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Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases
Accepted Manuscript Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases Maria Tsachaki, Alex Odermatt PII:
S0303-7207(18)30217-X
DOI:
10.1016/j.mce.2018.07.003
Reference:
MCE 10270
To appear in:
Molecular and Cellular Endocrinology
Received Date: 22 August 2017 Revised Date:
18 June 2018
Accepted Date: 3 July 2018
Please cite this article as: Tsachaki, M., Odermatt, A., Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases, Molecular and Cellular Endocrinology (2018), doi: 10.1016/ j.mce.2018.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases
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Maria Tsachakia and Alex Odermatta*
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of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University
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* Corresponding author: Prof. Alex Odermatt, Division of Molecular and Systems Toxicology,
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Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel,
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Switzerland. [email protected]
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Abstract
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The 17β-hydroxysteroid dehydrogenases (17β-HSDs) comprise enzymes initially identified by their ability
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to interconvert active and inactive forms of sex steroids, a vital process for the tissue-specific control of
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estrogen and androgen balance. However, most 17β-HSDs have now been shown to accept substrates
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other than sex steroids, including bile acids, retinoids and fatty acids, thereby playing unanticipated roles
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in cell physiology. This functional divergence is often reflected by their different subcellular localization,
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with 17β-HSDs found in the cytosol, peroxisome, mitochondria, endoplasmic reticulum and in lipid
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droplets. Moreover, a subset of 17β-HSDs are integral membrane proteins, with their specific topology
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dictating the cellular compartment in which they exert their enzymatic activity. Here, we summarize the
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present knowledge on the subcellular localization and membrane topology of the 17β-HSD enzymes and
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discuss the correlation with their biological functions.
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metabolism; steroid
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subcellular
localization;
membrane
topology;
17β-hydroxysteroid
dehydrogenases;
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Abbreviations: 3α-adiol, 5α-androstan-3α,17β-diol; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type
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1; 17β-HSD, 17β-hydroxysteroid dehydrogenase; A4, ∆4-androsten-3,17-dione; Aβ, amyloid peptide β;
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ACS3, acyl-CoA synthase 3; ADRP, adipose differentiation-related protein; AKR, aldo-keto reductase;
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AR, androgen receptor; CBR4, carbonyl reductase type 4; DHEA, dehydroepiandrosterone, DHT, 5α-
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dihydrotestosterone; ER, endoplasmic reticulum; ERAB, ER-associated Aβ peptide binding protein; FAS,
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fatty acid synthesis; G6PD, glucose 6-phosphate dehydrogenase; KAR, 3-ketoacyl-acyl carrier protein
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(ACP) reductase; LSS, lanosterol synthase; NAD, nicotinamide adenine dinucleotide; PAT, perlipin,
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ADRP and tail-interacting protein; PPAR, peroxisome proliferator-activated receptor; PSA, prostate-
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specific antigen; Ribonuclease P, RNase P; RODH, retinol dehydrogenase; roGFP, reduction-oxidation
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sensitive green fluorescent protein; SDR, short-chain dehydrogenase/reductase.
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1. Introduction The 17β-hydroxysteroid dehydrogenases (17β-HSDs) form an enzyme subfamily of the superfamily of
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short-chain dehydrogenases-reductases (SDRs), with the exception of 17β-HSD5 that belongs to the aldo-
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keto reductase (AKR) family (Moeller et al., 2009). At least 14 different 17β-HSDs have been described
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and they were named after their capacity to interconvert 17-oxo and 17-hydroxy sex steroids. They are
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numbered according to the chronological order of their discovery and the first three members (17β-HSD1,
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2 and 3) were recognized for their ability to interconvert the weaker 17-ketosteroid and the more potent
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17β-hydroxy forms of estrogens and/or androgens (Gast et al., 1989; Geissler et al., 1994; Wu et al.,
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1993). Although these enzymes act bi-directionally in vitro, they predominantly function as either
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oxoreductases or dehydrogenases in intact cells as well as in vivo (Khan et al., 2004), depending on the
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availability of a co-factor, which is preferably either nicotinamide adenine dinucleotide NADP(H) or
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NAD(H). Because of the intracellular abundance of these reduced and oxidized co-factors, typically,
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reductive 17β-HSDs use NADPH and oxidative enzymes NAD+ as co-factor (Hedeskov et al., 1987). The
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co-factor binding site is formed by a conserved structural motif called Rossmann fold (Buehner et al.,
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1973), encompassing the conserved sequence GxGxxxG. The Rossmann fold is a common co-factor
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binding element of SDRs. Notably, the AKR 17β-HSD5 does not contain a GxGxxxG motif in its
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sequence.
