Proteomics-Based Identification of Interaction Partners of the Xenobiotic Detoxification Enzyme FMO3 Reveals Involvement in Urea Cycle

The hepatic xenobiotic metabolizing enzyme flavin-containing monooxygenase 3 (FMO3) has been implicated in the development of cardiometabolic disease primarily due to its enzymatic product trimethylamine-N oxide (TMAO), which has recently been shown to be associated with multiple chronic diseases, including kidney and coronary artery diseases. Although TMAO may have causative roles as a pro-inflammatory mediator, the possibility for roles in metabolic disease for FMO3, irrespective of TMAO formation, does exist. We hypothesized that FMO3 may interact with other proteins known to be involved in cardiometabolic diseases and that modulating the expression of FMO3 may impact on these interaction partners. Here, we combine a co-immunoprecipitation strategy coupled to unbiased proteomic workflow to report a novel protein:protein interaction network for FMO3. We identified 51 FMO3 protein interaction partners, and through gene ontology analysis, have identified urea cycle as an enriched pathway. Using mice deficient in FMO3 on two separate backgrounds, we validated and further investigated expressional and functional associations between FMO3 and the identified urea cycle genes. FMO3-deficient mice showed hepatic overexpression of carbamoylphosphate synthetase (CPS1), the rate-limiting gene of urea cycle, and increased hepatic urea levels, especially in mice of FVB (Friend leukemia virus B strain) background. Finally, overexpression of FMO3 in murine AML12 hepatocytes led to downregulation of CPS1. Although there is past literature linking TMAO to urea cycle, this is the first published work showing that FMO3 and CPS1 may directly interact, implicating a role for FMO3 in chronic kidney disease irrespective of TMAO formation.