Molecular Mechanisms of Prion Protein Amyloid Formation

朊病毒蛋白淀粉样蛋白形成的分子机制

基本信息

批准号：
10272166
负责人：
SUZETTE Alise PRIOLA
金额：
$ 12.73万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10272166
关键词：
2019-nCoV Alzheimer&apos s Disease Amino Acids Amyloid Amyloid Proteins Amyloid beta-Protein Animals Annual Reports Biological Assay Biological Models Bovine Spongiform Encephalopathy Brain Brain region Cell-Free System Cells Chronic Wasting Disease Collaborations Complex Creutzfeldt-Jakob Syndrome Data Data Set Deer Deposition Development Diffuse Disease Endopeptidase K Equine mule Experimental Models Exposure to Familial Creutzfeldt-Jakob Disease Genetic Polymorphism Goals Human Iatrogenesis In Vitro Incidence Infection Infectious Agent Maps Mass Spectrum Analysis Medical Molecular Molecular Chaperones Mutation Nerve Degeneration Neurodegenerative Disorders Neurologic Parkinson Disease Pathogenesis Pathogenicity Pathway interactions Peptide Hydrolases Physiological Population Postdoctoral Fellow PrP PrP amyloid PrP gene PrPSc Proteins Prion Diseases Prions Process Proteins Proteomics Resistance Sampling Science Scrapie Sheep Site Statistical Data Interpretation Structure System Techniques Time Transgenic Mice Viral Work alpha synuclein amyloid formation base cell type design disease phenotype experimental study falls hyperphosphorylated tau in vivo Model inhibitor/antagonist insight instrument interest mass spectrometer misfolded protein mouse model nonhuman primate prion-like protein expression protein misfolding protein structure receptor tau Proteins uptake

项目摘要

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 TSE 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 whether or not 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 (A) protein, alpha synuclein and tau can propagate via a prion-like mechanism 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 A may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and A 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. 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 spread, 2) understanding how amyloid aggregation and disaggregation are controlled by the cell and, 3) studying how mutations and amino acid polymorphisms in PrP influence PrPSc amyloid formation in familial forms of prion disease. 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. Different proteinase K (PK) cleavage sites in the N-terminus of PrPSc are indicative of differences in its structure. Based on the PK cleavage sites, two major structural forms of PrPSc have been identified in sCJD: Type 1 and Type 2. Recently, it has been found that prions in many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc, suggesting that there may be a complex population of PrPSc molecules present many of which have different secondary structures (Brain 132: 2643 (2009)). Our project involves using LC-MS/MS Mass Spectrometry (MS) to precisely map the N-termini of PrPSc molecules associated with different neurological subtypes of CJD. We have completed about one-third of our experimental samples. In 2020, progress on this project was halted due to insurmountable design flaws in our Agilent 6550 iFunnel Q-TOF mass spectrometer. As a result, we have spent a considerable amount of time in 2020 arranging with Agilent for our current machine to be swapped for the much more reliable 6545XT Q-TOF. The new machine should be online in Fall 2020 and we should be able to resume sample analysis on this project. The ultimate goal of this study is to determine whether certain structural populations of PrPSc correlate with specific CJD phenotypes. We have an ongoing collaboration with Dr. Pedro Piccardo using MS to study BSE-infected non-human primates (NHP). These animals develop a neurodegenerative disease characterized by accumulation of PrPSc, hyper-phosphorylated tau, and alpha synuclein (J Gen Virol 95:1612-16-18 (2014)) in some brain regions but not others. We have used MS to do a proteomics study to try to determine the potential molecular mechanisms underlying the different disease pathogenesis observed in two different regions of the brain. Statistical analysis of the proteomics data revealed that some of the data sets were less robust than others, an issue directly related to the intractable problems associated with the Agilent 6550 iFunnel Q-TOF mass spectrometer noted in the previous paragraph. In 2020, we were unable to move forward with this project due to the problems with our mass spectrometer and the fact that other we cannot send prion-contaminated samples to outside entities for analysis by mass spectrometry. A new Orbitrap mass spectrometer is being installed at RML in Fall 2020 which will enable us to do a more accurate quantitation of these samples and identify differences in protein expression that will then be confirmed using non-MS based techniques. This experimental model will enable us to better understand the molecular mechanisms behind neurodegeneration in complex proteinopathies. The ordered aggregation of PrPSc, A, or other amyloid proteins during neurodegeneration is thought to be critical to the pathogenesis of 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 these aggregates remains poorly understood. In earlier studies of how prions interact with cells, we showed that the uptake and disaggregation of prions varied by 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. In 2020, post-doctoral fellow Dr. Daniel Shoup continued a project to study the processes involved in PrPSc aggregation and disaggregation using both cell-based and cell-free systems. He completed experiments looking at how cells disaggregate PrPSc from different prion strains during the initial stages of prion infection. In addition, he developed an in vitro protein re-folding assay using purified mammalian chaperones. He has begun to use this 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 how cellular chaperones interact with PrP aggregates. In addition, since chaperones can be cell type specific, his work may also help to elucidate why some cells are highly susceptible to prion infection while others are not. Finally, in 2020 Dr. Shoup also initiated studies to use his in vitro protein re-folding system to study the interactions between the SARS-CoV2 viral spike protein and its cellular receptor ACE2. These studies will help us to understand how changes in spike protein structure allow SARS-CoV2 to enter cells and provide an easily manipulatable in vitro system to identify inhibitors of the spike protein/ACE2 interaction.

