Mutant Exon1 Huntingtin Aggregation is Regulated by T3 Phosphorylation‐Induced Structural Changes and Crosstalk between T3 Phosphorylation and Acetylation at K6

Abstract

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 posttranslational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs. Main textSeveral lines of evidence suggest that the first N-terminal 17 amino acids of the Huntingtin protein (Htt) play important roles in modulating Htt structure, oligomerization, fibrils formation, membrane binding, subcellular localization and interactions with other proteins. [1] This, combined with the fact that this short N-terminal sequence harbors several residues that are subjected to diverse posttranslational modifications (PTMs), including phosphorylation, acetylation, ubiquitination and sumoylation (Scheme S1-A in the Supporting Information), suggests that PTMs may serve as reversible molecular switches for regulating Htt structure, interactome, cellular properties and toxicity. [2] However, the lack of knowledge about the enzymes involved in regulating these modifications has made it difficult to decipher their role in regulating the function(s) of Htt in health and disease.To address these limitations and enable deciphering of the Nt17 PTM code, our group has pursued the development of semisynthetic strategies that permit site-specific introduction of single or multiple PTMs within Nt17 of Huntingtin exon 1 (Httex1). Recently, we reported a semisynthetic strategy that permits site-specific phosphorylation at T3.[3] However, this strategy suffered from several limitations and was only suitable for introducing PTMs in the first N-terminal 9 residues of wild-type Httex1 (≤23 glutamine residues) (Scheme S2). [3] To allow investigations of all Nt17 PTMs, we developed and optimized two new modular semisynthetic protocols that permit the generation of wild-type and for the first time mutant Httex1 (mHttex1) proteins containing single or multiple PTMs (Scheme S3). The first strategy (Scheme S3-A) is only suitable for introducing PTMs in the N-terminal 9 residues of Httex1, whereas the second strategy allows the investigation of all the PTMs within the Nt17 domain. Additionally, the optimized conditions enabled the semisynthesis of mutant Httex1 (full details about these semisynthetic strategies are given in the Supporting Information). This optimized strategy enabled us, for...

TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models

“…Consistent with our previous observations, the mutant HTT proteins in the insoluble protein fraction were devoid of any pS13-phosphorylated HTT ( Fig EV3I). Together, these results combined with the previous observations that phosphorylation within N17 (pT3, pS13, pS16) inhibited mutant HTTex1 aggregation (Cariulo et al, 2017;Chiki et al, 2017;Deguire et al, 2018) indicate that reduced phosphorylation at these residues may facilitate HTT aggregation. In summary, our findings show that the TBK1-induced reduction in HTT levels and aggregation is mediated by its ability to promote the clearance or prevent the aggregation of monomeric or soluble HTT species, rather than by the phosphorylation and disaggregation of HTT fibrils or inclusions.…”

“…Previous studies indicated that phosphorylation or the introduction of phosphomimetic mutations at both S13 and S16 (Gu et al, 2009;Arbez et al, 2017;Branco-Santos et al, 2017;Chiki et al, 2017;Deguire et al, 2018) suppress mutant HTT aggregation in vitro and in cellular models. However, the effects of bona fide phosphorylation on mutant HTTex1 aggregation were evaluated only in vitro using semisynthetic proteins (Gu et al, 2009;Daldin et al, 2017;Deguire et al, 2018).…”

“…The first step towards assessing the therapeutic potential of targeting N17 PTMs for the treatment of HD is the identification of the enzymes that regulate these PTMs. This is important because increasing evidence shows that phosphorylation-mimicking mutations (e.g., serine (S) or threonine (T) to aspartate (D) or glutamate (E)) used to investigate the effects of protein phosphorylation may not fully phenocopy the effects of the bona fide modifications (Szczepanowska et al, 1998;Paleologou et al, 2008;Dephoure et al, 2013;Chiki et al, 2017;Deguire et al, 2018) and do not faithfully reproduce the dynamic nature of phosphorylation.…”

TBK1 regulates autophagic clearance of soluble mutant huntingtin and inhibits aggregation/toxicity in different models of Huntington’s disease

“…4I). Together, these results combined with the previous observations that phosphorylation within N17 (pT3, pS13, pS16) inhibited mutant HTTex1 aggregation Chiki et al, 2017;Deguire et al, 2018), indicate that reduced phosphorylation at these residues may facilitate HTT aggregation. In summary, our findings show that the TBK1-mediated reduction in HTT levels and aggregation is mediated by its ability to promote the clearance or prevent the aggregation of monomeric or soluble HTT species, rather than by the phosphorylation and disaggregation of HTT fibrils or inclusions.…”

“…Previous studies indicated that phosphorylation or the introduction of phosphomimetic mutations at both S13 and S16 (Anne et al, 2007;Arbez et al, 2017;Branco-Santos et al, 2017;Chiki et al, 2017;Deguire et al, 2018) could suppress mutant HTT aggregation in vitro and in cellular models.…”