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That 17β-HSD enzymes are not exclusively dedicated to sex steroid metabolism was realized after the
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discovery of 17β-HSD4, which was shown to participate in peroxisomal β-oxidation of fatty acids
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(Breitling et al., 2001; Leenders et al., 1996; Markus et al., 1996; Seedorf et al., 1995). Since then, the
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notion of a multifunctional 17β-HSD family was gradually established and experimentally supported. The
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diversity in substrate specificity and in vivo function is also mirrored in the subcellular localization of the
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different 17β-HSD enzymes. However, the presence of an enzyme in a specific organelle cannot always be
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deduced through the knowledge of its substrate. Although signal peptides and targeting sequences are
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present in some 17β-HSDs, definitive proof of the presence in a cell compartment requires the
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employment of biochemical methods (such as subcellular fractionation and immunoblotting) or indirect
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immunofluorescence. A critical issue in these studies is the utilization in each method of proper markers,
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in order to undoubtedly designate a certain protein to a cell organelle or the cytosol.
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Since all organelles are encircled by lipid mono- or bilayers, integral membrane proteins can either
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function in the lumen of an organelle or at its cytosolic side. Therefore, the topology (the orientation of
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different parts of a protein in relation to the membrane) is a fundamental feature of membrane proteins.
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All 17β-HSDs residing in the endoplasmic reticulum (ER) or lipid droplets are membrane-bound (17β-
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HSD2, 3, 6, 7, 11, 12 and 13). Different methods have been employed for topology determination of
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membrane-anchored 17β-HSDs, including selective permeabilization of cell membranes with digitonin
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followed by immunofluorescence detection, proteinase K protection assay followed by western blotting
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and, recently, live-cell measurements using reduction-oxidation sensitive green-fluorescent protein
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(roGFP) fusions with the respective 17β-HSD enzyme (Tsachaki et al., 2015).
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For subcellular localization and membrane topology determination, peptide tags, fusion or truncated
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proteins are frequently used. This raises the question of how such interventions could affect intracellular
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targeting. Given the complex nature of the above studies, erroneous initial allocation in a cell organelle or
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membrane topology have been reported, which has led to misconceptions regarding the physiological role
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of certain 17β-HSDs. In this review, we elaborate on current understanding of 17β-HSD subcellular
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localization and membrane topology (summarized in Fig. 1 and 2, respectively), based on previous
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investigations that include both in silico studies and experimental evidence. In addition, we elaborate on
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how this aspect of 17β-HSD protein physiology is potentially linked to their enzymatic function and the
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metabolic pathways in which they are involved.
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2. Subcellular localization of 17β-HSDs 2.1 Cytosolic 17β-HSD1, 5 and 14 4
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17β-HSD1 was the first enzyme identified in this family and its main function is to reduce the weakly
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active 17-keto estrogen estrone to the potent 17β-hydroxy estrogen estradiol and thereby stimulating the
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activity of estrogen receptors, a process particularly important in tissues such as the placenta, the breast
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and the ovaries (Luu The et al., 1989). An elevated expression of 17β-HSD1 has been associated with sex-
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specific malignancies, including breast and endometrial carcinomas (Gunnarsson et al., 2001; Oduwole et
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al., 2004), and inhibitors of this enzyme are considered for potential therapeutic applications to treat
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hormone-dependent cancer (Brozic et al., 2008; Cornel et al., 2012; Hilborn et al., 2017; Zhang et al.,
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2012). Although other 17β-HSDs (mainly 17β-HSD7 and 17β-HSD12) have been reported to be able to
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reduce estrone to estradiol, 17β-HSD1 clearly has the highest affinity, and combined with its expression
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pattern it is considered to be the main enzyme exerting this function (Laplante et al., 2009). It was
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suggested that 17β-HSD1 dwells in the cytosol, since this enzyme has no predicted membrane helices.
