TTR and Abeta: An Amyloid Regulatory Network?

TTR 和 Abeta：淀粉样蛋白监管网络？

• 批准号: 0930102
• 负责人: Regina Murphy
• 金额: $ 26.78万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0930102&HistoricalAwards=false
• 关键词: TTR; Abeta; Amyloid; Regulatory; Network

0930102MurphyAlzheimer's disease is the most common age-associated neurodegenerative disease. According to the "amyloid cascade hypothesis", aggregation of a protein, Abeta, is an essential step leading to death of neurons. Deposition of fibrillar aggregates of Abeta in the brain tissue of Alzheimer patients is considered one of the defining characteristic features of the disease. Recent evidence indicates that soluble Abeta oligomers that appear during the course of aggregation, rather than the final mature fibrils, are the most toxic. Studies with transgenic mice indicate that transthyretin, a protein present in blood and cerebrospinal fluid, protects neurons against the devastating damage caused by Abeta aggregates. These studies raise several questions: by what mechanism does transthyretin prevent Abeta toxicity? Is this protective mechanism lost when Alzheimer's disease develops? Are there pharmacological methods to restore transthyretin?s protective abilities?Preliminary data suggest that transthyretin inhibits Abeta toxicity by accelerating Abeta aggregation, thereby reducing the quantity of soluble toxic intermediates. Transthyretin in the blood or cerebrospinal fluid is normally chemically modified at a cysteine residue, and there are over 80 transthyretin mutants that are known to exist. Early data indicate that chemical modification or mutation can strongly influence the interaction of tranthyretin with Abeta, by changing the stability of the transthyretin tetramer. This research will further explore this hypothesis, and will elucidate the mechanism by which transthyretin affects Abeta aggregation. To accomplish this goal, transthyretin will be produced recombinantly and will be chemically modified at the cysteine residue, to mimic the normal blood protein. Additionally, several mutants will be generated by site-directed mutagenesis; these mutants differ by one or two amino acids from the wild-type but have different stabilities. The structure and stability of these transthyretin "variants" will be examined experimentally using mass spectrometry, circular dichroism, fluorescence spectroscopy and related techniques. Aggregation of Abeta in the presence of transthyretin will be measured by laser light scattering, size exclusion chromatography, and electron microscopy. The data will be used to develop mathematical models that describe the association of transthyretin and Abeta and the effect of these associations on Abeta aggregation kinetics. Additionally, small drug-like compounds that bind to transthyretin and increase its stability will be tested to determine if their use enhances the interaction between transthyretin and Abeta. Interestingly, transthyretin itself will aggregate under certain conditions; transthyretin fibril deposition is associated with an age-related disease called senile systemic amyloidosis. Thus, transthyretin is potentially amyloidogenic, yet it protects against the toxicity of another amyloidogenic protein, Abeta. Is it possible that other amyloidogenic proteins also interact with Abeta? Is there an amyloidogenic network of proteins, where proteins regulate each other's aggregation and toxicity? This novel concept will be explored by initiating studies with insulin and related proteins and Abeta. Insulin is a hexameric protein that will dissociate and form fibrils under certain conditions, and there is some limited data suggesting that insulin-related growth factors are, like transthyretin, protective against Abeta toxicity. Factors that regulate the efficacy of transthyretin as a protective agent against Abeta toxicity will be identified. Results from these studies could be used to design compounds with pharmacological activity that stabilize transthyretin and enhance its efficacy. Such compounds would represent a completely novel strategy for preventing Alzheimer's disease. On a more fundamental level, the research could lend support to the notion of a network of amyloidogenic and anti-amyloidogenic proteins that regulate each other's behavior. Such a concept is highly speculative, but identification of such a network, if it exists, would open up many new lines of inquiry and new approaches for preventing a broad range of aggregation-related diseases.Graduate students on this project are broadly educated in modern experimental approaches in protein folding and aggregation, and prepared to take leading roles in academia or in the biotechnology industry. Through active participation in a summer research program, underrepresented minority undergraduates will gain valuable experience and enjoy a sense of accomplishment that will contribute to their continued enthusiasm for research careers.

