Abstract
ABSTRACTDystonia is the third most common movement disorder, but its diagnosis and treatment remain challenging. One of the most severe types of dystonia is early-onset torsion dystonia (EOTD). The best studied and validated EOTD-associated mutation, torsinAΔE, is a deletion of a C-terminal glutamate residue in the AAA+ ATPase torsinA. TorsinA appears to be an endoplasmic reticulum (ER)/nuclear envelope chaperone with multiple roles in the secretory pathway and in determining subcellular architecture. Many functions are disabled in the torsinAΔE variant, and torsinAΔE is also less stable than wild-type torsinA and is a substrate for ER-associated degradation. Nevertheless, the molecular factors involved in the biogenesis and degradation of torsinA and torsinAΔE have not been fully explored. To identify conserved cellular factors that can alter torsinAΔE protein levels, we designed a new high-throughput, automated, genome-wide screen utilizing our validated Saccharomyces cerevisiae torsinA expression system. By analyzing the yeast non-essential gene deletion collection, we identified 365 deletion strains with altered torsinAΔE steady-state levels. One notable hit was EUG1, which encodes a member of the protein disulfide isomerase family (PDIs). PDIs reside in the ER and catalyze the formation of disulfide bonds, mediate protein quality control and aid in nascent protein folding. We validated the role of select human PDIs in torsinA biogenesis in mammalian cells and found that overexpression of PDIs reduced the levels of torsinA and torsinAΔE. Together, our data report the first genome-wide screen to identify cellular factors that alter expression levels of the EOTD-associated protein torsinAΔE. More generally, the identified hits help in dissecting the cellular machinery involved in folding and degrading a torsinA variant, and constitute potential therapeutic factors for EOTD. This screen can also be readily adapted to identify factors impacting the levels of any protein of interest, considerably expanding the applicability of yeast in both basic and applied research.
Highlights
Dystonia is a movement disorder characterized by sustained involuntary muscle contractions leading to abnormal, often repetitive movements and/or postures (Albanese et al, 2013)
Haploid cells containing only the deletion library chromosomes and the pRS425-GPD-torsinAΔEHA plasmid are generated by pinning the ‘diploid’ yeast onto medium supplemented with 5-fluoroorotic acid (5-FOA; which counter-selects for the URA3-expressing chromosomes) and galactose
We found that elevated levels of protein disulfide isomerase family (PDIs) and ERp72 significantly decreased the steady-state expression of both torsinA and torsinAΔE (Fig. 3): 48% of torsinA and 53% of torsinAΔE remained when PDI was co-overexpressed compared to cotransfection with an empty vector, and 68% of torsinA and 66% of torsinAΔE remained when ERp72 was co-overexpressed compared to co-transfection with an empty vector (P
Summary
Dystonia is a movement disorder characterized by sustained involuntary muscle contractions leading to abnormal, often repetitive movements and/or postures (Albanese et al, 2013). During biogenesis, torsinA acquires numerous post-translational modifications in the ER that require distinct enzymes: the oligosaccharyltransferase for N-linked glycosylation, the protein disulfide isomerases (PDIs) for disulfide bond formation, and the chaperone/lectin network for folding and assembly (Zacchi et al, 2016, 2014) Interactions with these ERresident machineries are crucial because the ΔE mutation decreases torsinA stability and targets torsinAΔE for degradation through a different pathway than wild-type torsinA (Giles et al, 2008; Gordon and Gonzalez-Alegre, 2008). Detailed exploration of additional hits from our screen may help us to understand how torsinA folds and assembles, how the torsinAΔE variant and wild-type torsinA are differentially degraded, and whether dedicated chaperones – some of which are being targeted pharmacologically (Brandvold and Morimoto, 2015; Brodsky and Chiosis, 2006; Balch et al, 2008) – impact torsinAΔE penetrance
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