, the protein activator of interferon-induced double-stranded RNA-activated protein kinase (PKR). PKR regulates the integrated stress response (ISR) via phosphorylation of the translation initiation factor eIF2α. This post-translational modification attenuates general protein synthesis while concomitantly triggering enhanced translation of a few specific transcripts leading either to recovery and homeostasis or cellular apoptosis depending on the intensity and duration of stress signals. PKR plays a regulatory role in determining the cellular response to viral infections, oxidative stress, endoplasmic reticulum (ER) stress, and growth factor deprivation. In the absence of stress, both PACT and PKR are bound by their inhibitor transactivation RNA-binding protein (TRBP) thereby keeping PKR inactive. Under conditions of cellular stress these inhibitory interactions dissociate facilitating PACT-PACT interactions critical for PKR activation. While both PACT-TRBP and PKR-TRBP interactions are pro-survival, PACT-PACT and PACT-PKR interactions are pro-apoptotic. In this study we evaluate if five DYT16 substitution mutations alter PKR activation and ISR. Our results indicate that the mutant DYT16 proteins show stronger PACT-PACT interactions and enhanced PKR activation. In DYT16 patient derived lymphoblasts the enhanced PACT-PKR interactions and heightened PKR activation leads to a dysregulation of ISR and increased apoptosis. More importantly, this enhanced sensitivity to ER stress can be rescued by luteolin, which disrupts PACT-PKR interactions. Our results not only demonstrate the impact of DYT16 mutations on regulation of ISR and DYT16 etiology but indicate that therapeutic interventions could be possible after a further evaluation of such strategies.

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

Protein Activator (PACT) activates the interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) in response to stress signals. Oxidative stress and endoplasmic reticulum (ER) stress causes PACT-mediated PKR activation, which leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. A dominantly inherited form of early-onset dystonia 16 (DYT16) has been identified to arise due to a frameshift (FS) mutation in PACT. To examine the effect of the resulting truncated mutant PACT protein on the PKR pathway, we examined the biochemical properties of the mutant protein and its effect on mammalian cells. Our results indicate that the FS mutant protein loses its ability to bind dsRNA as well as its ability to interact with PKR while surprisingly retaining the ability to interact with PACT and PKR-inhibitory protein TRBP. The truncated FS mutant protein, when expressed as a fusion protein with a N-terminal fluorescent mCherry tag aggregates in mammalian cells to induce apoptosis via activation of caspases both in a PKR- and PACT-dependent as well as independent manner. Our results indicate that interaction of FS mutant protein with PKR inhibitor TRBP can dissociate PACT from the TRBP-PACT complex resulting in PKR activation and consequent apoptosis. These findings are relevant to diseases resulting from protein aggregation especially since the PKR activation is a characteristic of several neurodegenerative conditions.

A frameshift mutation in the murine Prkra gene causes dystonia and exhibits abnormal cerebellar development and reduced eIF2α phosphorylation.

Mutations in Prkra gene, which encodes PACT/RAX cause early onset primary dystonia DYT-PRKRA, a movement disorder that disrupts coordinated muscle movements. PACT/RAX activates protein kinase R (PKR, aka EIF2AK2) by a direct interaction in response to cellular stressors to mediate phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α). Mice homozygous for a naturally arisen, recessively inherited frameshift mutation, Prkra lear-5J exhibit progressive dystonia. In the present study, we investigate the biochemical and developmental consequences of the Prkra lear-5J mutation. Our results indicate that the truncated PACT/RAX protein retains its ability to interact with PKR, however, it inhibits PKR activation. Furthermore, mice homozygous for the mutation have abnormalities in the cerebellar development as well as a severe lack of dendritic arborization of Purkinje neurons. Additionally, reduced eIF2α phosphorylation is noted in the cerebellums and Purkinje neurons of the homozygous Prkra lear-5J mice. These results indicate that PACT/RAX mediated regulation of PKR activity and eIF2α phosphorylation plays a role in cerebellar development and contributes to the dystonia phenotype resulting from this mutation.

DYT-PRKRA Mutation P222L Enhances PACT's Stimulatory Activity on Type I Interferon Induction.

DYT-PRKRA (dystonia 16 or DYT-PRKRA) is caused by mutations in the PRKRA gene that encodes PACT, the protein activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR). PACT participates in several cellular pathways, of which its role as a PKR activator protein during integrated stress response (ISR) is the best characterized. Previously, we have established that the DYT-PRKRA mutations cause enhanced activation of PKR during ISR to sensitize DYT-PRKRA cells to apoptosis. In this study, we evaluate if the most prevalent substitution mutation reported in DYT-PRKRA patients alters PACT's functional role in induction of type I IFNs via the retinoic acid-inducible gene I (RIG-I) signaling. Our results indicate that the P222L mutation augments PACT's ability to induce IFN ß in response to dsRNA and the basal expression of IFN ß and IFN-stimulated genes (ISGs) is higher in DYT-PRKRA patient cells compared to cells from the unaffected controls. Additionally, IFN ß and ISGs are also induced at higher levels in DYT-PRKRA cells in response to dsRNA. These results offer a new avenue for investigations directed towards understanding the underlying molecular pathomechanisms in DYT-PRKRA.