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Subsequently, immunofluorescence experiments using a specific antibody against human 17β-HSD1
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confirmed the cytosolic distribution (Tsachaki et al., 2015).
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The inactivation of estradiol to estrone can be performed by several 17β-HSDs (17β-HSD2, 4, 6, 8, 9, 10
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and 14), although the relative in vivo contribution of each of these enzymes to estradiol inactivation
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remains unclear. Among these enzymes, 17β-HSD14 is the only one residing in the cytosol, as shown by
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co-localization with the cytoplasmic protein phalloidin in immunofluorescence labelling experiments
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(Lukacik et al., 2007). In the same study, no co-localization with nuclei or mitochondria could be
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observed. 17β-HSD14 likely plays an important role in the local inactivation of estradiol in tissues where
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it is highly expressed, such as the breast, placenta and brain (Jansson et al., 2006; Lukacik et al., 2007;
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Sivik et al., 2012). Importantly, low expression of 17β-HSD14 was correlated with better prognosis, and
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its expression was shown to be predictive of response to tamoxifen treatment in estrogen-receptor positive
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breast cancer patients (Jansson et al., 2006; Sivik et al., 2012). In this case, the presence of 17β-HSD1 and
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17β-HSD14 in the cytosol could provide an efficient system for the intracellular regulation and
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maintenance of high estradiol concentrations. 17β-HSD14 is also able to oxidize other substrates, at least
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in vitro, including 5-androstene-3β,17β-diol and testosterone (Lukacik et al., 2007). Resolving the crystal
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structure of 17β-HSD14 revealed that the substrate binding pocket is wide enough to accommodate
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substrates other than steroids, raising the possibility that new roles for 17β-HSD14 will be uncovered in
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the future (Bertoletti et al., 2016; Lukacik et al., 2007).
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As mentioned above, all 17β-HSDs belong to the SDR superfamily with the exception of 17β-HSD5 (also
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known as AKR1C3) that is a member of the AKR superfamily (Penning, 2015). AKRs are dedicated in the
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reduction of carbonyl-containing substrates, including sugar aldehydes, keto-prostaglandins and keto-
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steroids. 17β-HSD5 catalyzes the reduction of the prostaglandins PGH2 and PGD2 to PGF2a and 9α,11β-
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PGF2, respectively, both products shown to promote cancer cell proliferation (Matsuura et al., 1998;
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Suzuki-Yamamoto et al., 1999). Importantly, 17β-HSD5 participates in metabolic cascades through which
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the most potent human androgen 5α-dihydrotestosterone (DHT) is synthesized, and has been considered as
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a potential target against prostate cancer (Adeniji et al., 2013). Upon binding of DHT to its cognate
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androgen receptor (AR) in the cytosol, it translocates into the nucleus where it regulates the expression of
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a plethora of target genes. Immunofluorescence experiments showed that in the absence of ligand
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activation 17β-HSD5 co-localizes with the AR in the cytosol, whereas treatment with the 17β-HSD5
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substrate ∆4-androsten-3,17-dione (A4) or the AR agonist R1881 led to translocation of both AR and 17β-
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HSD5 to the nucleus (Yepuru et al., 2013). Intriguingly, chromatin immunoprecipitation experiments
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demonstrated the binding of 17β-HSD5 to the promoter of the AR-target gene prostate specific antigen
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(PSA), when it was suggested to act as a coactivator of the AR. Nuclear localization has not been shown
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for any other 17β-HSD enzyme, and the specific function of a reductase binding to a gene regulatory
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region remains enigmatic.
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2.2 Peroxisomal 17β-HSD4
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The only enzyme of the 17β-HSDs localized in the peroxisomes is 17β-HSD4 (Adamski et al., 1995).
