Master of Science / Department of Biochemistry / Michal Zolkiewski / Early-onset dystonia (EOTD, also known as DYT1 or Oppenheim’s dystonia is the most severe and common form of hereditary dystonia, a neurological disorder characterized by abnormalities in the control of movement. It is linked to the deletion of a single GAG codon in the gene DYT1 that leads to the loss of a single glutamic acid residue in the C-terminal region of the protein torsinA (ΔE-torsinA). It is not known how the GAG deletion alters the torsinA structure and function. In this thesis, the expression and purification of recombinant torsinA variants from E. coli is reported. Wild type torsinA is not soluble after its expression in E. coli, possibly due to misfolding caused by cysteine. We produced Cys-less torsinA, and established a purification procedure to produce this mutant torsinA. Furthermore, because of the critical role likely to be played by the C-terminal domain of torsinA that contains the glutamate deletion, we produced fragments encoding the C-terminal domain of torsinA, and attempted to purify it. However, we failed to obtain appreciable amount of active proteins by both of the strategies. A novel SUMO fusion technology was also used in this study. We demonstrated that SUMO, when fused with torsinA variants, was able to enhance its expression and solubility in E. coli. A satisfactory yield of the fusion protein was successfully purified. Once we get appreciable quantities of folded torsinA variants, it is our future goal to study their function by using biochemical and high-resolution structural approaches.

The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.

DYT1 dystonia (DYT1) is a disabling inherited neurological disorder with juvenile onset. The genetic mutation in DYT1 leads to the deletion of a glutamic acid (E) residue in the protein torsinA. The function of torsinA and how the mutation leads to DYT1 is poorly understood. We hypothesize that how efficiently the disease-linked mutant protein is cleared may be critical for DYT1 pathogenesis. Therefore we explored mechanisms of torsinA catabolism, employing biochemical, cellular, and animal-based approaches. We asked if torsinA(wt) and torsinA(DE) are degraded preferentially through different catabolic mechanisms, specifically the ubiquitin proteasome pathway (UPP) and autophagy. We determined that torsinA(wt) is cleared by autophagy while torsinA(DE) is efficiently degraded by the UPP suggesting degradation processes can modulate torsinA(DE) levels. Proteins implicated in recognizing motifs on torsinA(DE) for targeting to the UPP represent candidate proteins that may modify DYT1 pathogenesis. We examined how removal of the hydrophobic domain and mutation of glycosylated asparagine residues on torsinA altered stability and catabolic mechanism. We found the glycosylation sites on torsinA are important for stability modulate its degradation through the UPP. F-box G-domain protein 1 (FBG1) has been implicated in degradation of glycosylated ER proteins. We hypothesized that FBG1 would promote torsinA degradation and demonstrated that FBG1 modulates levels of torsinA in a non-canonical manner through the UPP and autophagy. We examined if lack of FBG1 in a torsinA(DE) mouse model altered motor phenotypes. We saw no effect which suggests FBG1 does not alter DYT1 pathogenesis despite its promotion of torsinA(DE) degradation. In addition, we explored a potential mechanism for the previously described role of torsinA in modulating cytoplasmic protein aggregation. We hypothesized this endoplasmic reticulum (ER) resident protein would indirectly alter cytoplasmic protein aggregation through modulation of ER stress. We employed a poly-glutamine expanded repeat protein and pharmacological ER stressors to determine that torsinA does not alter poly-glutamine protein aggregation nor ER stress in a mammalian system. In summary, this thesis suggests proteins involved in the catabolism of torsinA(DE) may modify DYT1 pathogenesis and that torsinA and its DYT1-linked mutant are model proteins for investigating ER protein degradation by the UPP and autophagy.