I.3. F ATTY ACID AND STEROL / STEROID METABOLISM
I.3.3. S EX STEROID METABOLISM
Sex steroid hormones (gestagens, androgens, and estrogens) play a central role in life and represent extremely effective signaling molecules, e.g., for the sex steroid hormone receptor‐
regulated transcription of target genes. Because of their vital role in health (e.g., sexual differentiation and reproduction or modulation of the immune system) but also in diseases (e.g., cancer or autoimmune diseases), the metabolism of sex steroids, which is basically described in the following and illustrated in Figure I‐6, was and is intensely studied and reviewed by many researchers [78, 194–197].
The first steps of steroidogenesis start from the precursor cholesterol (1) and take place in mitochondria by the action of the mitochondrial type I cytochrome P450 enzyme CYP11A1 which catalyzes the formation of the C21‐steroid (gestagen) pregnenolone (2) through successive hydroxylation and oxidation reactions [198]. The subsequent oxidation of the pregnenolone’s 3β‐hydroxyl group and isomerization of the Δ5 double bond into a Δ4 double bond by a 3β‐hydroxysteroid dehydrogenase/Δ54‐isomerase (3β‐HSD) in the mitochondria or endoplasmic reticulum (ER) results in progesterone (3), the physiologically most active gestagen. Here, it should be noted that the pregnenolone exits the mitochondria in an unaided way for the subsequent steps of sex steroidogenesis in the ER [198]. In the ER, pregnenolone and progesterone become hydroxylated at position C17 of the steroid nucleus by the type II cytochrome P450 enzyme CYP17A1 leading to 17α‐hydroxypregnenolone (4) and 17α‐hydroxyprogesterone (5), respectively. Via the additional 17,20‐lyase activity of the CYP17A1, the 17α‐hydroxylated gestagens are converted into androgens (C19‐steroids) in the ER. Thereby, the cleavage of the C20‐C21 side chain from 17α‐hydroxypregnenolone and 17α‐hydroxyprogesterone results in the generation of dehydroepiandrosterone (DHEA) (8) and Δ4‐androstenedione (9), respectively. Beside the 3β‐HSDs, which catalyze the oxidation and Δ54‐isomerization of Δ5‐androgens, several enzymes belonging to the class of 3α‐HSDs and 17β‐HSDs, which catalyze redox reactions on the positions C3 and C17 of the steroid nucleus, respectively, are involved in the activation and inactivation of androgens [Figure I‐6]. By doing so, the most physiological active androgens testosterone (11) and dihydrotestosterone (DHT) (12) are built from but also inactivated to the less potent androgens Δ4‐androstenedione, Δ5‐androstene‐3β,17β‐diol (10), 3β,17β‐androstanediol (13), 3α,17β‐androstanediol (14), androstanedione (15), and androsterone (16). A key enzyme within the androgen pathway is the 5α‐reductase 2 (S5AR2). S5AR2 catalyzes the reduction of the Δ5 double bond in testosterone resulting in the even more potent saturated androgen DHT. However, active androgens can not only be synthesized via the classical pathway described above but also by a so‐called backdoor pathway [Figure I‐6]. Here, the Δ5 double bond of 17α‐hydroxypregnenolone is reduced by the Δ5‐reductase 1 (S5AR1) leading to 17α‐hydroxypregnane‐3,17‐dione (6) which is further reduced by 3α‐HSDs. The resulting pregnane‐3α,17α‐diol‐20‐one (7) is like the other 17α‐hydroxygestagens a good substrate for the CYP17A1 which catalyzes the generation of the androgen androsterone via its 17,20‐lyase activity [194, 195]. In vivo, the estrogens estrone (17) and the highly physiologically active estradiol (18) are generated from Δ4‐androstenedione and testosterone, respectively. These reactions are catalyzed by the aromatase CYP19A1, which belongs also to the type II cytochrome P450 enzymes, and takes place in the ER as all steps in sex steroidogenesis with the exception of the initial gestagen formation.
Figure I‐6: Sex steroid metabolism pathway.
Shown are the fundamental steps for the activation and inactivation of sex steroids together with the major enzymes catalyzing those reactions [78, 194–196]. Reactions of the so‐called backdoor pathway are indicated by dashed lines and the most physiologically active sex steroids are highlighted in yellow.
I.3.4. C
YTOCHROMEP450
ENZYMES(CYP
S)
AND THEIR ROLE IN VITAMIND
METABOLISMThe vitamin D metabolism and signaling represents another physiologically important pathway in mammalians in general and particularly in humans.
In humans, the metabolic vitamin D pathway [Figure I‐7], which can be divided into an activation and an inactivation (catabolic) sequence and is well described in literature, is mainly catalyzed by five enzymes belonging to the cytochrome P450 superfamily [199–201].
