Quantitative investigations into the molecular mechanisms of amyloid fibril fragmentation

淀粉样原纤维是蛋白质的一种形式，由于与许多毁灭性的人类大脑疾病有关，最近受到了广泛关注。例子包括阿尔茨海默病、克雅氏病、亨廷顿病和帕金森病。此外，淀粉样蛋白原纤维不寻常的物理特性意味着它们有潜力成为强大而稳定的工程纳米材料。如果我们要了解淀粉样蛋白原纤维在自然界中的正常功能，以及它们如何参与疾病，那么我们就可以开发针对淀粉样蛋白相关疾病的有效治疗方法，那么将淀粉样蛋白原纤维分解成更小的片段是一个必须完全理解的关键过程。然而，淀粉样蛋白纤维断裂的原因和后果仍然是一个很大程度上未探索的研究领域。该项目的长期目标是解决纤维断裂的分子和细胞机制。淀粉样蛋白原纤维是由正常蛋白质的全部或部分组装而成的，与淀粉样蛋白相关的破坏性人类疾病与淀粉样蛋白原纤维组装和沉积在大脑或人体其他部位的方式有关。然而，淀粉样蛋白原纤维也被认为是一类天然蛋白质形式，即所谓的“功能性淀粉样蛋白”。功能性淀粉样蛋白在细菌、酵母菌甚至人类中都扮演着重要的角色。淀粉样蛋白的一个亚类可以通过大原纤维的断裂形成小种子在生物体之间传播。这一亚类被称为朊病毒，它们存在于人类体内，可引起克雅氏病等疾病。在面包酵母中，它们赋予细胞特殊的细胞特性，并代代相传；一种“蛋白质基因”。为了充分了解朊病毒是如何形成和传播的，我们需要了解朊病毒种子（我们称之为繁殖体）是如何通过淀粉样纤维断裂产生的。不同来源的淀粉样蛋白原纤维的详细特征揭示了令人难以置信的强大结构，通常只有几十纳米厚，但有许多微米长。然而，淀粉样蛋白原纤维的断裂特性是其稳定性的主要因素。纤维断裂也是淀粉样蛋白相关疾病的一个重要因素，因为它影响与疾病相关的原纤维的大小和形状，这些原纤维通常是大团块的聚集体。与疾病相关的朊病毒传播的难易程度，淀粉样蛋白聚集体的聚集和沉积速度，以及这些聚集体对细胞的毒性是受淀粉样蛋白纤维断裂影响的重要疾病特性。为了解决这些问题，我们的目标是回答以下问题：淀粉样蛋白原纤维是如何断裂的，它们断裂的速度有多快，以及在活细胞中，断裂是如何与它们的特性联系在一起的。将结合实验、理论和计算方法，研究三种不同淀粉样蛋白的断裂特性：一种在酵母中形成朊病毒，一种与人类疾病有关，第三种是人工模型系统。从该项目中获得的见解也将对进一步探索淀粉样蛋白原纤维作为潜在纳米材料在技术应用中的作用至关重要，并将提供新的见解，这将有助于未来开发针对淀粉样蛋白相关疾病的治疗策略。

Amyloid fibrils are forms of protein that have received much recent attention through their association with numerous devastating human brain diseases. Examples include Alzheimer, Creutzfeldt-Jakob (CJD), Huntington and Parkinson diseases. Furthermore, the unusual physical characteristics of amyloid fibrils mean that they have the potential to become strong and stable engineered nanomaterials. Breaking amyloid fibrils into smaller pieces is a key process that must be fully understood if we are to understand how amyloid fibrils normally function in nature, and how they are involved in diseases so we can develop effective therapies against the amyloid-associated diseases. Nevertheless, the causes and consequences of amyloid fibril fragmentation remain a largely unexplored area of research. The long-term goal of this project is to resolve the molecular and cellular mechanisms of fibril fragmentation.Amyloid fibrils are assembled from whole or parts of normal proteins and the devastating human diseases associated with amyloid are linked to the way the amyloid fibrils are assembled and deposited in the brain or in other parts of the human body. However, amyloid fibrils have also been recognised as a class of natural protein forms, so-called 'functional amyloids'. Functional amyloids can play a number of important roles in bacteria, yeast and even humans. A sub-class of amyloids can spread between organisms by forming small seeds through the fragmentation of larger fibrils. This sub-class is referred to as prions and they exist in humans where they cause diseases such as CJD. In baker's yeast, they confer special cellular properties on the cells that are passed on from generation to generation; a form of 'protein gene'. To fully understand how prions are formed and transmitted requires that we understand how the prion seeds (which we call propagons) are generated through amyloid fibril fragmentation. Detailed characterisation of amyloid fibrils of different origins has revealed incredibly strong structures that are commonly only tens of nanometres thick but many micrometers long. The fragmentation property of amyloid fibrils is, however, the dominating factor for their stability. Fibril fragmentation is also an important factor in amyloid-associated disease because it influences the size and shape of the disease-associated forms of the fibrils, which are typically large clumps of aggregates. How easy do disease-associated prions spread, how fast are amyloid aggregates assembled and deposited, and how toxic are these aggregates to cells are important disease properties that are influenced by amyloid fibril fragmentation.To address these points, our goal is to answer the following questions: how are amyloid fibrils fragmented, how fast can they fragment, and how is fragmentation linked to their properties in living cells. Using a combination of experimental, theoretical and computational approaches, the fragmentation properties of three different amyloids will be studied: one that forms a prion in yeast, one that is associated with human disease, and the third is an artificial model system. The insights gained from this project will also be critical for further exploring amyloid fibrils as potential nanomaterial in technological applications, and will provide new insights that will facilitate the future development of therapeutic strategies against amyloid associated disease.