Crystallin aggregation and stabilization

描述(由申请人提供)：在美国，40岁以上的人中有1/6的人患有白内障，80岁前的大多数人都会患上白内障。它是世界范围内导致失明的主要原因。因此，人们对了解白内障的原因和形成机制很感兴趣。白内障被归类为一种错误折叠的疾病，晶状体蛋白的降解导致其聚集和沉淀。然而，缺乏关键的结构和机制信息，很大程度上是因为在实验上很难获得关于聚集蛋白质的结构信息，甚至更难确定导致沉淀的中间体的特征。在这项提案中，我们将使用2D IR光谱、同位素标记和质谱学的新组合来揭示γD-晶状体蛋白聚集的结构和动力学机制以及伴侣蛋白αB-晶状体蛋白抑制沉淀的方式的细节。利用表达的蛋白质连接来半合成γD-晶体蛋白，我们将对其各个结构域进行同位素标记，以便用2D IR光谱来监测它们的结构和动力学。使用UVB光模拟太阳辐射的共价损伤并引发聚集，我们将监测沉淀形成时的动力学和结构。我们从最初的文献中得知，酸诱导的γD-晶体蛋白变性只会导致淀粉样纤维的形成，C-末端结构域形成纤维核心，而不是以前认为的N-末端结构域。相反，UVB变性的初步结果显示，共价损伤导致纤维状和无定形聚集。显然，不同的途径之间存在竞争，这取决于蛋白质损伤的类型。一旦这些途径被表征，我们将研究它们是如何被伴侣蛋白αB-晶体蛋白修饰的。对于许多蛋白质来说，αB-晶体蛋白是一种比纤维状聚集体更好的伴侣，但白内障沉积物似乎大多是无定形聚集体，这意味着晶体蛋白的伴侣机制非常不同。最后，体外机制将针对从人类晶状体中收集的体内蛋白质提取物进行测试。我们想知道自然发生的损伤或晶体蛋白的组成是否会改变沉淀的体外机制和/或结构。这项提案中的几乎每一步都显示了初步结果。我们使用半合成和2D IR光谱的新方法提供了分子水平的见解，这对致力于了解αB-晶状体蛋白和白内障形成的伴侣机制的科学家群体非常重要。

DESCRIPTION (provided by applicant): Cataracts affect the vision of 1 in 6 people over the age of 40 in the United States and most people by the age of 80. It is the leading cause of blindness worldwide. As a result, there is much interest in understanding the cause of cataracts and the mechanism by which they form. Cataracts are classified as a misfolding disease whereby degradation of the crystallin lens proteins leads to their aggregation and precipitation. However, critical structural and mechanistic information is lacking largely because it is experimentally difficult to obtain structural information about aggregated proteins and even more difficult to characterize intermediates that are responsible for precipitation. In this proposal, w will use a novel combination of 2D IR spectroscopy, isotope labeling, and mass spectrometry to uncover details about the structure and kinetic mechanism by which γD- crystallin aggregates and the way in which the chaperone protein αB-crystallin inhibits precipitation. Using expressed protein ligation to semi-synthesize γD-crystallin, we will isotope label its individual domains so that their structures and kinetics can be monitored by 2D IR spectroscopy. Using UVB light to mimic covalent damage from solar radiation and initiate aggregation, we will monitor the kinetics and structures of the precipitates as they form. We know from our initial publications that acid-induced denaturation of γD-crystallin leads solely to amyloid fiber formation with the C-terminal domain forming the fibril core, not the N-terminal domain as was previously thought. In contrast, preliminary results on UVB-denaturation reveal that covalent damage leads to both fibrillar and amorphous aggregates. Clearly, there is a competition between pathways that depends on the type of protein damage. Once these pathways are characterized, we will study how they are modified by the chaperone protein αB-crystallin. For many proteins αB-crystallin is a better chaperone against amorphous than fibrillar aggregates, but cataract deposits appear to be mostly amorphous aggregates, implying that the chaperone mechanism is quite different for the crystallin proteins. Finally, the in vitro mechanisms will be tested against in vivo protein extracs collected from human lenses. We want to know if naturally occurring damage or composition of the crystallins alters the in vitro mechanisms and/or structure of the precipitates. Preliminary results are shown for nearly every step in this proposal. Our novel approach of using semi-synthesis and 2D IR spectroscopy is providing molecular-level insights that are important to the large community of scientists devoted to understanding the chaperone mechanisms of αB-crystallin and cataract formation.