p53 Inducible gene 6 (PIG6) encodes mitochondrial
proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation.
Proline dehydrogenase is proposed to generate radicals that contribute to
cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce
superoxide and/or H2O2, and it is unclear whether
proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six
cancer cell lines, ZR75-30 human
breast cancer cells had the highest basal
proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed
oxygen and produced H2O2 with
proline as sole substrate. Insects use
proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with
proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of
superoxide/H2O2 during
proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30
breast cancer cells the main sites were complex I and the
oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any
superoxide/H2O2 production from
proline dehydrogenase itself, there was no significant direct contribution of
proline dehydrogenase to the observed H2O2 production. Thus
proline oxidation by
proline dehydrogenase drives
superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic
carbon for other mitochondrial
dehydrogenases and not by producing
superoxide/H2O2 directly.