The induction of proline oxidase accompanying p53-dependent induction of apoptosis suggests that the proline metabolic pathway plays a role in programmed cell death. We are studying this metabolic pathway at the level of : a) proline oxidase - its induction and its role in apoptosis; b) pyrroline 5-carboxylate (P5C) and P5C reductase in cell signaling; and c) the function of prolidase in cell physiology. a) Proline oxidase - its induction and its role in apoptosis. In cells undergoing apoptosis, proline oxidase is induced and added proline stimulates the formation of reactive oxygen species (ROS). Importantly, in p53 negative cells transfected to overexpress proline oxidase, the addition of proline is sufficient to induce apoptosis. Recently, it has been shown that the expression of proline oxidase is silenced in certain tumors in the face of normal expression of p53, p21 and BAX suggesting that proline oxidase may act as a cancer suppressor. We are characterizing the proline oxidase promoter to determine its regulation on a molecular level. In addition, we are monitoring the expression of genes participating in this metabolic paradigm using real-time PCR and panels of cancer cells and tumors. Finally, we are investigating the developmental role for this metabolic pathway as can be inferred from findings in patients with deficiencies of these enzymes. One approach we have used is to study the development of Xenopus embryos. Ornithine aminotransferase (OAT), the enzyme that converts ornithine to P5C, may be important in neurodevelopment. From studies of in situ hybridization, the expression of OAT is seen predominantly in neural tissue in early embryonic development. Microinjection of OAT mRNA alters the tadpole morphology characterized by a short tail. The metabolic cascade responsible for this abnormality is being investigated. In summary, these studies show that proline and pyrroline-5-carboxylate may be important in metabolic signaling in development, proliferation, and apoptosis. b) Pyrroline 5-carboxylate (P5C) as a signaling and regulatory molecule. P5C, the product of proline oxidase, ornithine aminotransferase and glutamate synthase, has been shown to have regulatory activities. Recent work suggests that P5C reductase (P5CR) may play a role in receptor-mediated regulation. The two known isozymes of P5CR may serve distinct functions. P5CR1 is a NADH-dependent enzyme regulated to produce proline, whereas P5CR2 is a NADPH-dependent enzyme coupled with redox-related regulation. In lymphocytes activated by lectins, it appears that P5CR2 is induced. We are defining the interaction of P5CR2 with membrane receptors. c) Inhibition of prolidase by nickel. Prolidase hydrolyzes imidodipeptides with carboxyl terminus proline and is a metalloenzyme. Nickel which can cause pulmonary fibrosis and cancer in humans, is a potent inhibitor of prolidase activity. Kinetic studies show that nickel is a competitive inhibitor of the interaction with manganese, the most potent activator of catalytic activity. The inhibitory effect can be demonstrated not only in cell-free systems, but also in intact CHO-K1 cells which are proline auxotrophs but which also can grow on GLY-PRO. Nickel markedly inhibits growth of these cells on GLY-PRO but not on PRO. We are using these cells to perturb the processing of imidodipeptides and other proline containing peptides to investigate their potential role in cell signaling.
