Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
J Mol Biol. 2012 Nov 23;424(1-2):1-14. doi: 10.1016/j.jmb.2012.09.011. Epub 2012 Sep 18.
Aggregation of expanded polyglutamine repeat-containing fragments of the huntingtin (htt) protein may play a key role in Huntington's disease. Consistent with this hypothesis, two Ser-to-Asp mutations in the 17-amino-acid N-terminal htt(NT) segment abrogate both visible brain aggregates and disease symptoms in a full-length Q(97) htt mouse model while compromising aggregation kinetics and aggregate morphology in an htt fragment in vitro [Gu et al. (2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron64, 828-840]. The htt(NT) segment has been shown to play a critical role in facilitating nucleation of amyloid formation in htt N-terminal exon1 fragments. We show here how these Ser-to-Asp mutations dramatically affect aggregation kinetics and aggregate structural integrity. First, these negatively charged Ser replacements impair the assembly of the α-helical oligomers that play a critical role in htt amyloid nucleation, thus providing an explanation for reduced amyloid formation rates. Second, these sequence modifications alter aggregate morphology, decrease aggregate stability, and enhance the steric accessibility of the htt(NT) segment within the aggregates. Together, these changes make the sequence-modified peptides kinetically and thermodynamically less likely to aggregate and more susceptible, if they do, to posttranslational modifications and degradation. These effects also show how phosphorylation of a protein might achieve cellular effects via direct impacts on the protein's aggregation properties. In fact, preliminary studies on exon1-like molecules containing phosphoryl-Ser residues at positions 13 and 16 show that they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser-to-Asp mutants.
扩展聚谷氨酰胺重复的 huntingtin（htt）蛋白片段的聚集可能在亨廷顿病中起关键作用。与这一假说一致，htt（NT）片段中的 17 个氨基酸 N 端的两个 Ser-to-Asp 突变消除了全长 Q（97）htt 小鼠模型中的可见脑聚集物和疾病症状，同时在 htt 片段体外也改变了聚集动力学和聚集形态[Gu 等人（2009）。Ser13 和 Ser16 是全长人突变 huntingtin 在 HD 小鼠中诱导疾病发病机制的关键决定因素。神经元 64,828-840]。htt（NT）片段已被证明在促进 htt N 端外显子 1 片段中淀粉样形成的成核中起着关键作用。我们在这里展示了这些 Ser-to-Asp 突变如何显著影响聚集动力学和聚集结构完整性。首先，这些带负电荷的 Ser 取代物损害了在 htt 淀粉样形成核中起关键作用的α-螺旋寡聚物的组装，从而解释了形成淀粉样蛋白的速率降低。其次，这些序列修饰改变了聚集形态，降低了聚集稳定性，并增加了 htt（NT）片段在聚集物中的空间可达性。总之，这些变化使序列修饰的肽在动力学和热力学上更不容易聚集，如果聚集，也更容易发生翻译后修饰和降解。这些变化还展示了蛋白质磷酸化如何通过直接影响蛋白质的聚集特性来实现细胞效应。事实上，对含有 13 位和 16 位磷酸化 Ser 残基的exon1 样分子的初步研究表明，它们降低了聚集速率并产生了类似 Ser-to-Asp 突变体的非典型聚集形态。
