Abstract
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.
Highlights
To induce Flavin-containing monooxygenase 3 (FMO3) protein expression, we have used our established model of chemical inducibility by PCB126 exposure, which works through the Aryl hydrocarbon receptor to induce FMO3 in mice (Figure S1A)
The FMO3 antibody chosen successfully enriched for FMO3 protein during the pull down as it was detected in 9 out of 10 FMO3 IP samples and was not detected in normal IgG IP samples
One of the five core urea-cycle-related genes (UCGs), argininosuccinate lyase (ASL), did not pass the unbiased proteomic screening as a potential FMO3 protein interaction partners (PIPs), but we included it for completeness and observed a significant increase in the mice of both backgrounds (C57BL/6: 1.26-fold; p = 0.018 and FVB: 1.50-fold, p = 0.001) (Figure 2A,B)
Summary
We identified 51 FMO3 protein interaction partners, and through gene ontology analysis, have identified urea cycle as an enriched pathway. 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. 1. Introduction with regard to jurisdictional claims in Flavin-containing monooxygenase 3 (FMO3) is an endoplasmic reticulum-tethered protein primarily expressed in the liver of adult humans [1], which is known to induce Nor S-oxygenation of numerous drug substrates [1] and has recently gained much attention for its role in the formation of proatherogenic trimethylamine-N oxide (TMAO).
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