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。 TSE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是否主要作为淀粉样蛋白的病毒疾病遵循与PRPSC主要沉积为非淀粉样蛋白相同的致病过程。多项研究表明，由淀粉样蛋白（A）蛋白质，α-突触核蛋白和Tau形成的淀粉样蛋白可以通过类似prion的机制传播并从细胞基因中的细胞到细胞传播（例如Science 313：1781-1784（2006），2006年（2006年），NAT Cell Biol 11：909-913（2009），J.9799999。97999。9999。979999。9799。99（99）。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病中的淀粉样蛋白形成是通过类似prion的机制发生的，并且诸如AD相关A之类的蛋白质也可能是可传播的，感染性的蛋白质也可能是可传播的。神经退行性过程中错误折叠蛋白的共沉积，例如在某些SCJD的情况下，PRPSC和A对斑的共定位（ACTA Neuropathol 96：116：116-122（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病原体。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。该项目的重点是1）了解PRP淀粉样蛋白形成和扩散的途径，2）了解如何控制淀粉样蛋白的聚集和分裂，以及3）研究PRP中的突变和氨基酸多态性如何影响PRPSC PRPSC淀粉样蛋白在家族形式的prion疾病形式中。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。 PRPSC N末端中不同蛋白酶K（PK）切割位点表明其结构差异。基于PK裂解位点，在SCJD：1型和2型中已经确定了PRPSC的两种主要结构形式。最近，已经发现，在许多SCJD中，prions是1型和2型PRPSC的混合物，表明PRPSC分子可能存在许多不同的二级结构（Brain 132：2643：2643）（2009）（2009）。我们的项目涉及使用LC-MS/MS质谱法（MS）来精确绘制与CJD不同神经系统亚型相关的PRPSC分子的N末端。我们已经完成了大约三分之一的实验样本。在2020年，由于我们Agilent 6550 Ifunnel Q-TOF质谱仪的设计缺陷，该项目的进展被停止了。结果，我们在2020年花费了大量时间与安捷伦（Agilent）安排，以使我们当前的机器被交换为更可靠的6545xt Q-TOF。新机器应在2020年秋季在线，我们应该能够恢复该项目的样本分析。这项研究的最终目标是确定PRPSC的某些结构种群是否与特定的CJD表型相关。我们使用MS与Pedro Piccardo博士进行了持续的合作，以研究BSE感染的非人类灵长类动物（NHP）。这些动物发展出一种神经退行性疾病，其特征是PRPSC，高磷酸化的TAU和α突触核蛋白（J Gen Virol 95：1612-16-18（2014））在某些大脑区域而不是其他区域而不是其他。我们已经使用MS进行了一项蛋白质组学研究，以确定在大脑两个不同区域观察到的不同疾病发病机理的潜在分子机制。蛋白质组学数据的统计分析表明，某些数据集比其他数据集更强，这是与上一段中指出的与Agilent 6550 Ifunnel Q-TOF质谱仪相关的棘手问题直接相关的问题。在2020年，由于我们的质谱仪的问题以及其他我们无法将受prion污染的样品发送到外部实体以进行质谱分析，因此我们无法向前推进该项目。 2020年秋季，正在RML安装一个新的Orbitrap质谱仪，这将使我们能够对这些样品进行更精确的定量，并确定蛋白质表达的差异，然后使用基于非MS的技术进行确认。该实验模型将使我们能够更好地了解复杂蛋白质病中神经退行性背后的分子机制。神经退行性期间PRPSC，A或其他淀粉样蛋白的有序聚集被认为对蛋白质错误折叠疾病（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这些聚集体的机制仍然很少了解。在对王室如何与细胞相互作用的早期研究中，我们表明，prions的吸收和分裂因应变而异（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Prpsc crotegredecreded strains strains strains and strains and strains。 2020年，博士后研究员丹尼尔·舒普（Daniel Shoup）继续进行了一个项目，研究了使用基于细胞和无细胞的系统的PRPSC聚集和分类的过程。他完成了实验，研究细胞如何在prion感染的初始阶段将PRPSC与不同的prion菌株分解。此外，他使用纯化的哺乳动物伴侣开发了一种体外蛋白质重折叠测定。他已经开始使用这种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。这些研究将提供有关细胞伴侣如何与PRP聚集体相互作用的重要见解。此外，由于伴侣可能是特定于细胞类型的细胞类型，因此他的工作也可能有助于阐明某些细胞对prion感染的高度敏感，而另一些细胞则不容易受到感染。最后，Shoup博士在2020年还开始了研究，以使用其体外蛋白重折叠系统研究SARS-COV2病毒峰值蛋白与其细胞受体ACE2之间的相互作用。这些研究将帮助我们了解峰值蛋白结构的变化如何使SARS-COV2进入细胞，并提供易于操纵的体外系统，以鉴定峰值蛋白/ACE2相互作用的抑制剂。