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.
The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 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 Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.
Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne (Switzerland).
Annalisa
A
Ansaloni
Zhe-Ming
ZM
Wang
Jae Sun
JS
Jeong
Francesco Simone
FS
Ruggeri
Giovanni
G
Dietler
Hilal A
HA
Lashuel
D000595:Amino Acid Sequence; D005091:Exons; D006801:Humans; D000071058:Huntingtin Protein; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins
aggregation; huntingtin exon 1; huntington’s disease; phosphorylation; protein semisynthesis