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.
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