Date of this Version
Zhang, Lu. (2015). Reactive oxygen species homeostasis and proline catabolism. Dissertation, University of Nebraska-Lincoln.
The role of proline metabolism in regulating cellular redox status was first proposed three decades ago. Proline catabolism was then later found to induce programmed cell death and cell apoptosis by regulating ROS signaling. Proline oxidation was also found to promote cell survival under oxidative stress. Proline catabolism-mediated reactive oxygen species (ROS) were suggested to be involved in both cases by serving as a regulatory signal. In this work, the sources of proline oxidation-induced ROS production were explored in both bacteria and animal cells. Proline oxidation-induced ROS was found to be shared by bacteria (Escherichia coli) and animals (human and pig), suggesting it is a common occurence for many if not all organisms. The source of proline oxidation–mediated ROS was found to be the respiratory chain, where reducing equivalents generated during proline catabolism transfer electrons to molecular oxygen. Proline dehydrogenase (PRODH), the enzyme that catalyzes the first and rate limiting step of proline oxidation, does not directly contribute to proline oxidation-induced ROS formation. For instance, we found recombinant human PRODH1 has low reactivity with molecular oxygen (kcat = 0.06 min-1), which is 300-700 times slower than with an artificial electron acceptor (kcat =0.75 s-1) or the physiological electron acceptor ubiquinone-1 (kcat =0.35 s-1). We also studied the mechanism of proline treatment in regulating oxidative stress resistance, since proline supplementation was able to promote the survival of wild-type E. coli under oxidative stress. Depletion of PutA in E. coli resulted in increased sensitivity to oxidative stress, suggesting the role of proline oxidation in regulating oxidative stress resistivity. We found that ROS generated during proline oxidation activates the OxyR regulon leading to increased katG expression and oxidative stress tolerance. In addition, the level of proline oxidation induced ROS in E. coli are sufficient to serve as an adaptive signal to oxidative stress. In mitochondria, proline oxidation also led to ROS generation suggesting that a conserved feature of proline catabolism in different organisms in the formation of ROS as a by product.
Advisor: Donald Becker