Metabolic regulation of estrogen-synthesizing enzymes by post-translational modification

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In this study project, we focussed on the post-translational modification of aromatase, a rate-limiting enzyme of estrogen production, and analyzed molecular and regulatory mechanisms of the post-translational modification and physiological roles of this reaction. Western blotting analysis showed a possibility of post-translational modification of aromatase by phosphorylation in the cultured cells in the presence of various inducers and regulatory factors. Furthermore, ELISA assay of aromatase contents in the cultured cells suggested a possiblity that the intracellular degradation rate (the turnover rate) was modified by various factors. So, The in vitro assay system was introduced to analyze the process of the intracelluar degradation of the aromatase in the endoplasmic reticulum (microsomal fraction). The degradation of the microsomal aromatase content and activity required the presence of ATP-Mg2+ and the cytoplasmic fraction in the reaction. An aromatase antibody-immunoreactive protein with a high molecular weight (AR-ir protein) appeared in parallel with disappearance of the authentic aromatase with the molecular weight of 53, 000 by Western blotting analysis. The disappearance of the aromatase was inhibited by presence of Geldanamycin, an inhibitor of HSP90 chaperone protein. However, because the purified recombinant aromatase did not disappear and the AR-ir protein did not appear in the assay and also because the microsomal aromatase did not disappear when low concentration of cholic acid was added to the assay system to solubilize the microsomal membrane, it was suggested that the microsomal aromatase was quantitatively regulated by the microsomal membrane-dependent degradation system together with cytoplasmic regulatory proteins. We are now analyzing the constitutive factors participating in the microsomal membrane-dependent degradation by using FLAG-tagged aromatase.