老年痴呆症是最常见的年龄相关性神经退行性疾病。根据“淀粉样级联假说”，蛋白质Abeta的聚集是导致神经元死亡的重要步骤。阿尔茨海默病患者脑组织中Abeta纤维状聚集体的沉积被认为是该疾病的定义性特征之一。最近的证据表明，在聚集过程中出现的可溶性Abeta寡聚体，而不是最终成熟的原纤维，是最有毒的。对转基因小鼠的研究表明，存在于血液和脑脊液中的一种蛋白质--甲状腺素运载蛋白，可以保护神经元免受Abeta聚集体造成的破坏性损伤。这些研究提出了几个问题：甲状腺素运载蛋白通过什么机制预防Abeta毒性？当阿尔茨海默病发展时，这种保护机制会丧失吗？是否有药理学方法来恢复甲状腺素运载蛋白？保护能力？初步数据表明，甲状腺素运载蛋白通过加速Abeta聚集来抑制Abeta毒性，从而减少可溶性毒性中间体的量。血液或脑脊液中的甲状腺素运载蛋白通常在半胱氨酸残基处进行化学修饰，已知存在超过80种甲状腺素运载蛋白突变体。早期的数据表明，化学修饰或突变可以通过改变甲状腺素运载蛋白四聚体的稳定性来强烈影响甲状腺素运载蛋白与Abeta的相互作用。本研究将进一步探讨这一假说，并阐明甲状腺素运载蛋白影响Abeta聚集的机制。为了实现这一目标，将重组产生甲状腺素运载蛋白，并在半胱氨酸残基处进行化学修饰，以模拟正常的血液蛋白。此外，将通过定点诱变产生几种突变体;这些突变体与野生型相差一个或两个氨基酸，但具有不同的稳定性。这些甲状腺素运载蛋白“变体”的结构和稳定性将通过质谱、圆二色性、荧光光谱和相关技术进行实验研究。在存在甲状腺素运载蛋白的情况下，将通过激光散射、尺寸排阻色谱法和电子显微镜测量Abeta的聚集。这些数据将用于开发描述甲状腺素运载蛋白和Abeta的关联以及这些关联对Abeta聚集动力学的影响的数学模型。此外，将测试与甲状腺素运载蛋白结合并增加其稳定性的小药物样化合物，以确定它们的使用是否增强甲状腺素运载蛋白与Abeta之间的相互作用。有趣的是，甲状腺素运载蛋白本身在某些条件下会聚集;甲状腺素运载蛋白原纤维沉积与称为老年系统性淀粉样变性的年龄相关疾病有关。因此，甲状腺素运载蛋白是潜在的淀粉样蛋白，但它保护免受另一种淀粉样蛋白，A β的毒性。其他淀粉样蛋白也可能与Abeta相互作用吗？是否存在一个淀粉样蛋白网络，在那里蛋白质调节彼此的聚集和毒性？这一新概念将通过启动胰岛素和相关蛋白质以及Abeta的研究来探索。胰岛素是一种六聚体蛋白，在某些条件下会解离并形成原纤维，并且有一些有限的数据表明胰岛素相关生长因子如甲状腺素运载蛋白可保护免受Abeta毒性。将确定调节甲状腺素运载蛋白作为抗A β毒性的保护剂的功效的因素。这些研究的结果可用于设计具有稳定甲状腺素运载蛋白并增强其功效的药理活性的化合物。这些化合物将代表一种预防阿尔茨海默病的全新策略。在更基本的层面上，这项研究可以支持淀粉样蛋白和抗淀粉样蛋白网络的概念，这些网络可以调节彼此的行为。这样的概念是高度投机性的，但这样的网络的鉴定，如果它存在，将开辟许多新的调查路线和新的方法，以防止广泛的聚集相关的疾病。研究生在这个项目的广泛教育，在现代实验方法在蛋白质折叠和聚集，并准备在学术界或在生物技术产业的主导作用。通过积极参与暑期研究计划，代表性不足的少数民族本科生将获得宝贵的经验，并享受成就感，这将有助于他们对研究事业的持续热情。