Impact of R264C and R264H polymorphisms in human aromatase function

• Published in: The Journal of steroid biochemistry and molecular biology 2017-03, Vol.167, p.23-32
• Main Authors: Baravalle, Roberta, Di Nardo, Giovanna, Bandino, Andrea, Barone, Ines, Catalano, Stefania, Andò, Sebastiano, Gilardi, Gianfranco
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Androgens; Animals; Aromatase; Aromatase - chemistry; Aromatase - metabolism; Biosynthesis; Breast cancer; Breast Neoplasms - metabolism; Catalysis; Cell Line; Circular Dichroism; Coculture Techniques; Cyclic AMP-Dependent Protein Kinases - metabolism; Cyclic GMP-Dependent Protein Kinases - metabolism; Cytochrome P450; Estrogens; Estrogens - metabolism; Female; Hormones; Human aromatase; Humans; MCF-7 breast cancer cells; MCF-7 Cells; Molecular Conformation; Mutagenesis, Site-Directed; Mutation; Neurodegenerative diseases; Neurons - metabolism; Pathology; Phosphorylation; Polymorphism, Genetic; Protein kinase A; Proteins; Rats; Recombinant Proteins - metabolism; Sex hormones; Single-nucleotide polymorphism; SNPs; Spectrophotometry, Ultraviolet; Spectroscopy; ST14A neuronal cells; Structure-function relationships; Thermal denaturation
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2016.09.022

[Display omitted] •Human aromatase polymorphisms in R264 alter its catalytic efficiency.•R264 polymorphisms alter the consensus for phosphorylation by PKA/PKG on S267/T268.•In MCF-7 cells, the polymorphic variants show different activities compared to wild-type.•In neuronal progenitors, the activity of the polymorphic variants is similar to wild-type. The cytochrome P450 aromatase is involved in the last step of sex hormones biosynthesis by converting androgens into estrogens. The human enzyme is highly polymorphic and literature data correlate aromatase single nucleotide polymorphisms to the onset of pathologies such as breast cancer and neurodegenerative diseases. The aims of this study were i) to study the influence of the mutations R264C and R264H on the structure-function of the enzyme also upon phosphorylation by selected kinases and ii) to compare the activity of the variants to that of aromatase wild type in two different cell lines. Far-UV circular dichroism spectroscopy, thermal denaturation experiments and CO-binding assay showed that the two polymorphic variants are correctly folded. Steady-state kinetics experiments showed that rArom R264C and R264H exhibit a 1.5 and 3.4 folds lower catalytic efficiency, respectively, when compared to the wild type protein. Since R264 is part of the consensus motif of PKA and PKG1, phosphorylation experiments were performed to study the effect on aromatase function. Phosphorylation by PKA caused a decrease in activity by 36.2%, 49.3% and 27.9% in the wild type, R264C and R264H proteins respectively. Phosphorylation by PKG1 was also found to decrease the activity by 30.3%, 30.5% and 15.4% in the wild type, R264C and R264H proteins respectively. Experiments performed on the three full-length proteins expressed in human MCF-7 breast cancer cells and rat ST14A neuronal cells showed that, depending on the cell line used, the activity of the proteins is different, implicating different cellular mechanisms modulating aromatase activity. This work demonstrate that R264 polymorphism causes an intrinsic alteration of aromatase activity together with a different consensus for phosphorylation by different kinases, indicating that estrogen production can be different when such mutations are present. These findings are significant in understanding the onset and treatment of pathologies in which aromatase has been shown to be involved.