Endocrine Abstracts

Introduction: Aromatase cytochrome P450 is uniquely responsible for estrogen synthesis in vertebrates. The protein is expressed in the gonads and other extraglandular tissues. Deregulated expression of aromatase has pathological relevance and targeting the CYP19A1 product could be an effective strategy for treatment of affected tissues. Nevertheless, very little is known concerning the role of protein oligomerisation, glycosylation and degradation. Here we studied the aromatase cellular fate and the effects of different mutations in an in vitro setting.

Design: The coding region of CYP19A1 gene was cloned and mutations previously identified by us in patients (R365Q, E210K, M127R, R375H) were introduced by site-directed mutagenesis. Then HEK 293 cells, not expressing aromatase, were selected for in vitro transient expression. Aromatase cellular localization and protein expression were analyzed. Glycosylation and degradation were investigated with specific inhibitors, and aromatase activity was evaluated by estradiol assay.

Results: No differences in wild type and mutated aromatase protein cellular localization were found. The time course of protein expression by Western blotting showed the highest degradation rate for the E210K mutation followed by R365Q then M127R and R375H mutation. The same trend was observed for aromatase activity. Proteasome inhibition by MG132 resulted in the appearance of a non-glycosylated form, confirmed by tunicamycin treatment. Only the E210K mutation showed a degree of instability due to proteasome-mediated degradation. Other pathways were investigated but neither autophagy nor lysosomal inhibitors affected degradation, except for chloroquine (specific for lysosomes).

Conclusions: The reduced functional activity of the protein caused by different mutations appears to be due both to partially or completely inactivating effects, and to the increased instability of the mutated forms. Glycosylation confers stability to the protein, confirmed by MG132 and tunicamycin studies. Different degradation pathways could be involved depending on the degree of stability of the protein.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.