Functional characterization of the G162R and D216H genetic variants of human CYP17A1.

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.

Oxidative protein cross-linking reactions involving L-tyrosine in transforming growth factor-beta1-stimulated fibroblasts.

The mechanisms by which ligand-stimulated generation of reactive oxygen species in nonphagocytic cells mediate biologic effects are largely unknown. The profibrotic cytokine, transforming growth factor-beta1 (TGF-beta1), generates extracellular hydrogen peroxide (H2O2) in contrast to intracellular reactive oxygen species production by certain mitogenic growth factors in human lung fibroblasts. To determine whether tyrosine residues in fibroblast-derived extracellular matrix (ECM) proteins may be targets of H2O2-mediated dityrosine-dependent cross-linking reactions in response to TGF-beta1, we utilized fluorophore-labeled tyramide, a structurally related phenolic compound that forms dimers with tyrosine, as a probe to detect such reactions under dynamic cell culture conditions. With this approach, a distinct pattern of fluorescent labeling that seems to target ECM proteins preferentially was observed in TGF-beta1-treated cells but not in control cells. This reaction required the presence of a heme peroxidase and was inhibited by catalase or diphenyliodonium (a flavoenzyme inhibitor), similar to the effect on TGF-beta1-induced dityrosine formation. Exogenous addition of H2O2 to control cells that do not release extracellular H2O2 produced a similar fluorescent labeling reaction. These results support the concept that, in the presence of heme peroxidases in vivo, TGF-beta1-induced H2O2 production by fibroblasts may mediate oxidative dityrosine-dependent cross-linking of ECM protein(s). This effect may be important in the pathogenesis of human fibrotic diseases characterized by overexpression/activation of TGF-beta1.

Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-beta1.

Mitogenic growth factors and transforming growth factor beta1 (TGF-beta1) induce the generation of reactive oxygen species (ROS) in nonphagocytic cells, but their enzymatic source(s) and regulatory mechanisms are largely unknown. We previously reported on the ability of TGF-beta1 to activate a cell surface-associated NADH:flavin:O(2) oxidoreductase (NADH oxidase) that generates extracellular H(2)O(2). In this study, we compared the ROS-generating enzymatic systems activated by mitogenic growth factors and TGF-beta1 with respect to the primary reactive species produced (O(2)(.-) vs. H(2)O(2)), the site of generation (intracellular vs. extracellular) and regulation by Ras. We find that the mitogenic growth factors PDGF-BB, FGF-2, and TGF-alpha (an EGF receptor ligand) are able to rapidly (within 5 min) induce the generation of intracellular O(2)(.-) without detectable NADH oxidase activity or extracellular H(2)O(2) release. In contrast, TGF-beta1 does not stimulate intracellular O(2)(.-) production and the delayed induction of extracellular H(2)O(2) release is not associated with O(2)(.-) production. Expression of dominant-negative Ras (N17Ras) protein by herpes simplex virus-mediated gene transfer blocks mitogen-stimulated intracellular O(2)(.-) generation but has no effect on TGF-beta1-induced NADH oxidase activation/H(2)O(2) production. These results demonstrate that there are at least two distinctly different ROS-generating enzymatic systems in lung fibroblasts regulated by mitogenic growth factors and TGF-beta1 via Ras-dependent and -independent mechanisms, respectively. In addition, these findings suggest that endogenous production of ROS by growth factors/cytokines may have different biological effects depending on the primary reactive species generated and site of production.

[Decile distribution of risk of mortality from ischemic cardiopathy in the male population registered at a health care center]. / Distribución según deciles de riesgos de mortalidad por cardiopatía isquémica de la población masculina adscrita a un centro de salud.

OBJECTIVE: To distribute the male population registered at our health centre into deciles of risk of death from ischaemic heart disease, using only the data in the clinical history. DESIGN: A crossover, retrospective and observational study, without random distribution. SETTING: Urban health centre. PATIENTS AND OTHER PARTICIPANTS: The work material was 2848 clinical histories of men aged between 25 and 55 1420 of these were histories with up-to-date data because the men had had a consultation during the preceding year. MEASUREMENTS AND MAIN RESULTS: The method proposed by Shaper to evaluate cardiovascular risk was used. The most prevalent risk factor was tobacco dependency, at 50.1% (CI 95%, 47.5-52.7), whereas hypertension and diabetes did not exceed 14.1% (CI, 12.3-15.9) and 2.5% (CI, 0.016-3.31), respectively, 90% of those under 35 were in the 10-30 deciles, while about half the over-45s were in the 40-90 deciles. CONCLUSIONS: Use of the Shaper method enabled us to distribute our male patients according to risk and identify 4% (2.56-5.37) as being at high risk of ischaemic heart disease.