As shown in Figure I‐7, the endogenous vitamin D molecule vitamin D3 (3), which is like the nutritional vitamin D2 (4) a direct precursor for all subsequent enzymatic vitamin D activation and inactivation steps of the vitamin D pathway, is formed in the skin from the vitamin D3 precursor 7‐dehydrocholesterol (1) by two successive non‐enzymatic reactions. Here, 7‐dehydrocholesterol is first transformed to provitamin D (2) by an UV‐light triggered photochemical reaction. This intermediate is then isomerized to vitamin D3 by an isothermal reaction. For the generation of the major biologically active vitamin D derivatives 1α,25‐dihydroxyvitamin D3 (7) and 1α,25‐dihydroxyvitamin D2 (combined in the term 1α,25‐dihydroxyvitamin D), vitamin D3 as well as vitamin D2 are generally in the liver first hydroxylated at position C25 by the 25‐hydroxylases CYP27A1, CYP2R1, and CYP3A4 forming the vitamin D storage forms 25‐hydroxyvitamin D3 (5) and 25‐hydroxyvitamin D2. In the second activation step which takes place in the kidneys but also other target tissues, these storage forms are further hydroxylated at position C1 by the 1α‐hydroxylase CYP27B1 to generate the potent 1α,25‐dihydroxyvitamin D. However, 25‐hydroxyvitamin D3 and 25‐hydroxyvitamin D2 can also become hydroxylated at position C24 by the 24‐hydroxylase CYP24A1 which results in 24,25‐dihydroxyvitamin D3 (6) and 24,25‐dihydroxyvitamin D2, respectively. The active vitamin D metabolites are inactivated and catabolized by successive hydroxylation and/or oxidation reactions, again catalyzed by the CYP24A1. Thereby, the initial hydroxylation occurs either at position C24 (8) or at position C23 (10) and initiates the calcitroic acid (9) or lactone (11) degradation pathway, respectively. Besides, the C3 epimerization of vitamin D metabolites via a yet unknown mechanism seems to be another pathway for the modulation of vitamin D signaling in vivo [202–205].
The major biologically active vitamin D metabolite 1α,25‐dihydroxyvitamin D is involved in a plethora of physiological processes in a vitamin D receptor dependent or independent manner. Thus, for example, 1α,25‐dihydroxyvitamin D effects the bone health by regulating the calcium and phosphorus homeostasis, increases the muscle strength and mass via neuromuscular effects, mediates favorable immunomodulatory effects in diseases (e.g., in multiple sclerosis, type 1 diabetes, psoriasis, rheumatoid arthritis, or asthma), mediates cardiovascular effects via the regulation of the renin‐angiotensin system and an increased insulin sensitivity, and reduces the risk of cancer by antiproliferative, antiangiogenetic, prodifferentiative, and apoptotic effects [206–208]. Although 1α,25‐dihydroxyvitamin D had been considered to be the most important biologically active vitamin D metabolite for a long time, recent studies predicted also a physiological role for the vitamin D metabolite 24,25‐dihydroxyvitamin D [209, 210]. Because of the deep impact of the levels of vitamin D metabolites in health and disease, cytochrome P450 enzymes of the metabolic vitamin D pathway as well as vitamin D metabolites themselves are promising targets in the therapy of multiple diseases, like osteoporosis, inflammatory diseases, and cancer [199, 206, 211].
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Figure I‐7: Vitamin D metabolism pathway.
The vitamin D pathway is exemplarily shown for endogenous vitamin D3 metabolites; but it is also valid for nutritional vitamin D2 derivatives [199–201]. The major vitamin D3 storage form 25‐hydroxyvitamin D3 (5) and the most potent biologically active metabolite 1α,25‐dihydroxyvitamin D3 (7) are highlighted in yellow.
I.4. A IMS OF THIS THESIS
AKRs and SDRs are large and steadily growing superfamilies which are characterized by their typical cofactor binding structures: (α/β)8‐barrel structure and Rossmann fold, respectively. Despite these central, conserved structures, members of both superfamilies reveal highly variable and mostly broad substrate spectra [14, 122]. Because of their crucial roles in vivo and their association with numerous diseases, members of these superfamilies have been intensely studied for a long time.
Bioinformatic improvements lead to the permanent identification of novel family members.
Thus, a novel human AKR member with unknown features and biological roles – called AKR1B15 – was identified and annotated in 2011 [22]. This thesis aimed to characterize the novel human AKR1B15 fundamentally in order to get an idea about its function in vivo.
For this, the expression of AKR1B15 on transcript and protein level as well as the subcellular localization of AKR1B15 isoforms, demanding also the generation of specific antibodies, were analyzed. Furthermore, substrates belonging mainly to the group of steroids or fatty acids were identified and enzymatic parameters determined. Finally, as starting point for future in vivo analyses, all necessary constructs for an AKR1B15 knock‐out in cell lines expressing AKR1B15 endogenously via the TALEN technology were prepared and tested.
In contrast, some members belonging to the AKR or SDR family have been known for a long time, however, their actual functions in vivo are still unclear. One of those family members is the human SDR HSD17B12. Although there is strong evidence that the human 17β‐HSD12 is involved in the fatty acid metabolism in vivo, a role in the steroid metabolism cannot be completely excluded [139, 140]. Here, a method for the solubilization and purification of the presumed membrane‐bound protein from the heterologous expression system Pichia pastoris was established, allowing for a closer characterization of its enzymatic activities.
Vitamin D metabolites, especially 1α,25‐dihydroxyvitamin D, are known to have numerous effects in vivo. Because of its assumed anticancer effects, 1α,25‐dihydroxyvitamin D itself but also enzymes belonging to the superfamily of CYPs, which catalyze the metabolism of vitamin D metabolites and thus control the levels of active vitamin D metabolites in vivo, represent promising targets for cancer therapy [208]. A comprehensive determination of the levels of all metabolites of the vitamin D pathway in biological samples could enable an in‐depth assessment of the vitamin D status in samples but would also help to answer open questions of basis research. Thus, an analytical tool which could measure more than only one or two vitamin D metabolites at a time (as it is common for most commercially available methods at the moment) would facilitate such diagnostic and research.
Hence, another aim of this thesis was to develop, establish, and validate a LC‐MS/MS based method for the simultaneous detection and quantification of as many important components of the vitamin D metabolism pathway as possible in biological samples, especially in samples from cell culture experiments. By the use of this novel method, it was aimed to explore the uptake and metabolism of vitamin D metabolites in cultured cells as well as to analyze the effect of azoles (e.g., itraconazole), which are known to be CYP inhibitors, on the vitamin D metabolism (especially on the inactivation of 1α,25‐dihydroxyvitamin D).