TTR and Abeta: An Amyloid Regulatory Network?

阿尔茨海默病是最常见的与年龄相关的神经退行性疾病。根据“淀粉样蛋白级联假说”，一种名为Abeta的蛋白质的聚集是导致神经元死亡的关键步骤。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病的决定性特征之一。最近的证据表明，聚集过程中出现的可溶性Abeta寡聚体，而不是最终成熟的纤维，毒性最大。对转基因小鼠的研究表明，转甲肾上腺素是一种存在于血液和脑脊液中的蛋白质，它可以保护神经元免受Abeta聚集体造成的破坏性损害。这些研究提出了几个问题：转甲肾上腺素是通过什么机制防止Abeta毒性的？当阿尔茨海默病发展时，这种保护机制会消失吗？有没有药理方法可以恢复转甲状腺素？S的保护能力？初步数据表明，转甲状腺素通过促进Abeta的聚集来抑制Abeta的毒性，从而减少可溶性毒性中间体的数量。血液或脑脊液中的转甲状腺素通常是半胱氨酸残基的化学修饰，已知存在80多个转甲状腺素突变体。早期数据表明，化学修饰或突变可以通过改变转甲状腺素四聚体的稳定性来强烈影响转甲状腺素与Abeta的相互作用。这项研究将进一步探索这一假说，并将阐明转甲状腺素影响Abeta聚集的机制。为了实现这一目标，转甲肾上腺素将被重组生产，并将在半胱氨酸残基上进行化学修饰，以模拟正常的血液蛋白。此外，还将通过定点突变产生几个突变体；这些突变体与野生型相差一到两个氨基酸，但具有不同的稳定性。我们将利用质谱学、圆二色谱、荧光光谱学和相关技术对这些反式甲腺素变异体的结构和稳定性进行实验研究。用激光光散射法、尺寸排除层析法和电子显微镜可以测量Abeta在转甲状腺素存在下的聚集性。这些数据将被用来开发数学模型，描述转甲状腺素和Abeta的关联，以及这些关联对Abeta聚集动力学的影响。此外，还将测试与转甲状腺素结合并提高其稳定性的小型类药物化合物，以确定它们的使用是否增强了转甲状腺素和Abeta之间的相互作用。有趣的是，在某些条件下，甲状腺激素本身会聚集；甲状腺激素原纤维沉积与一种称为老年性系统性淀粉样变性的年龄相关疾病有关。因此，转甲状腺素具有潜在的淀粉样变性，但它可以保护另一种淀粉样变性蛋白Abeta的毒性。有没有可能其他淀粉样蛋白也与Abeta相互作用？是否存在蛋白质的淀粉样变性网络，其中蛋白质相互调节彼此的聚集和毒性？这一新的概念将通过启动对胰岛素及其相关蛋白质和Abeta的研究来探索。胰岛素是一种六聚体蛋白质，在特定条件下会解离并形成纤维，一些有限的数据表明，胰岛素相关生长因子与转甲状腺激素一样，对Abeta毒性具有保护作用。调节转甲状腺素作为Abeta毒性保护剂的有效性的因素将被确定。这些研究的结果可以用来设计具有药理活性的化合物，以稳定转甲状腺素并提高其疗效。这种化合物将代表着一种全新的预防阿尔茨海默病的策略。在更基本的层面上，这项研究可以支持由致淀粉样蛋白和抗淀粉样蛋白组成的网络的概念，这些蛋白相互调节彼此的行为。这样的概念是高度推测的，但识别这样一个网络，如果它存在，将打开许多新的研究路线和新的方法来预防广泛的聚集相关疾病。这个项目的研究生在蛋白质折叠和聚集的现代实验方法方面受到广泛的教育，并准备在学术界或生物技术行业担任领导角色。通过积极参与暑期研究项目，未被充分代表的少数族裔本科生将获得宝贵的经验和成就感，这将有助于他们继续热衷于研究事业。

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.