Molecular Mechanisms of Prion Protein Amyloid Formation

朊病毒蛋白淀粉样蛋白形成的分子机制

• 批准号: 10692139
• 负责人: SUZETTE Alise PRIOLA
• 金额: $ 38.83万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10692139
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid Proteins; Amyloid beta-Protein; Annual Reports; Biochemistry; Biological Assay; Biological Models; Bovine Spongiform Encephalopathy; Brain; Cell-Free System; Cells; Chronic Wasting Disease; Complex; Creutzfeldt-Jakob Syndrome; Data; Deer; Deposition; Diffuse; Disease; Equine mule; Exposure to; Familial Creutzfeldt-Jakob Disease; Human; Iatrogenesis; In Vitro; Incidence; Infection; Infectious Agent; Laboratories; Manuscripts; Medical; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Parkinson Disease; Pathogenesis; Pathogenicity; Pathway interactions; Peptide Hydrolases; Persons; Physiological; Postdoctoral Fellow; PrP; PrP amyloid; PrP gene; PrPSc Proteins; Prion Diseases; Prions; Process; Proteins; Resistance; Science; Scrapie; Sheep; Transgenic Mice; Work; Writing; alpha synuclein; amyloid formation; base; experimental study; in vivo Model; insight; instrument; interest; misfolded protein; mouse model; prion-like; protein aggregation; protein folding; protein misfolding; tau Proteins; uptake

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of CJD cases and occurs randomly at an incidence of 1-2 per million people worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. The infectious agent of prion diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. PrPSc can be deposited in the brain as either diffuse amyloid negative deposits or as dense amyloid positive deposits. For reasons that are not yet clear, amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unknown if prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. Multiple studies have shown that amyloid formed from amyloid beta (Abeta) protein, alpha synuclein and tau also propagate via prion-like mechanisms and spread from cell-to-cell in transgenic mouse models (e.g. Science 313: 1781-1784 (2006), Nat Cell Biol 11: 909-913 (2009), J Exp Med 209: 975-986 (2012)). Based on these data, it has been suggested that amyloid formation in neurodegenerative proteinopathies such as Alzheimers Disease (AD) and Parkinsons disease (PD) occurs via prion-like mechanisms and that proteins such as AD-associated Abeta may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and Abeta to plaques in some cases of sCJD (ACTA Neuropathol 96:116-122 (1998)), also suggest that interactions between these proteins could contribute to disease pathogenesis. Laboratory models of prion infection therefore represent a way of studying prion and prion-like mechanisms of disease that can be applied to other neurodegenerative diseases triggered by misfolded proteins. 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 primarily on 1) understanding the pathways of PrP amyloid formation and spread and, 2) understanding how protein aggregation and disaggregation are controlled by the cell. Since PrPSc formation and spread appear to be mechanistically similar to the formation and spread of amyloid in other neurodegenerative diseases, the results of our prion studies will likely be broadly applicable to other diseases of protein misfolding and deposition. The ordered aggregation of PrPSc, Abeta, and other amyloid proteins during neurodegeneration is thought to be critical to the pathogenesis of neurodegenerative protein misfolding diseases such as prion disease and AD. However, the processes by which these aggregates form and the mechanisms by which the cell can degrade them remains poorly understood. In earlier studies of how prions interact with cells, we showed that the uptake and disaggregation of prions varied by prion strain (J. Virol. 87: 11552-61 (2013), Annual Report 2013; Am. J. Pathol. 184: 3299-3307 (2014), Annual Report 2014) suggesting that the composition of PrPSc aggregates differed between strains. The post-doctoral fellow in the lab, Dr. Daniel Shoup, has demonstrated that the sizes and stabilities of PrPSc change during cellular uptake and degradation and that these changes vary with the prion strain, potentially impacting the ability of a given prion strain to infect cells (Shoup and Priola, Biochemistry 60: 398-411 (2021), Annual Report 2021). In 2022, Dr. Shoup continued his work on this project. His new data show that the conformations of both amyloid and non-amyloid forms of PrPSc differ but that they are similarly altered upon initial uptake and degradation by the cell. His data also suggest that these structural changes occur in different cellular microenvironments. He is currently writing a manuscript based on these results which should be submitted before the end of the year. In 2022, Dr. Shoup continued experiments developing an in vitro protein re-folding assay using purified mammalian chaperones. He successfully purified large amounts of several different chaperone and co-chaperone proteins. He has also purified PrPSc from two different prion strains, one that forms amyloid and one that does not. Dr. Shoup is now optimizing the conditions for using one of these chaperones in a protein folding/re-folding assay. His initial experiments have shown that the chaperone can unfold non-amyloid PrPSc in vitro. He will continue to use his cell-free system to study how PrPSc aggregates from different prion strains are unfolded and refolded by cellular chaperones under physiological conditions. These studies will provide important insights into which chaperones can interact with PrPSc as well as how that interaction leads to disassembly and degradation of PrPSc aggregates.

