Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence

Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence.

Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and –bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

SAT-736 Dissecting the Relative Role of Estrogen and Androgen in Fibrosis, Skeletal Muscle Atrophy, and Inguinal Hernia Formation

Introduction: More than one in four men develop symptomatic inguinal hernia, and hernia repair is the most commonly performed general surgical procedure in the US. Despite its prevalence, the molecular mechanisms causing inguinal hernia remain unclear. Aromatase, the key enzyme for the conversion of testosterone (T) to estradiol (E2), is present in human but not mouse skeletal muscle tissue. We recently demonstrated that robustly increased local E2 levels in lower abdominal muscle (LAM) tissue and decreased circulating T levels were associated with fibrosis and myocyte atrophy in LAM tissue, leading to severe scrotal (inguinal) hernia formation in a humanized aromatase transgenic mouse model (Aromhum) with a high LAM human aromatase expression. To further determine the relative role of estrogen and androgen in the development of inguinal hernia, we generated a novel mild Aromhum mouse model with lower LAM aromatase expression compared with the severe model. Methods: Mild Aromhum mice were followed for 6 months to determine hernia incidence and measure hernia size (n=30). We treated mild Aromhum mice with the aromatase inhibitor, letrozole (n=12) for 12 weeks. Circulating and LAM E2 levels in mice were measured using mass spectrometry. LAM tissue fibrosis and myocyte size were determined by Masson’s trichrome staining and H&E staining, respectively. Results: The mild Aromhum mice contain a single copy of the human aromatase genomic fragment with a truncated regulatory region, giving rise to significant but mildly elevated LAM E2 levels (2.5-fold) at 15 weeks of age. Interestingly, these mice maintain normal circulating T levels. Furthermore, we show that mildly increased LAM E2 without decreased circulating T levels cause hernia formation in about 88% of mild Aromhum mice in contrast to 100% hernia formation in mice containing the full-length human aromatase regulatory region (severe Aromhum model), suggesting that higher LAM estrogen and low serum T levels contribute to this severe phenotype. Treatment with an aromatase inhibitor restores LAM E2 levels to normal levels and completely prevents inguinal hernia formation in the mild Aromhum mice. In LAM fibroblasts of mild Aromhum mice, we find very high levels of estrogen receptor-α expression, which possibly mediates estrogen-induced hernia formation. Conclusion: Taken together, our findings from the mild Aromhum mouse model suggest that lower levels of estrogen excess in LAM are the primary driver of muscle atrophy and hernia formation because this mouse model do not exhibit circulating T deficiency. Our findings will constitute a starting point for dissecting the relative roles of estrogen and androgen action in inguinal hernia development. This has the potential to facilitate drug development to prevent and treat hernias, especially recurrent hernias after primary hernia repairs in vulnerable populations such as elderly men.

Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†

Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.

Towards an Understanding of the Mode of Action of Human Aromatase Activity for Azoles through Quantum Chemical Descriptors-Based Regression and Structure Activity Relationship Modeling Analysis

Aromatase is an enzyme member of the cytochrome P450 superfamily coded by the CYP19A1 gene. Its main action is the conversion of androgens into estrogens, transforming androstenedione into estrone and testosterone into estradiol. This enzyme is present in several tissues and it has a key role in the maintenance of the balance of androgens and estrogens, and therefore in the regulation of the endocrine system. With regard to chemical safety and human health, azoles, which are used as agrochemicals and pharmaceuticals, are potential endocrine disruptors due to their agonist or antagonist interactions with the human aromatase enzyme. This theoretical study investigated the active agonist and antagonist properties of “chemical classes of azoles” to determine the relationships of azole interaction with CYP19A1, using stereochemical and electronic properties of the molecules through classification and multilinear regression (MLR) modeling. The antagonist activities for the same substituent on diazoles and triazoles vary with its chemical composition and its position and both heterocyclic systems require aromatic substituents. The triazoles require the spherical shape and diazoles have to be in proper proportion of the branching index and the number of ring systems for the inhibition. Considering the electronic aspects, triazole antagonist activity depends on the electrophilicity index that originates from interelectronic exchange interaction (ωHF) and the LUMO energy ( E LUMO PM 7 ), and the diazole antagonist activity originates from the penultimate orbital ( E HOMONL PM 7 ) of diazoles. The regression models for agonist activity show that it is opposed by the static charges but favored by the delocalized charges on the diazoles and thiazoles. This study proposes that the electron penetration of azoles toward heme group decides the binding behavior and stereochemistry requirement for antagonist activity against CYP19A1 enzyme.