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 and/or Ser-16 or phosphomimetic substitutions at both 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 residues 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 cross-talk 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, and underscore the critical importance of identifying the enzymes responsible for regulating Htt phosphorylation, and their potential as therapeutic targets for managing Huntington's disease.
Highlights
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)
We show that phosphorylation at either Ser-13 and/or Ser-16 or phosphomimetic substitutions at both these residues inhibit the aggregation of mutant Httex1, but that only phosphorylation strongly disrupts the amphipathic ␣-helix of the N terminus and prompts the internalization and nuclear targeting of preformed Httex1 aggregates
N-terminal fragments of Htt have been found in nuclear inclusion bodies in Huntington’s disease (HD) patients [12], and overexpression of exon 1 of Htt (Httex1) with expanded polyQ repeats alone is sufficient to a variable extent recapitulate many characteristics of HD in mice [13], suggesting that N-terminal Htt fragments may play a critical role in the pathogenesis of HD [14, 15]
Summary
Expressed protein ligation requires a nucleophilic cysteine residue at the N terminus of the C-terminal ligation fragment and a thioester at the C terminus of the N-terminal fragment. After 48 h, the height of the Ser 3 Asp mutant Httex aggregates had increased notably to sizes comparable with unmodified Httex1-Q42, suggesting that the introduction of phosphomimetic mutation at a single serine residue only slightly influences the final morphology of the fibrils, consistent with the ability of the WT, S13D, and S16D Httex1-Q42 to form -sheet–rich aggregates (Fig. 4) [51]. Residues did not influence the ability of Nt17 to adopt an ␣-helical conformation in the presence of POPG or 1,2-dimyristoylsn-glycero-3-phosphocholine LUVs (Fig. 9 (B and C) and Fig. S1) These results demonstrate that the phosphomimetics do not reproduce the effect of phosphorylation on Nt17 conformation upon membrane binding, highlighting the importance of using bona fide phosphorylated proteins/peptides to study the effect of this modification on the structural properties of Httex. No statistically significant differences were noted among the three phosphorylated variants, indicating that a single phosphorylation event at either Ser-13 or Ser-16 is sufficient to prompt the nuclear enrichment of Httex1-Q22 or Httex1-Q42 aggregates
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