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Evidence for peroxisomal localization of 17β-HSD4 was provided by immunogold electron microscopy,
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where 17β-HSD4 co-localized with the peroxisomal-resident enzymes catalase and acyl-CoA oxidase
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(Markus et al., 1995). Later, the subcellular localization of 17β-HSD4 was also deduced from the presence
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at its C-terminus of the peroxisomal targeting tripeptide Ala-Lys-Leu (AKL) (Moller et al., 1999), which
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represents an alternate version of the classical Ser-Lys-Leu (SKL) peroxisomal targeting signal. In the
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porcine enzyme this sequence is varied as Ala-Lys-Ile, and its deletion from the protein causes cytosolic
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mislocalization (Moller et al., 1999).
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Although the N-terminal part of the protein is reminiscent of other SDRs, the amino acid sequence of 17β-
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HSD4 points towards a multi-functional enzyme. The diverse roles of 17β-HSD4 in peroxisomes have
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been well-documented and are attributed to distinct protein domains (Breitling et al., 2001). A sterol and
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phospholipid transfer domain present at the C-terminus of 17β-HSD4 is related to sterol carrier protein 2
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(SCP2), and such activity has been clearly demonstrated for the enzyme (Leenders et al., 1996; Markus et
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al., 1996; Seedorf et al., 1995). Additionally, based on sequence similarity to the yeast FOX2 proteins,
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17β-HSD4 was suggested to participate in peroxisomal β-oxidation of fatty acids and bile acids (Leenders
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et al., 1996). Indeed, acyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities of 17β-HSD4 are
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supported by strong experimental evidence (Caira et al., 1998; Leenders et al., 1996; Markus et al., 1996;
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Qin et al., 1997). Knockout of 17β-HSD4 in mice showed severe impairments in β-oxidation of 2-methyl-
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branched fatty acids, the bile acid intermediates di- and trihydroxycoprostanic acid, and very long-chain
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fatty acids (Baes et al., 2000). The importance of 17β-HSD4 for peroxisomal function is further
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underlined by reports for mutations of the HSD17B4 gene, leading to conditions reminiscent of the
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Zellweger syndrome (Suzuki et al., 1997; van Grunsven et al., 1998; van Grunsven et al., 1999).
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Despite the above functions attributed to 17β-HSD4, it has also been considered important for estrogen
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metabolism, since it was first described as an estradiol-inactivating enzyme in porcine uterus (Adamski et
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al., 1992). Luteneization of equine ovarian follicles, a process accompanied by a decrease in estradiol and
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increase in progesterone levels, was linked to increase in the expression of 17β-HSD4 (Brown et al.,
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2004). Additionally, single nucleotide polymorphisms in the HSD17B4 gene were well-correlated with
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endometrial cancer risk (Karageorgi et al., 2011). According to its amino acid sequence, 17β-HSD4 is
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unlikely to contain any transmembrane helices and is thus expected to be a soluble protein within the
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peroxisomal matrix. Since estrogen metabolism in this compartment has not been demonstrated, and the
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presence of 17β-HSD4 in the cytosol has never been shown, the actual involvement of 17β-HSD4 in
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estrogen metabolism remains obscure.
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2.3 Mitochondrial 17β-HSD8 and 17β-HSD10
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Fatty acid synthesis (FAS) in mitochondria is responsible for producing a number of long-chain fatty
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acids, including the respiratory complex I component 3-hydroxymyristoyl-ACP and lipoic acid (Hiltunen
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et al., 2010). The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) has been recently
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discovered to be 17β-HSD8. The presence of 17β-HSD8 in mitochondria was demonstrated by co-
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localization with the MitoTracker Red dye in immunofluorescence labeling experiments (Chen et al.,
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2009). The primary sequence of 17β-HSD8 indicates that it is a soluble enzyme and should thus reside in
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the mitochondrial matrix. Moreover, it has been shown to form a heteroterameric complex with carbonyl
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reductase type 4 (HsCBR4), where it acts as a scaffold to the functional subunit CBR4 (Venkatesan et al.,
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2014). Based on these findings and the fact that it likely acts as an oxidase in vivo, it was suggested that
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17β-HSD8 could function in channeling 3R-hydroxyacyl-CoA intermediates to β-oxidation in
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mitochondria. Three-dimensional models of 17β-HSD8 in complex with NAD+ showed that it accepts
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substrates similar to those of the β-ketoacyl-ACP reductases of Brassica napus, further supporting a role
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in fatty acid metabolism (Pletnev et al., 2005).
