Effect of small peptide binders on huntingtin fibrillization and toxicity

项目总结 亨廷顿病是一种进行性神经退行性疾病，由基因外显子1突变引起。 亨廷顿蛋白(MHttex 1)，导致蛋白质凝结和聚集。而确切的遗传基础 这种疾病已经知道几十年了，至今仍没有治愈或有效的临床治疗方法。治疗性的 由于不清楚mHttex 1是如何发挥其毒性作用的，这一事实使开发变得复杂。结构研究 以循序渐进的方式显示mHttex 1单体聚集体，错误折叠成几个中间体 结构，最后形成具有这种疾病特征的纤维状聚集体。之前的研究已经 还表明，其中一些中间构象，如原纤维，是有毒的。为了更好地理解 这些结构，兰根实验室与理查德·罗伯茨博士合作，为 原纤维。我们的第一组多肽结合剂HD1和HD8不仅对这些有毒物质表现出高度的亲和力 原纤维，但也含有mHttex 1寡聚体，另一种早期的mHttex 1构象被认为是有毒的。甚至更多 令人兴奋的是，我们的粘合剂似乎可以防止一种被称为“播种”的现象，即引入少量的 错误折叠的mHttex 1将通过模板辅助的错误折叠来加速mHttex 1单体的纤化。在……里面 在这项提议中，我将利用我们的新型多肽结合剂来了解HD1和HD8如何阻止mHttex 1的种子 以及这些多肽结合剂对mHttex 1聚集和毒性有何影响。在《目标1》中，我将 使用生物物理方法的组合，如电子顺磁共振(EPR)、固态核 核磁共振和一种新的荧光显微镜应用于实时监测纤维生长 时间到了。这些实验将揭示HD1和HD8的播种抑制机制以及结构 这些多肽结合剂的信息，这将是未来开发治疗的重要信息。另外， 初步证据表明，在细胞培养中，mHttex 1与HD1或HD8共表达可减少 整体mHttex 1。利用细胞培养和亨廷顿病小鼠模型，Aim 2将探索如何 HD1和HD8在细胞内与mHttex 1相互作用以及这些肽结合物是否可以拯救有害的 MHttex 1的影响。这些实验将提供HD1和HD8如何确切地在 MHttex 1，并评估这些多肽结合剂的治疗潜力。

PROJECT SUMMARY Huntington’s disease is a progressive neurodegenerative disorder caused by a mutation in exon1 of the huntingtin protein (mHttex1), which causes the protein to fibrillize and aggregate. While the exact genetic basis of this disease has been known for decades, there is still no cure or effective clinical treatment. Therapeutic development is complicated by the fact that it is unclear how mHttex1 exerts its toxic effects. Structural studies have revealed mHttex1 monomer aggregates in a stepwise manner, misfolding into several intermediate structures before eventually forming the fibrillar aggregates characteristic of the disease. Previous studies have also shown that some of these intermediate conformers, such as protofibrils, are toxic. To better understand these structures, the Langen lab collaborated with Dr. Richard Roberts to make small peptide binders for protofibrils. Our first set of peptide binders, HD1 and HD8, have shown a high affinity for not only these toxic protofibrils, but also with mHttex1 oligomers, another early mHttex1 conformer posited to be toxic. Even more exciting, our binders appear to prevent a phenomenon known as “seeding,” where introducing a small amount of misfolded mHttex1 will accelerate fibrillization of mHttex1 monomer through template-assisted misfolding. In this proposal, I will leverage our novel peptide binders to learn how HD1 and HD8 prevent seeding of mHttex1 monomer, and what effect these peptide binders will have on mHttex1 aggregation and toxicity. In Aim 1, I will use a combination of biophysical methods such as electron paramagnetic resonance (EPR), solid-state nuclear magnetic resonance (ssNMR), and a new fluorescence microscopy application to monitor fibril growth in real time. These experiments will reveal both the seeding inhibition mechanism of HD1 and HD8 as well as structural information of these peptide binders, which will be important for developing future treatments. Additionally, preliminary evidence shows that co-expressing mHttex1 with HD1 or HD8 in cell culture decreases the amount of overall mHttex1. Using both cell culture and mouse models of Huntington’s disease, Aim 2 will explore how HD1 and HD8 interact with mHttex1 within the cell and whether these peptide binders can rescue the deleterious effects of mHttex1. These experiments will provide a picture of how exactly HD1 and HD8 exert their effects on mHttex1 and evaluate the therapeutic potential of these peptide binders.