AGE RELATED CONFORMATIONAL MODIFICATIONS OF PROTEIN

修饰蛋白在老年动物组织中的积累是生物衰老的一个有充分证据的症状。在被发现在老化过程中发生修饰的蛋白质中，有些只会发生构象变化，没有共价修饰。这些错误折叠的蛋白质经常在组织中聚集和沉淀，有时会造成毁灭性的后果。对这些修饰的分子起源、导致这些改变发展的相互作用、有助于老化构象稳定的因素以及这种现象强烈的年龄依赖性的原因的理解仍然不完整。这项拟议研究的广泛目标是深入了解蛋白质转化为老化构象异构体的基础因素。我们将关注两个蛋白质，糖酵解酶磷酸甘油酸激酶(PGK)和纤溶相关蛋白纤溶酶原激活物抑制物-1(PAI-1)。我们实验室对老化过程中的PGK修饰进行了广泛的研究，并证明其起源于构象变化。PAI-1被证明经历了一个自发的构象变化，从其生物活性状态到非活性形式，称为潜伏期，从而显示出一个分子老化过程。PAI-1还与老年人血栓形成频率的增加有关，也可能与癌症发生率的增加有关。本研究的实验目的如下：1.探讨PGK和PAI-1的衰老机制。这些实验将建立在关于两种蛋白质中每一种的老化机理方面的大量现有知识的基础上，并将涉及使用新的生物物理方法，包括单分子光谱学，以解决整体测量中模糊的问题。先前的研究表明，半胱氨酸氧化极大地促进了PGK的老化，并将主要努力解释这一现象。对于PAI-1，我们将探索特定的分子下相互作用，这些相互作用是其转化为潜在形式的特征。2.为了验证我们的假设，即PGK和PAI-1的老化是折叠过程中的晚期事件。这将通过对这两种蛋白质年轻和年老形式的热力学稳定性的比较研究，以及通过研究伴侣蛋白如何影响分子老化过程来实现。3.表征分子老化过程中的结构变化。这一表征将通过X射线结晶学和使用室温磷光实时检测构象变化来实现。

The accumulation of modified proteins in tissues of old animals is a well documented symptom of biological aging. Among the proteins found to become modified during aging, some are altered only conformationally and possess no covalent modifications. These misfolded proteins frequently aggregate and precipitate in tissues, sometimes with devastating results. The understanding of the molecular origin of these modifications, of the interactions which lead to the development of these alterations, of factors which contribute to the stabilization of the aged conformation, and of the reasons for the strong age-dependence of this phenomenon, are still incomplete. The broadly defined goal of this proposed study is to gain insight into the factors that underlie the conversion of proteins into aged conformational isomers. We will focus on two proteins, the glycolytic enzyme phosphoglycerate kinase (PGK) and the fibrinolysis-associated protein plasminogen activator inhibitor-1 (PAI-1). PGK modifications during aging have been extensively studied in our laboratory and were demonstrated to originate in conformational alterations. PAI-1 has been shown to undergo a spontaneous conformational change from its biologically active state to an inactive form, termed latent, thus displaying a molecular aging process. PAI-1 has also been implicated in the increased frequency of thrombosis in the elderly, and potentially also in the increased rate of cancer. The experiments for this study will address the following aims: 1. To explore the mechanisms involved in the aging of PGK and PAI-1. These experiments will build on the significant amount of existing knowledge about mechanistic aspects of the aging of each of the two proteins and will involve the use of new biophysical methodology including single molecule spectroscopy to address issues obscured in ensemble measurements. Previous studies have shown that cysteine oxidation greatly facilitates the rate of PGK aging and a major effort will be to explain this phenomenon. With PAI-1 we will explore specific infra-molecular interactions that feature in its conversion to the latent form. 2. To test our hypothesis that the aging of PGK and PAI-1 represents a late event in folding. This will be achieved in comparative studies of the thermodynamic stabilization of the young and old forms of each of these two proteins, and by studying how chaperone proteins affect the molecular aging process. 3. To characterize structural alterations involved in the molecular aging. This characterization will be achieved by X-ray crystallography, and by using room temperature phosphorescence to detect conformational changes in real time.