hormone in the circulation is DHEAS, the sulfoconjugated form of DHEA. The daily DHEAS production in adult women of reproductive age is up to 20 mg, compared to 3-6 mg for DHEA, 1-6 mg for androstenedione and 0.1-0.4 mg for testosterone (Burger, 2002).
DHEAS is in the context of AR activation an inactive metabolite and cannot directly be converted to active androgens. DHEAS has strikingly different biochemical properties than DHEA: as a hydrophilic molecule, it is not able to cross lipid membranes freely as most other steroids and has to rely on active transport mechanisms. DHEAS is bound to plasma proteins (mainly albumin) and thus has a much longer plasma half-life of about 20 hours compared to 3-6 hours for DHEA. The sulfation of DHEA is catalysed by the enzyme DHEA sulfotransferase (SULT2A1) and predominantly occurs within the ZR (Falany et al., 1989; Strott, 2002). The enzyme steroid sulfatase (STS), which is mainly expressed in peripheral tissues, cleaves the sulfate moiety off the DHEAS molecule to regenerate DHEA, which can be converted further downstream to active sex steroids. Hence, through the counter- actions of SULT2A1 and STS, circulating, albumin-bound DHEAS has been thought to represent a storage pool for amplification of sex steroid signalling in peripheral target tissues. However, little is known about the exact impact of the DHEA sulfation system and its role during childhood development.
1.1.9.1. DHEA sulfotransferase (SULT2A1) and 3’-phosphoadenosine-5’-
phosphosulfate (PAPS) synthases (PAPSS)
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development (Strott, 2002; Dawson, 2011). Sulfation is a catalytic reaction where a sulfate group (SO42-) is transferred to an acceptor molecule. The introduction of a
sulfate group into a molecule results in striking alterations of the molecule’s biochemical properties, mainly an increase in water solubility as well as conformational changes.
Sulfation reactions are catalysed by sulfotransferases (SULTs), and more than 50 SULTs have been cloned and characterized in humans (Strott, 2002).
Depending on their cellular localization and chemical properties, two super- families can be distinguished: 1) soluble, cytosolic SULTs and 2) membrane-bound SULTs that are usually located within the Golgi apparatus (Strott, 2002). Members of the cytosolic group of SULTs are involved in steroid and drug metabolism, the membrane-associated SULTs sulfo-conjugate carbohydrates, peptides and proteins.
SULT2A1, a member of the subfamily 2 of cytosolic SULTs, has been traditionally named DHEA sulfotransferase, as it utilizes DHEA as its major substrate (Kong et al., 1992). However, it has a broad substrate specificity and also sulfates other Δ4 steroids, like pregnenolone or 17-hydroxypregnenolone, but also testosterone, oestradiol and bile acids (Radominska et al., 1990; Falany, 1997). Usually, the 3α/β- or the 17β-hydroxyl-groups of these molecules undergo sulfation by SULT2A1. Other SULTs involved in steroid metabolism have been identified; in particular SULT1B1 with its two isoforms SULT1B1a and SULT1B1b is able to sulfate hydroxysteroids (Whitnall et al., 1993; Strott, 2002).
SULT2A1 is robustly expressed in the cytosol of many steroidogenic tissues, in particular tissues involved in androgen production and metabolism, which are adrenal cortex, testicles, ovaries, prostate and liver but also the digestive tract (Javitt et al., 2001).
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The sulfate group for sulfation reactions mediated by SULTs needs to be ‘activated’ and is delivered by an organic compound named 3’-phosphoadenosine-5’- phosphosulfate (PAPS). In fact, PAPS serves as the universal sulfate donor for all sulfation reactions, and all tissues in mammals are able to produce PAPS from ATP and inorganic sulfate (Mulder, 2003). The generation of PAPS requires two distinct catalytic reactions carried out by the bi-functional enzyme PAPS synthase (PAPSS): First, ATP is sulfurylated yielding adenosine-5’-phosphosulfate (APS) as an intermediate, which is secondly further phosphorylated to form PAPS. While both enzymatic reactions, ATP sulfurylase and APS kinase, are located in plants, yeast and bacteria on different polypeptide chains, they are fused to one enzyme in metazoans forming the PAPSS enzyme (Strott, 2002; Mueller and Shafqat, 2013). Two PAPSS isoforms exist in humans, and their genes are located on two separate chromosomes: PAPSS1 (chromosome 4q25-26) is highly conserved amongst different species, including rodents, guinea pig and drosophila (Venkatachalam, 2003; Yanagisawa et al., 1998). It has about 80% amino acid identity with the human PAPSS2 isoform (chromosome 10q23-56) (Strott, 2002), which was discovered in a search for the genetic cause of a bone malformation disorder in a large consanguineous pedigree with affected members suffering from distinct bone malformations called spondylepimatephyseal dysplasia (SEMD) (Haque et al., 1998). Both isoforms have a very similar structure, genomic arrangement (12 exons with similar intro/exon boundaries), length (614 and 624 amino acids, respectively) and distribution of catalytic domains with the ATP sulfurylase domain being located at the N-terminus and the APS kinase domain at the carboxy-terminus (Xu et al., 2000). Two splice variants exist for the human PAPSS2 isoform, PAPSS2a and PAPSS2b: PAPSS2b has an additional exon comprising 5 amino acids (‘GMALP’) within the
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ATP sulfurylase domain (van den Boom et al., 2012; Fuda et al., 2002). However, there are differences between the two PAPSS isoforms in regards to their tissue expression: PAPSS1 is virtually ubiquitously expressed, while PAPSS2 is mainly in cartilage, adrenal, gonads, placenta, colon and liver (Fuda et al., 2002; Stelzer et al., 2007). Furthermore, a different subcellular distribution of PAPSS isoforms has been reported. Sulfation reactions are essentially taking place within the cytoplasm for most substrates, including steroids; however, sulfation of oestrogens has been reported to occur in the nucleus for rodents and guinea pig (Mancini et al., 1992; Whitnall et al., 1993). Strikingly, Besset et al. reported that the human PAPSS1 isoform is located in the nucleus whereas PAPSS2 is expressed in the cytoplasm of mammalian cells (Besset et al., 2000). Recently, Schröder et al. showed that the distribution pattern of both PAPSS isoforms is much more variable and both enyzmes are expressed dynamically in the cytosol and the nucleus of mammalian cells (Schröder et al., 2012). Interestingly, work from the same group has shown that PAPSS2 is less stable with a half-life of several minutes for the unfolding protein, whereas PAPSS1 remains structurally intact at physiological temperatures (van den Boom et al., 2012). In addition, Grum et al. has shown that PAPSS isoforms associate as hetero-dimers, suggesting the possibility of co-synergistic regulation of intracellular sulfation pathways (Grum et al., 2010).