可传播的海绵状脑病（TSE或PRION疾病）是一组罕见的神经退行性疾病，包括绵羊中的刮饼，牛海绵状脑病（BSE）和M子鹿和Elk中的牛皮脑病（BSE）和慢性浪费疾病（CWD）。在人类中，最常见的prion病类型是可以以几种形式发生的克鲁特兹菲尔特 - 贾科布疾病（CJD）。 零星的CJD（SCJD）构成了大多数CJD案件，并且在全球范围内以每百万人民为1-2人的发病率随机发生。医源性CJD（ICJD）与暴露于Prion受污染的医疗工具或产品有关，而家族性CJD（FCJD）与Prion蛋白基因中的突变有关。 Prion疾病的感染因子称为prion，主要由正常的，蛋白酶敏感的prion蛋白PRPC的异常重塑，抗蛋白酶抗性形式（PRPSC）组成。 PRPSC可以作为弥漫性淀粉样蛋白负沉积物或致密淀粉样蛋白阳性沉积物沉积在大脑中。由于尚不清楚的原因，淀粉样蛋白的疾病形式似乎不如非淀粉样蛋白形式传播。此外，尚不清楚PRPSC主要作为淀粉样蛋白的pr疾病是否遵循与PRPSC主要沉积为非淀粉样蛋白的prion疾病相同的致病过程。 多项研究表明，由淀粉样蛋白（ABETA）蛋白，α-突触核蛋白和Tau形成的淀粉样也通过类似prion的机制传播并从细胞到细胞中传播，在转基因小鼠模型中（例如Science 313：1781-1784（2006），NAT Cell Biol 11：909-913-913（2009），J 909-99（2009）。 基于这些数据，已经提出，诸如阿尔茨海默氏病（AD）和帕金森氏病（PD）的神经退行性蛋白质疾病中的淀粉样蛋白形成是通过类似prion的机制以及诸如AD相关的Abeta之类的蛋白质发生的。 神经退行性期间错误折叠蛋白的共沉积，例如在某些SCJD的情况下，PRPSC和ABETA与斑块共定位（ACTA神经性疾病96：116-122（1998）），也表明这些蛋白质之间的相互作用可能有助于疾病病原体。因此，prion感染的实验室模型代表了一种研究疾病的prion和prion样机制的方法，可应用于其他神经退行性疾病，这是由错误折叠蛋白触发的其他神经退行性疾病。 我们有兴趣了解PRP淀粉样蛋白形成的分子机制，并开始使用体外和体内模型系统来解决此问题。该项目主要关注1）了解PRP淀粉样蛋白形成和扩散的途径，以及2）了解蛋白质聚集和分解如何由细胞控制。 由于PRPSC的形成和扩散似乎在机械上与淀粉样蛋白在其他神经退行性疾病中的形成和扩散相似，因此我们的prion研究结果可能广泛适用于其他蛋白质错误折叠和沉积的疾病。 神经退行性过程中PRPSC，ABETA和其他淀粉样蛋白的有序聚集被认为对神经退行性蛋白误折叠疾病（如prion病和AD）的发病机理至关重要。 但是，这些聚集体形成的过程以及细胞可以降解它们的机制仍然很少理解。 在对王子如何与细胞相互作用的早期研究中，我们表明，prion毒的摄取和分裂因pr菌株而异（J.Virol。87：11552-61（2013），2013年年度报告；Am。J.Pathol。184：3299-3307（2014），2014年年度报告，2014年年度报告，表明PRPS PRPC PRECC的组成不同。 实验室的博士后研究员丹尼尔·舒普（Daniel Shoup）博士证明，PRPSC在细胞摄取和降解过程中的大小和稳定性随着prion菌的菌株的变化而变化，可能会影响给定的prion菌菌株对感染细胞的能力（Shoup和Priola，Priola，BioChemistry，Biochemistry，Biochemistry 60：398-411）（20221），每年2021年），2021年），2021年），2021年），2021年）。 2022年，Shoup博士继续对该项目进行工作。他的新数据表明，PRPSC的淀粉样蛋白和非淀粉样蛋白形式的构象不同，但在细胞初始摄取和降解时，它们类似地改变了它们。他的数据还表明，这些结构变化发生在不同的细胞微环境中。他目前正在根据这些结果撰写手稿，该结果应在年底之前提交。 2022年，Shoup博士继续实验使用纯化的哺乳动物伴侣开发体外蛋白质重折叠测定。他成功地纯化了大量几个不同的伴侣和伴侣蛋白。他还从两种不同的prion菌株中纯化了PRPSC，一种菌株形成淀粉样蛋白，另一种不形成淀粉样蛋白。 Shoup博士现在正在优化在蛋白质折叠/重折叠测定中使用这些伴侣之一的条件。他的最初实验表明，伴侣可以在体外展开非淀粉样蛋白PRPSC。他将继续使用他的无细胞系统来研究如何在生理条件下通过细胞伴侣展开和重塑来自不同prion菌株的PRPSC聚集体。这些研究将提供重要的见解，伴侣可以与PRPSC相互作用，以及这种相互作用如何导致PRPSC聚集体的分解和降解。