The recognition and removal of aged or aberrant proteins is vital to cellular health and longevity. Cells in culture (models of cells in vivo) exhibit a maximum life span potential (MLSP) before ceasing to replicate, unless a malignant transformation occurs resulting in immortality. Although the cause of the finite MLSP is unknown, it is possible than an accumulation of aberrant proteins (most likely due to oxygen radical damage) reduces the specific activities of certain biochemical processes to below a threshold level required for the performance of some vital cellular function. Aging of a multicellular organism (i.e., man) is simply an integral of the age-related dysfunction of individual cells. In analogy with the cellular system, life continues until the activity of cells controlling some vital function drops below a threshold level required to sustain the homeostasis of the whole organism. The processes of recognition and removal of aberrant proteins within the cell are, therefore, extremely important in maintaining cellular vitality and, hence, the vitality of the organism as a whole. Nevertheless, very little is known about intracellular protein turnover, and in particular, membrane protein turnover. This proposal addresses the question of biochemical mechanisms of membrane protein turnover using a model system recently characterized by the principal investigator. The fundamental goals of the work are to: Identify sites and mechanisms of the interaction of active oxygen species with a membrane protein Assess the efficacy of lipophilic antioxidants (i.e., Vitamin E) to prevent the damage and alteration of membrane proteins Elucidate the biochemical mechanisms of recognition of an age-altered or biochemically-altered protein Characterize the mechanisms of removal and catabolism of an altered membrane protein. Protein turnover in cells grown under various oxygen concentrations or in the presence of deuterated water (to stabilize singlet oxygen) will be assayed by radioisotopic techniques and a novel, nondestructive fluorescence method. Since protein turnover was previously shown to occur in isolated membranes, the effects of extrinsically added antioxidants (both lipophilic and hydrophilic) will be tested. The oxidatively modified residues in the protein will be identified by amino acid analysis, and a step-by-step purification procedure will be undertaken to identify the protein (and cofactors) required for membrane protein turnover. Completion of this project should constitute the first description of the biochemical mechanism(s) involved in the recognition and removal of aberrant membrane proteins.
