One-Pot Semisynthesis of Exon 1 of the Huntingtin Protein: New Tools for Elucidating the Role of Posttranslational Modifications in the Pathogenesis of Huntington's Disease

摘要

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Ntl7) of Huntingtin exon 1 (Httexl) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Ntl 7 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httexl containing 23 glutamine residues (Httexl-23Q). Our studies show thatpT3 significantly slows the oligomeriza-tion and fibrillization of Httexl-23Q and that Httexl variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httexl aggregation. The ability to produce wt or site-specifically modified tag-free Httexl should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions ofHtt in health and disease.