title: Impact of R264C and R264H polymorphisms in human aromatase function.
abstract: 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.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Baravalle
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy; CrisDi, Interdepartmental Center for Crystallography, via Pietro Giuria 7, 10125, Torino, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.affiliation: Department of Medicine and Experimental Oncology, University of Torino, Via Michelangelo 27, 10126, Torino, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bandino
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, CS, Italy.
authors.forename: Ines
authors.initials: I
authors.lastname: Barone
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, CS, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Catalano
authors.affiliation: Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Arcavacata di Rende, CS, Italy.
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Andò
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, via Accademia Albertina 13, 10123 Torino, Italy; CrisDi, Interdepartmental Center for Crystallography, via Pietro Giuria 7, 10125, Torino, Italy. Electronic address: gianfranco.gilardi@unito.it.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001141:Aromatase / Q000737:chemistry / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002942:Circular Dichroism; D018920:Coculture Techniques; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004967:Estrogens; D011994:Recombinant Proteins; D001141:Aromatase; C561410:CYP19A1 protein, human; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases
keywords: Human aromatase; MCF-7 breast cancer cells; Phosphorylation; SNPs; ST14A neuronal cells