疏水性N端信号肽及C端GPI定位信号序列对朊病毒蛋白错误折叠的影响

Prion diseases are a group of degeneration diseases which caused by misfolding of prion protein (PrP). Till now, the understanding of molecular mechanism of prion protein conformational conversion is limited. The cellular nascent synthetic Pre-pro-PrP contains N-terminal signal peptide (N-SP) and GPI anchor peptide signal sequence (GPI-PSS) which can be removed in endoplasmic reticulum (ER). However, the abnormal translocation of Pre-pro-PrP always leads to the intracellular retaining of prion protein. Ctm/Ntm-PrP are transmembrane forms of PrP located on ER membrane and cyto-PrP is located in cytoplasm. Both of the Ctm-PrP and cyto-PrP retains the N-terminal signal peptide, and cyto-PrP also contains the GPI-anchor peptide. Little is known about how the hydrophobic N-signal peptide and GPI-anchor signal sequence influence the conformational change of prion protein. In current project, we aim to study the misfolding behavior of intracellular retained prion protein. The thermal stability and secondary structure of prion proteins with or without the sequences mentioned above will be studied. Meanwhile, the aggregation dynamics will be detected by ThT fluorescence or UV turbidity assay. The PK resistance and detergent solubility assay will be applied to compare the classic biochemical feature of PrPSc-like aggregates. Furthermore, we plan to investigate the intracellular retain, location and the neuro toxicity of misfolded prion protein with N-SP or/and GPS-PSS. Combining these results, we expect to clarify the impact of N-SP and GPI-PSS on the misfolding of prion protein. These studies may provide experimental evidences for understanding the biochemical basis of sporadic and familial prion diseases, and may provide new clues for prevention and treatment of prion diseases and other neuro-degenerative diseases.

朊病毒病是一类由朊病毒蛋白错误折叠引起的神经退变性疾病，然而朊病毒蛋白PrPC最初自发变构形成异常的具有神经毒性的病理型构象PrPSc的原因目前仍不清楚。细胞内合成的前体朊病毒蛋白在分泌过程中的异常通常会导致其在细胞内滞留，形成跨内质网膜的Ctm/Ntm-PrP或游离在胞浆中的cyto-PrP形式。我们前期的研究也发现朊病毒蛋白病理突变体E200K在细胞内的滞留及聚集（Wang Z, Xiao GF & Zhou Z, BBA 2016）。胞内滞留型朊病毒蛋白通常保留N端信号肽区域或/及C端GPI定位信号序列，这些疏水序列对于朊病毒蛋白错误折叠的影响目前还鲜有研究。本项目拟结合蛋白质生化技术研究N端信号肽及C端GPI定位信号序列等在朊病毒蛋白错误折叠中的作用，并在细胞层面研究这些区域对神经细胞中朊病毒蛋白聚集、定位及毒性的影响。从上述角度为理解朊病毒蛋白自发错误折叠致病的分子机制提供线索。

朊病毒（Prion）又称蛋白质感染颗粒，是只含有蛋白质不含有核酸的亚病毒。朊病毒蛋白由α-螺旋为主的天然构象转变为以β-折叠为主的异常构象与朊病毒疾病的发生发展密切相关。朊病毒蛋白信号肽区域主要由疏水性氨基酸组成，其对于朊病毒蛋白错误折叠的影响目前研究相对较少。圆二色谱结果表明信号肽PrP1-28在六氟异丙醇中显示出典型α-螺旋结构特征，而在水溶液中其α-螺旋含量显著降低而β-折叠含量显著增加。水溶液中PrP1-28具有一定的蛋白酶K抗性且其ThT结合荧光信号有显著的增强，表现出淀粉样纤维的结构特征。PrP1-28与PrP23-231共孵育一定程度上影响了PrP23-231的折叠途径，略微缩短了纤维化的延滞期，且增强了纤维化聚集的ThT荧光强度。PrP1-28与PrP23-232共孵育形成的纤维化聚集具有较强的蛋白酶K抗性。圆二色谱及微量热泳动实验表明PrP1-28能与带负电POPS脂质体特异性结合，且导致其二级结构进一步转变为β-折叠占主构象。朊病毒蛋白PrP23-231同样可以与POPS特异性结合且导致α-螺旋丧失及β-折叠含量的显著增加。同时，POPS的结合能够不同程度增强PrP23-231及信号肽PrP1-28样品的ThT荧光信号，反映出类淀粉样纤维化聚集的生成。然而，电中性POPC脂质体对PrP1-28及PrP23-231的结构均无显著影响。在细胞水平研究结果表明经内质网转运抑制剂处理后，朊病毒蛋白在细胞内的聚集表现出显著的细胞毒性。此外，研究发现没食子酸衍生物鞣花酸及五没食子酰葡萄糖能够与朊病毒蛋白结合并抑制朊病毒蛋白纤维化聚集的形成。在鞣花酸或五没食子酰葡萄糖存在下朊病毒蛋白形成的聚集体蛋白酶K抗性较弱且能够被去垢剂降解。上述研究结果为理解疏水信号肽对朊病毒蛋白错误折叠聚集的影响提供了一定的实验证据，为朊病毒疾病及相关蛋白质构象病的治疗提供了重要线索。