However, the functional consequences of the differences regarding their dynamic sub-cellular localization patterns, in vitro protein stability and dimerisation, in particular in the context of androgen pre-receptor metabolism, are still unknown and remain to be established.
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Figure 10: Interconversion of DHEA and DHEAS. The sulfation of DHEA is catalysed by the enzyme
DHEA sulfotransferase (SULT2A1); the opposite direction, the hydrolysis of the sulfate moiety from DHEAS, is performed by steroid sulfatase (STS). Both enzymes depend on co-factors: SULT2A1 requires the activated sulfate compound PAPS, generated by the enzyme PAPS synthase (PAPSS) via two catalytic activities: (1) ATP is sulfurylated to APS by the ATP sulfurylase activity, (2) APS is further phosphorylated to form PAPS. A cysteine residue of the STS enzyme needs to be post- translationally activated to a formylglycine (FGly) by the mono-oxigenase formylglycine-generating enzyme (FGE), encoded by the SUMF gene. FGly is further hydroxylated, crucial for the hydrolysis of the sulfate moiety catalysed by STS.
1.1.9.2. Steroid sulfatase (STS) and sulfatase-modifing factors (SUMFs)
Steroid sulfatase (STS, aka as arylsulfatase C) is a membrane-bound microsomal enzyme and member of a highly conserved family of arylsulfatases (ARS) (Reed et al., 2005). Sulfatase enzymes act in the opposite direction of sulfotransferases and catalyse the cleavage of the sulfate moiety from a variety of substrates, including conjugated steroids and other hormones, proteoglycans, post- translationally modified proteins and aromatic compounds.
The action of STS is implicated in the regeneration of steroids and its precursors from their inactive sulfo-conjugated esters, thereby enhancing their availability in peripheral target cells on the pre-receptor level. It hydrolyses the sulfate
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group of 3β-hydroxysteroid sulfates, including DHEAS, pregnenolone sulfate, 17- Preg sulfate, sulfated oestrogens, but also of cholesterol sulfate (Reed et al., 2005).
The STS gene is localized on the short arm of the X-chromosome (Xp22.3), which is part of the pseudo-autosomal region escaping X-inactivation (Ballabio et al., 1987a). It encodes a protein of 583 amino acids and has a mass of about 65kDA (Stein et al., 1989).
Genetic abnormalities of the STS gene causes steroid sulfatase deficiency (STSD) resulting in the skin condition X-linked ichthyosis, a common inborn error of metabolism with a prevalence of 1:6,000 (Fernandes et al., 2010). It is characterized by thickening of the epidermis and large, brown scales of the skin; the pathomechanism is thought to be due to accumulation of cholesterol sulfate in the stratum corneum of the epidermis. Abnormal steroid metabolism has been reported in small cohorts of patients with STSD as well as elevated levels of sulfated steroids, like DHEAS and oestrogen sulfates (Lykkesfeldt et al., 1985a; Milone et al., 1991; Delfino et al., 1998).
The molecular mechanisms underlying STS catalytic activity have been studied in detail and are highly conserved amongst different human sulfatase enzymes (Reed et al., 2005; Ghosh, 2007): a highly conserved cysteine residue resides in the catalytic centre of the sulfatase, which is post-translationally modified to form a formylglycine (FGly) residue. FGly is catalytically active and ‘attacks’ the sulfate moiety of substrates and is essential to hydrolyse the sulfate ester bond (Recksiek et al., 1998; Dierks et al., 1998; Ghosh, 2007) (Figure 10). The modification of the cysteine to form the FGly is mediated by the co-enzyme formylglycine generating enzyme (FGE), which is encoded by the gene sulfatase-
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deficiency, a rare and fatal autosomal recessive disorder characterized by absent activity of all sulfatase enzymes (Cosma et al., 2003; Dierks et al., 2003).
STS is believed to be expressed in virtually all human adult and foetal tissues in very small amounts (Strott, 2002). Immunohistochemistry studies have demonstrated that STS is localized in the endoplasmic reticulum and Golgi apparatus (Willemsen et al., 1988). A systemic expression analysis employing semi-quantitative RT-PCR, immunohistochemistry and biochemical detection by activity assays detected either absent or very low STS expression and activity in a variety of human foetal and adult tissues, with low-moderate STS detection in lung, aorta, adrenal gland, thyroid, mammary gland, testis, prostate and endometrium (Miki et al., 2002). However, the richest source for STS is the syncytiotrophoblast of the human placenta.
1.2. A
DRENAL ANDROGENS DURING HUMAN PRE-
AND POSTNATALDEVELOPMENT