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The gene encoding 17β-HSD10 is located on the X chromosome and several mutations of the enzyme are
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linked with mental retardation (Yang et al., 2011). The function of this enzyme remains to be further
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investigated. Elevated 17β-HSD10 levels have been reported in Alzheimer’s patients (He et al., 2002). It
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has been shown to bind the amyloid β (Aβ) peptide, which accumulates in the brain of Alzheimer’s
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patients and is thought to play a major role in the disease progression (Kissinger et al., 2004). 17β-HSD10
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is also important for isoleucine metabolism and oxidative inactivation of the potent neurosteroid
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allopregnanolone (He et al., 2005; Ofman et al., 2003). Besides, 17β-HSD10 was shown to catalyze the
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conversion of the inactive androgen 5α-androstan-3α,17β-diol (3α-adiol) to the potent androgen DHT,
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with a potential role in the backdoor pathway of androgen generation that is thought to promote prostate
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cancer (Auchus, 2004; Mohler, 2014; Penning, 2010).
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Initially, 17β-HSD10 was reported to reside in the ER in HeLa cells (giving rise to the suggested name
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ER-associated Aβ peptide binding protein-ERAB), and addition of Aβ peptide led to its re-distribution to
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the plasma membrane in the human neuroblastoma cell line SK-N-SH (Yan et al., 1997), an observation
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that was supported by confocal microscopy and subcellular fractionation studies. However, in the
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immunofluorescence experiments presented in this work no plasma membrane marker was used to
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confirm such localization. Additionally, no compartment-specific marker was demonstrated to validate
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proper separation of all cell fractions. In a follow-up work by the same group, this localization could be
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reproduced, however the additional presence of 17β-HSD10 in the mitochondria of the same cells was
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shown (Yan et al., 1999). Although it cannot be excluded that 17β-HSD10 also localizes in the ER or at
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mitochondria/ER contact sites in the cell lines tested above, all subsequent studies using specific markers
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for both immunofluorescence labeling and subcellular fractionation experiments have reported exclusively
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mitochondrial localization (He et al., 2001; He et al., 1999; He et al., 2000). Interestingly, an early study
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identified bovine 17β-HSD10 as the 3-hydroxyacyl-CoA dehydrogenase of the mitochondrial fatty acid β-
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oxidation (Furuta et al., 1997). It was also demonstrated that its protein sequence contains a 16-amino acid
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N-terminal non-cleaved mitochondrial signal peptide, similar to that of human and rat thiolases.
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Nevertheless, fusion of the 15 first amino acids of human 17β-HSD10 to GFP failed to result in
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mitochondrial localization of the fusion protein, whereas fusion of the first 34 amino acids was sufficient
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(Shafqat et al., 2003). This implies that additional elements following this signal peptide are necessary for
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mitochondrial targeting.
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An important role of 17β-HSD10, directly linked to its subcellular localization, is in mitochondrial tRNA
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processing, as an essential component of Ribonuclease P (RNase P) (Holzmann et al., 2008; Vilardo et al.,
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2012). The role of 17β-HSD10 in mitochondrial tRNA processing is believed to underlie the etiology of
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the HSD10 disease, which is characterized by severe neurological defects and cardiomyopathy
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(Deutschmann et al., 2014; Falk et al., 2016; Vilardo et al., 2015). The plethora of substrates and functions
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attributed to 17β-HSD10 points towards a multifunctional enzyme (Yang et al., 2014; Yang et al., 2005),
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and the precise mechanisms by which its malfunction or absence leads to disease remain to be more
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clearly determined.
