AGE RELATED CONFORMATIONAL MODIFICATIONS OF PROTEIN

老年动物组织中修饰蛋白质的积累是生物衰老的一个有据可查的症状。 在老化过程中被修饰的蛋白质中，有些蛋白质仅在构象上发生改变，而没有共价修饰。这些错误折叠的蛋白质经常在组织中聚集和沉淀，有时会产生破坏性的结果。 对这些改变的分子起源、导致这些改变的发展的相互作用、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是不完整的。 这项拟议研究的广义目标是深入了解蛋白质转化为老化构象异构体的因素。 我们将集中在两个蛋白质，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剂-1（派-1）。 在我们的实验室中已经广泛研究了老化过程中PGK的修饰，并证明其起源于构象改变。 派-1已显示经历从其生物活性状态到称为潜伏的非活性形式的自发构象变化，从而显示分子老化过程。派-1还与老年人血栓形成频率增加有关，并且还可能与癌症发病率增加有关。 本研究的实验将达到以下目的：1。探讨PGK和派-1参与衰老的机制。 这些实验将建立在大量的现有知识的机制方面的老化的两种蛋白质，并将涉及使用新的生物物理方法，包括单分子光谱学，以解决问题模糊的合奏测量。 以前的研究表明，半胱氨酸氧化大大促进PGK老化的速度，一个主要的努力将是解释这一现象。 与派-1，我们将探索特定的分子内相互作用，其特点是在其转换为潜在的形式。2.为了验证我们的假设，PGK和派-1的老化代表了折叠的晚期事件。 这将在这两种蛋白质的年轻和年老形式的热力学稳定性的比较研究中实现，并通过研究伴侣蛋白如何影响分子衰老过程。3.以表征分子老化过程中的结构变化。 该表征将通过X射线晶体学和通过使用室温磷光来实现，以真实的时间检测构象变化。

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.