The Interactome at the N17 Domain of Huntingtin the Interactome at the N17 Domain of Huntingtin

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein. Recent research demonstrates that post-translational modifications of huntingtin could be an important determinant of mutant huntingtin’s toxicity in HD. In particular, phosphorylation at residues serine 13 and 16 within the first 17 amino acids of huntingtin (N17), have been shown to be critical modulators of mutant huntingtin’s toxicity and localization, and can be triggered by stress. This project aims to study how phosphorylation within N17 alters the interactome at this site and what physiological stress results in the nuclear localization of N17 and huntingtin. The initial search to identify potential interactors was conducted through an affinity chromatography assay using a wild type striatal cell line derived from knock in mouse model of HD. Fluorescent lifetime imaging microscopy (FLIM) to determine Fӧrester resonance energy transfer (FRET), co-immunoprecipitation and coimmunofluorescence assays were then used to validate real interactors of N17. Analysis from this project has validated two previously unidentified interactors of N17; SET, a small nucleo-oncoprotein, and vimentin, a type 3 intermediate filament. Both interactors have suggested two potentially novel roles for N17 within huntingtin, in cell cycle regulation and intermediate filament dynamics. Finally, smart screening techniques using stress-inducing compounds reveal that the sensitivity of N17 to stress and its subsequent nuclear localization can be attributed in part to activation of oxidative stress pathways. Data shown here can be expanded upon to elucidate how huntingtin function and response to cell stress are regulated and mediated via N17 and potentially how this mechanism is disrupted within