Critical Significance of the Region between Helix 1 and 2 for Efficient Dominant-Negative Inhibition by Conversion-Incompetent Prion Protein

Prion diseases are fatal infectious neurodegenerative disorders in man and animals associated with the accumulation of the pathogenic isoform PrPSc of the host-encoded prion protein (PrPc). A profound conformational change of PrPc underlies formation of PrPSc and prion propagation involves conversion of PrPc substrate by direct interaction with PrPSc template. Identifying the interfaces and modalities of inter-molecular interactions of PrPs will highly advance our understanding of prion propagation in particular and of prion-like mechanisms in general. To identify the region critical for inter-molecular interactions of PrP, we exploited here dominant-negative inhibition (DNI) effects of conversion-incompetent, internally-deleted PrP (ΔPrP) on co-expressed conversion-competent PrP. We created a series of ΔPrPs with different lengths of deletions in the region between first and second α-helix (H1∼H2) which was recently postulated to be of importance in prion species barrier and PrP fibril formation. As previously reported, ΔPrPs uniformly exhibited aberrant properties including detergent insolubility, limited protease digestion resistance, high-mannose type N-linked glycans, and intracellular localization. Although formerly controversial, we demonstrate here that ΔPrPs have a GPI anchor attached. Surprisingly, despite very similar biochemical and cell-biological properties, DNI efficiencies of ΔPrPs varied significantly, dependant on location and inversely correlated with the size of deletion. This data demonstrates that H1∼H2 and the region C-terminal to it are critically important for efficient DNI. It also suggests that this region is involved in PrP-PrP interaction and conversion of PrPC into PrPSc. To reconcile the paradox of how an intracellular PrP can exert DNI, we demonstrate that ΔPrPs are subject to both proteasomal and lysosomal/autophagic degradation pathways. Using autophagy pathways ΔPrPs obtain access to the locale of prion conversion and PrPSc recycling and can exert DNI there. This shows that the intracellular trafficking of PrPs is more complex than previously anticipated.

朊病毒疾病（Prion diseases）是一类发生于人类与动物体内的致命性传染性神经退行性疾病，其发病与宿主编码的朊蛋白（PrPc）的致病型异构体PrPSc的异常蓄积密切相关。PrPc发生显著构象变化是PrPSc形成的分子基础，而朊病毒的增殖过程则依赖于PrPc底物与PrPSc模板的直接相互作用，进而介导构象转化。阐明PrP分子间相互作用的界面与模式，将极大推动我们对朊病毒增殖机制的深入理解，同时也有助于拓展对类朊病毒机制的整体认知。

为明确PrP分子间相互作用的关键区域，本研究利用了无构象转化能力的内部缺失型PrP（ΔPrP）对共表达的具有构象转化能力的PrP所产生的显性负抑制（dominant-negative inhibition, DNI）效应。我们在第一与第二α螺旋（α-helix, H1~H2）区域构建了一系列不同缺失长度的ΔPrP，该区域此前被推测与朊病毒物种屏障及PrP原纤维形成密切相关。

如既往研究报道，所有ΔPrP均表现出异常特性：包括去污剂不溶性、有限的蛋白酶消化抗性、高甘露糖型N-连接糖基化修饰，以及细胞内定位。尽管此前存在学术争议，但本研究证实ΔPrP均带有GPI锚（GPI anchor）修饰。令人意外的是，尽管ΔPrP的生化与细胞生物学特性极为相似，但其DNI效率却存在显著差异：该效率取决于缺失位点的位置，且与缺失片段的大小呈负相关。本研究数据表明，H1~H2区域及其C端区域对于高效发挥DNI效应至关重要。该结果同时提示，该区域参与了PrP分子间相互作用以及PrPC向PrPSc的构象转化过程。

为解释细胞内定位的PrP如何发挥DNI效应这一悖论，本研究证实ΔPrP可同时通过蛋白酶体途径与溶酶体/自噬途径完成降解。通过利用自噬途径，ΔPrP能够抵达朊病毒构象转化及PrPSc循环的亚细胞位点，并在此处发挥DNI效应。该研究结果表明，PrP的细胞内运输过程远比此前预想的更为复杂。