Abstract
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.
Highlights
Huntington’s disease (HD) is a fatal, autosomal neurodegenerative disease characterized by motor (Huntington, 1872; Ross and Tabrizi, 2011) and cognitive declines (Marder et al, 2000) as well as psychiatric symptoms (Paulsen et al, 2001)
HD is caused by a mutation in the huntingtin gene (HTT), resulting in an expansion in the CAG repeat within its first exon (Gusella et al, 1983; MacDonald, 1993), which is translated into an extended polyglutamine repeat in the huntingtin protein (Htt) (Kremer et al, 1994)
Investigating the biological role of M8 oxidation is more challenging than other Nt17 Post-translational modifications (PTMs), such as phosphorylation, because there are no natural amino acids that mimic methionine oxidation and factors that are known to induce protein oxidation, such as oxidative stress, are not always chemoselective
Summary
Huntington’s disease (HD) is a fatal, autosomal neurodegenerative disease characterized by motor (Huntington, 1872; Ross and Tabrizi, 2011) and cognitive declines (Marder et al, 2000) as well as psychiatric symptoms (Paulsen et al, 2001). HD is characterized by neuronal degeneration in the striatum and the cortex (Reiner et al, 1988; Rosas et al, 2003) and the formation and accumulation of nuclear inclusions composed of mutant Htt (polyQ repeat ≥36) aggregates and other proteins (DiFiglia et al, 1997; Hodgson et al, 1999). Overexpression of mutant Httex alone with polyQ length ranging from 80 to 175 reproduces many aspects of HD pathology in various animal and cellular models, including the formation of huntingtin inclusions (Mangiarini et al, 1996; Martindale et al, 1998; Scherzinger et al, 1999; Barbaro et al, 2015)
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