Title
N-terminal Huntingtin (Htt) phosphorylation is a molecular switch regulating Htt aggregation, helical conformation, internalization, and nuclear targeting
Author
Sean M. Deguire, Francesco S. Ruggeri, Mohamed-Bilal Fares, Anass Chiki, Urszula Cendrowska, Giovanni Dietler, Hilal A. Lashuel
Year
2018
Journal
Journal of Biological Chemistry
Abstract
Huntington’s disease is a fatal neurodegenerative disorder resulting from a CAG repeat expansion in the first exon of the gene encoding the Huntingtin protein (Htt). Phosphorylation of this protein region (Httex1) has been shown to play important roles in regulating the structure, toxicity, and cellular properties of N-terminal fragments and full-length Htt. However, increasing evidence suggests that phosphomimetic substitutions in Htt result in inconsistent findings and do not reproduce all aspects of true phosphorylation. Here, we investigated the effects of bona fide phosphorylation at Ser-13 or Ser-16 on the structure, aggregation, membrane binding, and subcellular properties of the Httex1-Q18A variant and compared these effects with those of phosphomimetic substitutions. We show that phosphorylation at either Ser-13 or Ser-16 or phosphomimetic substitutions at these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic α-helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates. In synthetic peptides, phosphorylation at Ser-13, Ser-16, or both strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Experiments with peptides bearing different combinations of phosphorylation sites within Nt17 revealed a phosphorylation-dependent switch that regulates the Httex1 structure, involving crosstalk between phosphorylation at Thr-3 and Ser-13 or Ser-16. Our results provide crucial insights into the role of phosphorylation in regulating Httex1 structure and function. They underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation and their potential as therapeutic targets for managing Huntington’s disease.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Aggregation, Chemical stability, Biochemistry