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2.4 Lipid droplet-associated 17β-HSD11 and 17β-HSD13
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Lipid droplets are cytoplasmic organelles dedicated to lipid storage and are regarded to be formed by
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budding off from ER membranes (Pol et al., 2014). They contain a lipid monolayer and they are mostly
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found in adipocytes, although they are also present in metabolically active tissues, such as the liver and the
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muscle. 17β-HSD11 was the first enzyme in the 17β-HSD family shown to be located in lipid droplets. In
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the first study describing its subcellular localization, N-terminally myc-tagged 17β-HSD11 transiently
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transfected into the mouse adrenal Y1 cell line was regarded to be in the cytoplasm based on
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immunofluorescence labeling (Chai et al., 2003). However, no organelle or cytoplasmic markers were
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employed in this study, and the microscopic image was likely misinterpreted. Also, the N-terminal tag
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might have affected the localization of the protein due to its close proximity to the membrane spanning
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helix. Later, it was shown that 17β-HSD11 is one of the most abundant proteins in the lipid droplet
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fraction of the human hepatoma cell line HuH7, together with Adipose differentiation-related protein
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(ADRP) and acyl-CoA synthase 3 (ACS3). This was demonstrated by mass spectrometric analysis and
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confirmed by immunoblotting using a specific antibody (Fujimoto et al., 2004). When cells were treated
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with oleic acid, which promotes lipid droplet formation, the lipid droplet localization became more
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evident. In this study, 17β-HSD11 exhibited an exclusive lipid droplet localization; it could not be
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detected by immunoblotting in the ER cellular protein fraction nor did it show any ER labeling in
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fluorescence labeling experiments. Interestingly, 17β-HSD11 expression was found to be upregulated in
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the mouse intestine upon activation of peroxisome proliferator-activated receptor alpha (PPARα), which
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has major roles in lipid metabolism (Motojima, 2004). In subcellular fractionation studies 17β-HSD11 was
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present in both the ER and the lipid droplet fraction when mice were fed normal diet, and specifically
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localized in the lipid droplet fraction when they were treated with a PPARα agonist (Yokoi et al., 2007).
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Moreover, in the Chinese hamster ovary cell line 17β-HSD11 was found to localize in the ER under
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normal conditions and to be transferred from the ER to the lipid droplets after oleic acid treatment.
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The N-terminal hydrophobic part of mouse 17β-HSD11 (aminoacids 4-16) seems to be important for
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correct intracellular targeting, since deletion of this region leads to mitochondrial localization (Horiguchi
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et al., 2008). Based on in silico predictions this should be part of a transmembrane region, which also
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contains the lipid droplet-targeting motif of the PAT family (perilipin, ADRP and tail-interacting protein).
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Deletion of this motif or of two of its conserved amino acids causes cytosolic mislocalization of mouse
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17β-HSD11 (Horiguchi et al., 2008). Analysis of the membrane topology of 17β-HSD11 suggested that it
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contains a membrane helix close to the N-terminus, followed by the largest part of the protein, containing
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the Rossmann fold and active sites, which faces the cytosol (Tsachaki et al., 2015).
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Although 17β-HSD11 has been shown to be able to convert several substrates, including estradiol and 3α-
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adiol (Brereton et al., 2001; Li et al., 1998; Lundova et al., 2016), the in vivo enzymatic activity is far from
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being resolved and has surely not been correlated with its subcellular localization. Even less is known
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about the other lipid droplet-associated enzyme 17β-HSD13 (Liu et al., 2007), which has a surprisingly
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similar primary amino acid sequence to 17β-HSD11 (65% identical amino acids). The N-terminus of 17β-
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HSD13, which is also highly hydrophobic and contains a motif similar to the PAT of 17β-HSD11, has
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been shown to be sufficient for correct targeting of the protein in lipid droplets (Horiguchi et al., 2008).
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Topology studies showed similar results than those obtained for 17β-HSD11, suggesting that the enzyme
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transverses the membrane once and its active site protrudes into the cytosol (Tsachaki et al., 2015). The
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recent DiscovEHR human genetics study revealed an association of a 17β-HSD13 splice variant
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(rs72613567:TA) resulting in an unstable truncated protein with a reduced risk of nonalcoholic and
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alcoholic liver disease (Abul-Husn et al., 2018). This splice variant was associated with a lower risk of
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nonalcoholic steatohepatitis, but not with simple steatosis, suggesting that this variant inhibits disease
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progression. Wild-type 17β-HSD13 and the truncated variant were found to be expressed on the surface of
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the lipid droplet membrane. This study also provided evidence for several bioactive lipids, including
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leukotriene B3 and B4, as well as eicosanoids, as potential physiologically relevant substrates.
