Molecular Mechanisms of Prion Protein Amyloid Formation

传染性海绵状脑病(TSE)是一组罕见的神经退行性疾病，包括人类的克雅氏病(CJD)、绵羊的瘙痒病、牛海绵状脑病(BSE)和马鹿、麋鹿的慢性衰弱病(CWD)。寨卡病毒的传染性可以跨越物种障碍。疯牛病已在英国感染人类，并担心慢性萎缩性胃病在美国可能会有类似的表现，这一事实突显了了解TSE发病机制和开发有效的抗TSE疗法的重要性。TSE疾病的感染源的确切性质尚不清楚。感染的易感性可能受到正常宿主蛋白(PrP-sen)和该蛋白的异常蛋白酶K抗性形式PrP-res之间的氨基酸同源性的影响。PrP-RES的形成与传染性密切相关，PrP-RES被认为是TSE疾病的感染源。然而，PrP-res可以以弥漫的淀粉样阴性沉积或致密的淀粉样阳性沉积的形式沉积在大脑中。淀粉样型的TSE似乎比非淀粉样型的传染性更差，这表明它们引发疾病的方式有根本的不同。此外，目前尚不清楚PrP-RES主要以淀粉样蛋白沉积的TSE疾病是否遵循与PrP-RES以非淀粉样蛋白形式沉积的TSE疾病相同的致病过程。我们对了解PrP淀粉样蛋白形成的分子机制很感兴趣，并已开始使用体外和体内模型系统来探讨这一问题。本项目的重点是：1)了解PrP淀粉样蛋白的形成途径；2)研究PrP基因突变如何影响家族性TSE病PrP-res淀粉样蛋白的形成。 2009年，我们在无细胞纤化系统中使用PrP多肽来研究PrP分子的不同区域如何影响PrP淀粉样蛋白的形成。我们还用不同类型的人TSE感染了表达没有GPI锚点的人PrP-sen的小鼠。我们的期望是，如果这些小鼠发展成PrP淀粉样蛋白，我们可以利用它们作为人类淀粉样蛋白疾病的体内模型系统。 遗传性TSE病与PrP基因内的突变有关。这些突变之一是在PrP中插入额外的8个氨基酸基序的拷贝(八肽重复)。我们使用了这种遗传突变的体外纤化模型来研究重复区域如何影响PrP-RES和PrP淀粉样蛋白的形成。去年，我们证明了环境条件可以改变纤维形成的机制，进而改变淀粉样蛋白的超微结构特性。2009年，我们收集的数据表明，八肽重复区域可以显著影响PrP-RES和PrP淀粉样蛋白的最终结构。八肽重复区域的结构影响似乎是菌株特有的，因此可能有助于解释与不同类型的TSE感染性相关的不同病理。

Transmissible spongiform encephalopathies (TSE) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. TSE infectivity can cross species barriers. The fact that BSE has infected humans in Great Britain and concerns that CWD may act similarly in the US underscores the importance of understanding TSE pathogenesis and developing effective anti-TSE therapeutics. The precise nature of the infectious agent of the TSE diseases is unknown. Susceptibility to infection can be influenced by amino acid homology between a normal host protein (PrP-sen) and the abnormal proteinase K-resistant form of this protein, PrP-res. Formation of PrP-res is closely associated with infectivity and PrP-res has been hypothesized to be the infectious agent in the TSE diseases. However, PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of TSE appear to be less transmissible than non-amyloid forms, suggesting that there is a fundamental difference in how they trigger disease. Furthermore, it is unclear whether or not TSE diseases where PrP-res is deposited primarily as amyloid follow the same pathogenic processes as TSE diseases where PrP-res is deposited as non-amyloid forms. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrP-res amyloid formation in familial forms of TSE disease. In 2009, we used PrP peptides in a cell-free system of fibrillization to examine how different regions of the PrP molecule may influence PrP amyloid formation. We have also infected mice that express human PrP-sen without the GPI anchor with different types of human TSE. Our expectation is that, if these mice develop PrP amyloid, we can utilize them as an in vivo model system for human amyloid disease. Hereditary forms of TSE disease are associated with mutations within the PrP gene. One of these mutations is the insertion of extra copies of an eight amino acid motif (octapeptide repeat) into PrP. We have used an in vitro fibrillization model of this hereditary mutation to study how the repeat region influences the formation of both PrP-res and PrP amyloid. Last year, we demonstrated that environmental conditions could change the mechanism of fibril formation which in turn altered the ultrastructural properties of the amyloid. In 2009, we have gathered data suggesting that the octapeptide repeat region can significantly influence the final structure of both PrP-res and PrP amyloid. The structural influence of the octapeptide repeat region appears to be strain specific and thus may help to explain the different pathologies associated with different types of TSE infectivity.