Discovery of Protein‐protein Interaction Partners of FMO3 and Relationship with Cell Signaling Pathways

Abstract

Background A critical role of FMO3 is to convert trimethylamine (TMA), a gut microbial metabolite, into trimethylamine-N-oxide (TMAO). As a metabolic product of FMO3, TMAO is a biomarker of cardiometabolic diseases, such as atherosclerosis, diabetes, and kidney disease. Although TMAO may itself be a causative mediator of inflammatory diseases, there is some evidence that FMO3 may roles in metabolic diseases irrespective of TMAO formation. We hypothesized that FMO3, a primarily microsomal protein, can impact on cardiometabolic disease through protein/protein interactions with other important mediators of disease risk. Aims In our study, we aim to analyze protein-protein interaction partners of FMO3 by co-immunoprecipitation techniques coupled with untargeted proteomics to further understand a larger regulatory network impacted on by FMO3. Methods Using a Co-IP strategy we determined FMO3 protein interaction partners in the livers from male mice as well as in mice where FMO3 was induced by a chemical, polychlorinated biphenyl 126 (PCB126); Transcription of FMO3 can activated by the xenobiotic response transcription factor aryl hydrocarbon receptor (AhR) which PCB 126 is known to bind. Furthermore, we investigated mRNA and protein level changes by qPCR and western blot respectively of these interaction partners in vitro and in vivo through genetic manipulation to elucidate physiological and pathological implications of disruption of FMO3 interaction network. Results Our study identified 34 protein interaction partners of FMO3, in which 29 partners increased their interaction when FMO3 was induced. Many of these interaction partners relate to inflammation, metabolism, or mitochondrial function. FMO3 deletion in the hepatocyte cell line AML12 via siRNA surprisingly resulted in significant changes at the mRNA level of many of these interaction partners. These observations have been validated in mice deficient in FMO3, suggesting FMO3 may have some impact on the expression level of its interaction partners and on potential biological process of these partners. For instance, intermediate filament protein Vimentin (VIM) shows stronger binding with FMO3 in the PCB 126 treated group. RT-qPCR and immunoblotting results of VIM indicate that in the female FMO3 -/- mouse liver, expression of both mRNA and protein of VIM is significantly increased. This finding reveals a previously unknown regulatory pathway in which the xenobiotic metabolism enzyme FMO3 associates with and perhaps regulates hepatic cytoskeleton protein expression. Further studies investigating FMO3’s role in intracellular trafficking and tissue remodeling are ongoing.