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

Abstract

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.

EIF2AK2 Missense Variants Associated with Early Onset Generalized Dystonia

“…Evidence for a disturbed integrated stress response (ISR) has been previously reported in other monogenic causes of dystonia, including DYT‐PRKRA, DYT‐TOR1A, DYT‐THAP1, and DYT‐SGCE. Several studies have shown that variants in the PRKRA gene enhanced the susceptibility to endoplasmic reticulum stress leading to heightened EIF2AK2 activation, dysregulation of ISR, and increased apoptosis 32,34,35 . ISR dysregulation has been reported to play a central role in DYT‐TOR1A 10,11,36 .…”

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

“…A form of early-onset dystonia (DYT16) has been linked to mutations in PACT. A recessive inherited disease results from the point mutation P222L, while a dominant inherited disease arises from a frameshift-induced missense mutation, both of which leads to robust spontaneous PKR activation and apoptosis [230,231]. These mutant PACT proteins also display altered affinity for TRBP, and release of wild-type PACT from the inhibitory PACT-TRBP complex upon cellular stress signals led to longer-lasting PKR and caspase activation in patient-derived lymphoblasts [231,232].…”

Uncovering Tumour Heterogeneity through PKR and nc886 Analysis in Metastatic Colon Cancer Patients Treated with 5-FU-Based Chemotherapy

“…However, the high level of post-translational modifications and regulation of the protein indicated that PKR activity does not necessarily have to correspond to the amount of its messenger RNA. In fact, numerous proteins have been described as regulating their activity (e.g., PACT, trans-activation response (TAR) RNA binding protein (TRBP), nucleophosmin (NPM)) [4,7,8,[46][47][48] and several post-translational modifications have been showed by SUMOylation and ISGylation, among others [49,50]. Nc886 has been described as a PKR inhibitor, being the inhibition of PKR/NF-kB in correlation with its tumour suppressor activity.…”