DataSheet1_Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.pdf

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.

已有研究证实，亨廷顿蛋白（Htt）N端前17个氨基酸残基（Nt17）区域内的翻译后修饰（PTMs）可在体外及多种亨廷顿病模型中抑制突变型Htt蛋白的聚集并减轻其毒性。本研究在此基础上，进一步探究了8位甲硫氨酸氧化（oxM8）及其与6位赖氨酸乙酰化（AcK6）或3位苏氨酸磷酸化（pT3）的串扰对突变型Httex1（mHttex1）聚集的影响。研究结果显示，M8氧化可延缓但无法完全抑制mHttex1的聚集，且对其聚集产物的最终形态无显著影响。当oxM8与AcK6同时存在时，可显著抑制Httex1的原纤维形成。圆二色谱分析与分子动力学模拟研究表明，能够降低mHttex1聚集速率的PTMs（包括oxM8、AcK6/oxM8、pT3、pT3/oxM8及13位丝氨酸磷酸化（pS13）），可增加Nt17区域内短N端螺旋（前8个氨基酸残基）的丰度，或降低其他螺旋结构（包括长螺旋及短C端螺旋）的丰度。而未对mHttex1聚集速率产生影响的PTMs（如AcK6），其对应的肽段螺旋构象分布与未修饰肽段基本一致。上述结果表明，相较于Nt17区域的整体螺旋性，N端与C端螺旋构象及长螺旋的相对丰度更能阐释不同Nt17 PTMs对mHttex1聚集的调控效应，这也阐明了体外实验中PTMs对Nt17整体螺旋性的影响与mHttex1聚集效应之间缺乏相关性的原因。综上，本研究为单一PTMs及不同PTMs间的串扰调控mHttex1聚集的差异化作用提供了全新的结构学见解。