oa One-pot Semisynthesis Of Exon1 Of The Mutant Huntingtin Protein: An Important Advance Towards Elucidating The Molecular And Structural Determinants Of Huntingtin's Aggregation And Toxicity

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG expansion gene which is translated into a polyglutamine stretch within the first exon of the Huntingtin protein (Htt). HD patients suffer from motor impairments, cognitive decline and depression. A hallmark of HD pathogenesis is the loss of neurons in the striatum and cortex which is closely linked to the formation of large cytoplasmic and especially nuclear aggregates composed of various N-terminal fragments of the mutant Huntingtin protein. In HD patient brains, Htt abnormally aggregates due to the expansion of its polyQ tract (more than 37 glutamines). Moreover, the severity of the disease increases with the number of glutamines, and the age of onset lowers as the polyQ tract expands. Several experimental observations suggest that overexpression of exon 1 of the mutant Huntingtin protein (Httex1) alone in transgenic mice is sufficient to reproduce HD pathology. Httex1, among other N-terminal Htt fragments, has also been consistently found in post-mortem HD brains. However, the molecular determinants of Httex1 aggregation and toxicity in HD remain unknown. Httex1 undergoes a wide range of post-translational modifications (PTMs) (such as phosphorylation, acetylation, ubiquitination and sumoylation) which were shown to modulate the toxicity and aggregation properties of the protein. Nevertheless, a comprehensive understanding of the effects of these PTMs on the biophysical and biochemical properties of Httex1 remains challenging since: 1) production and purification of recombinant mutant Httex1 protein is complicated by its high aggregation propensity; 2) most of the enzymes involved in regulating Httex1 PTMs remain unknown. In this study, we developed a novel semisynthetic strategy for the production of the highly aggregation-prone mutant Htt. Using specific chemical ligation and desulfurization conditions; we were able to produce for the first time, tag-free and pure mutant Httex1 in mg quantities. This advance will enable for the first time investigation of mutant Httex1 in the absence of large protein fusions, thus allowing for accurate determination of the role of the polyQ repeat length and post-translational modifications in modulating the aggregation kinetics and toxicity. Furthermore, the availability of highly pure WT and post-translationally modified mutant Httex1 proteins should facilitate structural studies aimed at determining the structural basis of Htt misfolding, aggregation and the identification and validation of novel HD biomarkers.