The CYP11A1 assays contained 0

The CYP11A1 assays contained 0.1 M CYP11A1, 1.6 M Fdx, and 0.4 M FdR, whereas the protein concentrations in the CYP27A1 assays were 0.02 M CYP27A1, 0.6 M Fdx and 0.08 M FdR. cholesterol, 5-cholestenoic acid, 22R-hydroxycholesterol, phospholipids, ferredoxin, ferredoxin reductase, A2E == Introduction == In all extrahepatic organs, the first step in cholesterol biotransformations, the hydroxylation reaction, is usually always carried out by cytochrome P450 enzymes (CYP or P450) 27A1, 46A1, and 11A1 that generate more polar forms of cholesterol, oxysterols, for elimination or regulation (reviewed in [1]). CYP27A1 is usually expressed in the inner mitochondrial membranes of many tissues where it converts cholesterol to 27-hydroxycholesterol, the first step in a pathway of cholesterol removal that complements lipoprotein-mediated reverse cholesterol transport [24]. In the lung, macrophages and vascular endothelium, however, CYP27A1 performs additional oxidations of 27-hydroxycholesterol yielding 5-cholestenoic acid [57], whose production is usually favored by the high mitochondrial CYP27A1 to cholesterol ratio and the presence of the extracellular acceptor, albumin, outside the cell [6]. Lack of CYP27A1 underlies a slowly progressive, if untreated, disease cerebrotendinous xanthomatosis characterized by multiple symptoms including tendon xanthomas, neurological and retinal abnormalities, juvenile bilateral cataracts, and premature atherosclerosis [811]. Microsomal CYP46A1 is usually a brain-specific enzyme that catalyzes cholesterol 24-hydroxylation, the major mechanism whereby cholesterol is usually disposed from the brain [1214]. Medical significance of CYP46A1 is not yet established but could involve effects on memory and cognition as indicated by animal studies [15,16]. CYP11A1 is usually abundant in the inner mitochondrial membranes of steroidogenic tissues where it initiates the production of all steroid hormones by sequential metabolism of cholesterol to 22R-hydroxycholesterol, 20, 22R-dihydroxycholesterol and pregnenolone [17,18]. Partial or complete deficiency of CYP11A1 leads to congenital lipoid adrenal hyperplasia [1921], a severe endocrine disorder, lethal without a lifetime hormone replacement therapy. In the adrenal cortex, mitochondria contain cholesterol at subsaturating for CYP11A1 levels [22]; hence cholesterol availability to CYP11A1 (i.e. transfer from the outer to the inner mitochondrial membrane) is the rate-limiting step in the overall steroidogenesis in this tissue [23]. In contrast, in placental mitochondria, cholesterol is at near-saturating concentrations for CYP11A1 [24], yet another factor, electron supply by the P450 redox partners, limits the rate at which cholesterol is usually metabolized to pregnenolone in the placenta [25,26]. Thus, activity of CYP11A1 as well as CYP27A1 is usually affected by tissue-specific environment which, along with other mechanisms, controls the rate of enzyme-mediated cholesterol metabolism. The retina is usually a light-sensitive tissue lining the inner surface of the eye. Crenolanib (CP-868596) The retina has a complex multilayered structure and is composed of the neural retina (NR), formed by 9 layers of different cell types, and a single layer of the underlying epithelial cells called retinal pigment epithelium (RPE). RPE is considered a part of the retina because of its close association with the NR. The retina was found to express all extrahepatic cholesterol-metabolizing P450s as exhibited initially by immunohistochemical stainings by others [2731] and then by us by mass spectrometry quantifications [32,33]. We measured CYPs 27A1 and 46A1 in human NR and CYP11A1 in bovine NR and found that CYP27A1 is the most abundant cholesterol-metabolizing P450 in the NR expressed at ~8-fold higher levels than CYP46A1 and ~70-fold higher levels than CYP11A1 [32,33]. Parallel evaluation Crenolanib (CP-868596) of the oxysterol profiles exhibited that in human Crenolanib (CP-868596) and bovine NR and RPE the product of CYP27A1 activity is usually a rare metabolite 5-cholestenoic acid rather than a more common metabolite 27-hydroxycholesterol [34]. Enzymatic products of CYP46A1 and CYP11A1 (24-hydroxycholesterol and pregnenolone, respectively) were detected in the NR and RPE as well [34], at comparable levels despite CYP11A1 is usually a low abundance retinal P450. Interestingly, NR and RPE contained the CYP11A1 reaction intermediate and biologically active oxysterol 22R-hydroxycholesterol, usually not released from the enzyme active site during the formation of pregnenolone [35,36]. The unexpected conversion of retinal cholesterol to 5-cholestenoic acid and detection of 22R-hydroxycholesterol are important findings because they may pertain to the mechanisms whereby the retina regulates cholesterol Rabbit polyclonal to Dynamin-1.Dynamins represent one of the subfamilies of GTP-binding proteins.These proteins share considerable sequence similarity over the N-terminal portion of the molecule, which contains the GTPase domain.Dynamins are associated with microtubules. elimination. Therefore, in the present study, we investigated how organ-specific lipid and cholesterol content and the P450-redox partner ratio affect the activities and product profile of cholesterol-metabolizing mitochondrial CYPs 27A1 and 11A1. Our results establish the biochemical basis for the production of 5-cholestenoic acid and 22R-hydroxycholesterol in the retina and identify key retinal features that affect enzyme-mediated cholesterol removal from the NR and RPE. == Materials and Methods == == Materials == Cholesterol and [3H]cholesterol were from Steraloids. Inc.