Biochemical characterization of the normal and pathogenic forms of the MJD1 gene product, ataxin -3

The polyglutamine expansion diseases are a class of inherited neurodegenerative disorders in which expansion of a CAG repeat encoding a polyglutamine stretch in the target protein results in a toxic gain of function and subsequent disease in affected individuals. Numerous cell culture and animal studies have implicated polyglutamine expansion as a potential cause of protein aggregation and subsequent disease progression and neurodegeneration. While there are many possible mechanisms for toxicity, all of them involve a change in the proteins caused by glutamine expansion, suggesting that abnormal conformation and aggregation are part of the disease mechanism. Studies of pathogenicity have been limited by an inability to acquire full length protein, and have used instead truncated proteins with exposed polyglutamine tails. I present here methods for preparing large quantities of pure, intact human protein in both the normal and expanded polyglutamine form. Furthermore, I propose a potential model for domain structure containing regions of ataxin-3 outside the polyglutamine region. Utilizing ataxin-3 protein with both normal and expanded polyglutamine regions I have demonstrated that the native structure of the ataxin-3 protein is destabilized upon expansion of the polyglutamine region. Concomitant with this destabilization is the ability of the expanded glutamine protein to form fibrils which share morphologic and tinctorial properties with amyloid fibrils first characterized in Alzheimer\u27s disease. Conformational studies of these fibrils reveals an acquisition of β-sheet secondary structure that may be parallel in nature. Previous models of polyglutamine pathogenesis proposed a polar zipper structure comprised of polyglutamine regions forming stable antiparallel β-sheets; our data suggest an alternative structure may be present. Construction of a three dimensional model of polyglutamine β-sheets in both parallel and antiparallel form showed that the parallel β-sheet model is allowed conformationally and is at least as energetically favorable as the antiparallel model