Proline oxidase (POX) is an inner mitochondrial membrane protein that oxidizes proline to generate superoxide as well as ATP. Work in our laboratory has documented that POX activity is highly regulated during genotoxic and metabolic stress. Inducers of POX include p53, and proline-dependent ROS play a role in p53- and mitochondrial-mediated apoptosis. Succinate dehydrogenase, also known as Complex II of the electron transport chain (ETC), oxidizes the TCA cycle metabolite succinate to fumarate as it reduces ubiquinone to ubiquinol, generating ATP through oxidative respiration. Complex II and the p53-DNA damage repair pathway are intimately linked. Mutations in Complex II have been shown to increase risk of breast and thyroid cancer through destabilization of p53, and tocopherol-succinate has been demonstrated to reduce radiation-induced apoptosis. Thus, the ETC and activities of p53 appear to be linked through Complex II.

Because POX can generate ATP from proline, we explored the relationship between POX and the ETC. We found that POX binds directly to ubiquinone, and that ubiquinone is a very effective acceptor of proline-derived electrons. Increased POX catalytic activity due to ubiquinone required Complex III and Complex IV of the ETC to be functional, indicating that ATP generated by POX is through oxidative respiration. In addition, experiments using the Seahorse XF24 analyzer confirmed that while POX can utilize proline as a short term source of electrons during nutrient stress conditions, long term expression of POX results in reactive oxygen species (ROS)-dependent suppression of respiration, consistent with its role in mitochondrial-mediated apoptosis. We found that the POX and Complex II were linked functionally, and that the presence of succinate dramatically affects POX activity. Lineweaver-Burk analysis showed that succinate inhibits POX catalytic activity using anti-competitive inhibition, a rare mechanism of enzymatic inhibition. Two highly selective inhibitors of the Complex II holoenzyme, TTFA and carboxin, inhibit the ubiquinone-mediated activities of POX; in addition, POX co-immunoprecipitates with Complex II, indicating a physical interaction between these two enzymes. Interestingly, proline appears to stimulate oxidation of succinate by Complex II in vitro, and acute co-administration of proline and succinate to POX-expressing cells results in a burst of oxidative respiration well above that seen with either substrate alone. Finally, addition of succinate to cell culture inhibits ROS generation by POX and inhibits the long-term effects of POX on respiration.

Together, these data support a model in which POX is an integral contributor in oxidative respiration during nutrient stress, and in which respiration is regulated by level and duration of POX expression, as well as levels of proline relative to the TCA cycle metabolite, and Complex II substrate, succinate.

Citation Format: Chad N. Hancock, James M. Phang. Effects of co-regulation of proline oxidase and succinate dehydrogenase on oxidative respiration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4613. doi:10.1158/1538-7445.AM2013-4613