Characterization of Dystonia Gene and Protein

Massachusetts General Hospital, Boston MA

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Abstract

DESCRIPTION (provided by applicant): The long term goal of this research is to understand the function of the novel protein, torsinA, which is mutated in early onset torsion dysconia. This neurologic disease is inherited as an autosomal dominant condition with reduced penetrance and a developmental window of susceptibility in childhood and adolescence. The torsins are members of the AAA+ superfamily of chaperone proteins involved in protein configurational changes. Studies to date suggest that torsinA may be involved in vectorial membrane movement and/or response of cells to oxidative stress. Only two in-frame mutations resulting in loss of amino acids in the carboxy terminal of this protein have been found in patients with early onset dystonia. In these studies we will screen for additional mutations in patients at the genomic and transcript levels. This analysis will be complemented by generation and expression of targeted mutations in the protein to elucidate structure/function determinants of ATPase activity, oligomerization, and posttranslational modifications. Cellular correlates will include the formation of whorled membrane inclusions in cells overexpressing the GAG-deleted form of torsinA found in most patients. In parallel, a search will be undertaken to identify partner proteins and their binding domains to torsinA, using the yeast two hybrid system, co-immunoprecipitation, affinity binding to purified protein, and protein chip assays. Immunocytochemistry will be used to visualize the cellular location of partner proteins relative to torsins.The predicted function of torsinA as a sensor to oxidative Stress will be examined by characterizing the posttranslational modifications to torsins which result from exposure to hydrogen peroxide and by evaluating whether expression of wild type or mutant forms of torsin act to protect or sensitize cells to this oxidative stress. TorsinA's predicted function in membrane and protein movement will be assessed by monitoring the morphology of the endoplasmic reticulum and vesicles, as well as constitutive and regulated secretion of proteins and vesicle recycling in cultured cells and neurons expressing mutant forms. These studies should help elucidate the function of this novel class of chaperone proteins and reveal how specific mutations in torsinA can disrupt cell function. Dystonia represents a special class of neurologic diseases, which do not manifest apparent neurodegeneration. This class of diseases may be amenable to therapy informed by the molecular etiology of dysfunction at the cellular level.