Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington’s disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.