Estrogen Receptor Alpha Interacts with Mitochondrial Protein HADHB and Affects Beta-oxidation Activity

It is known that estrogen receptors can function as nuclear receptors and transcription factors in the nucleus and as signaling molecules in the plasma membrane. In addition, the localization of the receptors in mitochondria suggests that they may play important roles in mitochondria. In order to identify novel proteins that are involved in ERα-mediated actions of estrogens, we used a proteomic method that integrated affinity purification, two-dimensional gel electrophoresis, and mass spectrometry to isolate and identify cellular proteins that interact with ERα. One of the proteins identified was trifunctional protein β-subunit (HADHB), a mitochondrial protein that is required for β-oxidation of fatty acids in mitochondria. We have verified the interaction between ERα and HADHB by coimmunoprecipitation and established that ERα directly binds to HADHB by performing an in vitro binding assay. In addition, we have shown that ERα colocalizes with HADHB in the mitochondria by confocal microscopy, and the two proteins interact with each other within mitochondria by performing coimmunoprecipitation using purified mitochondria as starting materials. We have demonstrated that the expression of ERα affects HADHB activity, and a combination of 17β-estrodiol and tamoxifen affects the activity of HADHB prepared from human breast cancer cells that express ERα but not from the cells that are ERα deficient. Furthermore, we have demonstrated that 17β-estrodiol plus tamoxifen affects the association of ERα with HADHB in human cell extract. Our results suggest that HADHB is a functional molecular target of ERα in the mitochondria, and the interaction may play an important role in the estrogen-mediated lipid metabolism in animals and humans.