The molecular mechanisms determining the onset of protein aggregation revealed by single molecule force-clamp spectroscopy

我们身体中的每个器官都是由大量的单个细胞以协调的方式共同工作组成的。在每个细胞内，有成千上万种不同的蛋白质，它们以一种非常完善和同步的方式发挥作用。一般来说，这些蛋白质中的每一种都可以以两种不同的形状存在-折叠和未折叠状态。一旦蛋白质在核糖体中表达，它们就会在我们的身体中不断地展开和重新折叠，核糖体是生产它们的小工厂。大多数蛋白质只有在处于折叠状态时才具有“活性”或“功能”。不能折叠会引起无数的毁灭性疾病，如阿尔茨海默氏症，帕金森氏症，疯牛病，白内障和许多其他疾病。当单个分子经历构象变化并且不再能够折叠回其天然结构时，这些疾病具有它们的起源。这是毒性的起源之一;一旦蛋白质被展开并且不能回到其折叠状态，它就会以相当快的方式粘附到相邻的未折叠蛋白质上。这会产生一个成核种子，最终导致聚集体的存在，称为淀粉样蛋白，这是几种疾病的标志。例如，在阿尔茨海默病和帕金森病患者的大脑中发现了聚集体斑块。也许最明显的淀粉样蛋白疾病是人眼中的白内障形成，其中淀粉样蛋白可以通过观察受影响的眼睛直接观察到。不幸的是，一旦检测到这些淀粉样蛋白聚集体，往往为时已晚。因此，要发现这一聚合过程为何以及何时开始，确实具有挑战性。该项目旨在开发一种新的策略，能够实验性地操纵单个蛋白质的状态，并探测每个蛋白质在其折叠轨迹期间所采用的不同形状和构象的时间演变。现在的挑战是在单个分子的水平上检测蛋白质无法折叠的机制。在单分子水平上对聚集蛋白质的研究很少，而且只有最近的研究。我将使用一种新的技术，命名为单分子力钳光谱，研究不同的轨迹所遵循的未折叠的蛋白质在其旅程的天然状态。这种技术已经被证明是成功的，第一次，通过进化设计折叠的蛋白质所采用的不同构象。我将首先进一步研究，以完全了解单个蛋白质折叠之前的不同轨迹。然后，我将扩展这种方法来研究导致各种疾病的蛋白质的折叠行为，例如阿尔茨海默氏症（Abeta42多肽），帕金森氏症（由α-突触核蛋白的聚集引起）以及眼睛的白内障，这是由蛋白质gammaD晶体蛋白的错误折叠引发的。在所有这些情况下，我将比较这些淀粉样蛋白形成蛋白与那些成功折叠的蛋白的行为。我将确定构象（折叠，未折叠或中间），其中每一个这些淀粉样蛋白形成蛋白从“功能性”折叠路线出发。最后，我将研究其他伴侣蛋白（称为伴侣蛋白）的存在是否有助于聚集蛋白的折叠机制。总之，这些单分子技术现在已经达到了成熟的水平，它们可以用于攻击生物学中更重要的挑战，例如导致蛋白质聚集的基本生物学机制，起源于单分子水平。我抓住了一个非凡的机会，扩大目前的应用力钳，以解决这些疾病的共同之谜。这些基本的生物物理学研究对影响若干研究领域具有很大的希望，例如对目前无法治愈的这种毁灭性人类疾病的分子理解。

Each organ in our body is composed of a large number of individual cells working together in a coordinated fashion. Inside each cell, there are thousands of different proteins that perform their function in a very well-established and synchronized way. In general, each of these proteins can be found in two different shapes -the folded and the unfolded states. Proteins unfold and refold continuously in our bodies once they are expressed in the ribosomes, which are the small factories where they are produced. Most proteins are 'active' or 'functional' only when they are in their folded state. Failing to fold gives rise to a myriad of devastating diseases such as Alzhemier's, Parkinson's, Mad Cow, eye's cataracts and many others. These diseases have their origin when a single molecule undergoes a conformational change and is not able to fold back into its native structure anymore. This is one of the origins of toxicity; once a protein is unfolded and cannot get back to its folded state, it sticks to a neighboring unfolded protein in a rather fast way. This creates a nucleation seed that eventually results in the presence of aggregates, called amyloids, which are the signature of several diseases. For example, plaques of aggregates are found in the brains of Alzhemier's and Parkinson's disease patients. Perhaps the most conspicuous amyloid disease is cataract formation in the human eye, where the amyloids can be observed directly by looking into an affected eye. Unfortunately, once these amyloid aggregates are detected, it is often too late to act. It is therefore really challenging to discover why and when this aggregative process starts. This project aims to develop a new strategy to able to experimentally manipulate the state of a single protein and probe the time evolution of the different shapes and conformations adopted by each protein during its folding trajectory. The challenge is now to detect the mechanisms why proteins fail to fold, at the level of a single molecule. There are very few and only recent studies of aggregating proteins at the single molecule level. I will use a novel technique, named single molecule force-clamp spectroscopy, to study the different trajectories followed by an unfolded protein in its journey to the native state. This technique has already proved successful at identifying, for the first time, the different conformations adopted by a protein that has been evolutionary designed to fold. I will first further the investigations to completely understand the different trajectories followed by an individual protein until it folds. I will then expand this methodology to study the folding behaviour of proteins that cause a great variety of diseases such as Alzheimer's (the Abeta42 polypeptide), Parkinson's (caused by the aggregation of the alpha-synuclein protein) and also the eye's cataract, which is triggered by the misfolding of the protein gammaD crystallin. In all these cases, I will compare the behaviour of these amyloid-forming proteins with those proteins that succeed to fold. I will identify the conformation (folded, unfolded or intermediate) where each of these amyloid-forming proteins departs from the 'functional' folding route. I will finally study if the presence of other companion proteins, called chaperones, assists in the folding mechanism of the aggregating proteins. Altogether, these single molecule techniques have now reached a level of maturity where they can be used to attack more significant challenges in biology such as the basic biological mechanisms leading to protein aggregation, originating at the single molecule level. I seize on the remarkable opportunity of expanding the current applications of force-clamp to solving the common riddle of these diseases. These basic biophysical studies hold great promise for impacting several fields of research, such as the molecular understanding of such devastating human diseases for which there is, at the present time, no cure.