Effect of small peptide binders on huntingtin fibrillization and toxicity

项目摘要 亨廷顿氏病是一种进行性神经退行性疾病，由亨廷顿基因外显子1突变引起。 亨廷顿蛋白（mHttex1），它导致蛋白质软化和聚集。虽然确切的遗传基础 这种疾病已经知道了几十年，仍然没有治愈或有效的临床治疗方法。治疗 由于mHttex 1如何发挥其毒性作用尚不清楚，因此发展变得复杂。结构研究 已经揭示了mHttex1单体聚集体以逐步的方式，错误折叠成几个中间体， 在最终形成疾病特征性的纤维状聚集体之前，先前的研究 还表明，这些中间构象，如原纤维，是有毒的。更好地了解 这些结构，兰根实验室与理查德罗伯茨博士合作，使小肽结合剂， 原纤维我们的第一组肽结合剂，HD1和HD8，不仅对这些有毒的 原纤维，但也与mHttex1寡聚体，另一个早期mHttex1构象假定是有毒的。更 令人兴奋的是，我们的粘合剂似乎可以防止一种称为“播种”的现象， 错折叠的mHttex 1将通过模板辅助的错折叠加速mHttex 1单体的氟化。在 在这个提议中，我将利用我们的新型肽结合剂来了解HD1和HD8如何阻止mHttex1的播种 单体，以及这些肽结合剂对mHttex1聚集和毒性的影响。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 磁共振（ssNMR），和一个新的荧光显微镜应用，以监测纤维生长在真实的 时间这些实验将揭示HD 1和HD 8的接种抑制机制以及结构 这些肽结合剂的信息，这将是重要的开发未来的治疗。此外，本发明还 初步证据显示，在细胞培养物中共表达mHttex1与HD1或HD8降低了 整体mHttex1。Aim 2将使用细胞培养和亨廷顿病小鼠模型来探索如何 HD1和HD8在细胞内与mHttex1相互作用，以及这些肽结合剂是否可以拯救有害的 mhtex1的影响。这些实验将提供一幅关于HD1和HD8如何确切地对 mHttex1，并评估这些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.