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Interestingly, ablation of 17β-HSD13 in mice led to hepatic steatosis and inflammation, with evidence for
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impaired mitochondrial β-oxidation, without alterations in circulating steroid concentrations or
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reproductive performance (Adam et al., 2018). The above observations should stimulate further research
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to uncover the physiologically most relevant substrates of 17β-HSD11 and 17β-HSD13, as well as the role
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of these two enzymes in lipid metabolism, control of lipid storage and energy homeostasis.
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2.5 17β-HSD2, 3, 6, 7, 9 and 12 at the ER membrane
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Besides 17β-HSD11 and 17β-HSD13, both located at the ER and in lipid droplets, six other 17β-HSDs
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have been shown to localize at the ER. All are transmembrane proteins, with their substrate and co-factor
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binding site facing either the ER lumen or the cytosol. Until recently, attempts to resolve the membrane
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topology of these enzymes involved mainly proteinase K digestion of isolated microsomes followed by
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immunoblotting to determine whether the exposed protein part is protected (indicating luminal
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orientation) or not (supporting cytosolic orientation). This biochemical method relies on the proper
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microsomal preparation, yielding >90% inside-out microsomes (ER luminal part protected, cytoplasmic
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side facing the solution). The activity and purity of the proteinase K batch needs to be carefully evaluated.
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Proteinase K is still active in the presence of detergent and at 4°C, thus proper inactivation to terminate the
295
reaction is essential. Another method is the selective permeabilization of plasma membranes using
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detergents such as digitonin, followed by indirect immunofluorescence to determine whether a specific
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epitope of the protein of interest faces the cytosolic or luminal side of the ER. Similarly, the plasma
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membrane-specific pore forming agent streptolysin-O allows selective access to the cytosolic
299
compartment. Importantly, suitable positive and negative controls must be included in the above
300
mentioned methods.
301
Recently, we employed a method for membrane topology determination of SDRs in living cells, based on
302
fusing part or the SDR sequence with the redox-sensitive roGFP2 (Tsachaki et al., 2015). Depending on
303
the oxidation conditions of the compartment, the emission intensity of roGFP2 when excited at two
304
different wavelengths (405 nm and 480 nm) changes. The intensity ratio at the two different wavelengths
305
allows calculating the degree of roGFP2 oxidation. A high oxidation rate of roGFP2 indicates ER luminal
306
localization and a low oxidation rate a cytoplasmic one. By fusing roGFP2 either to the very C-terminus
307
of the different 17β-HSD enzymes or immediately upstream of the co-factor binding and active sites, we
308
could solve the topology of 17β-HSD2, 17β-HSD6, 17β-HSD7 and 17β-HSD12, along with the ER/lipid
309
droplet-localized 17β-HSD11 and 17β-HSD13. These results were corroborated with selective
310
permeabilization of cell membranes using digitonin.
311
For the above methods, fusion proteins of the respective 17β-HSD enzyme with GFP or other epitope tags
312
(for instance myc or flag) at either the C- or N-terminus can be used. Given that many proteins contain at
313
the N-terminus signal sequences for proper targeting, modifications at the C-terminus are usually
314
preferred. Proper localization of the fusion protein at the expected cellular compartment has to be
315
confirmed to avoid artifacts. Unfortunately, even with careful experimental design and validation,
316
structural changes introduced by protein modifications that could inverse proper membrane insertion can
317
never be fully excluded. Below, we revise our present understanding on the membrane topology of ER-
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resident 17β-HSDs.
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17β-HSD2 is responsible for oxidizing the active 17β-hydroxy forms of many sex steroids to their inactive
322
or weakly active 11-keto forms, exerting a protective function in peripheral tissues to avoid excessive
323
action of these steroids (Moghrabi et al., 1997). Importantly, it exhibits opposite effects to 17β-HSD1 by
324
inactivating estradiol to estrone (Miettinen et al., 1996), and a decreased expression has been associated
325
with breast cancer (Gunnarsson et al., 2001). Additionally, it has a role in androgen inactivation by
326
mediating the conversion of testosterone to A4, androstenediol to dehydroepiandrosterone (DHEA), DHT
327
to androstanedione and 3α-adiol to androsterone (Elo et al., 1996; Wu et al., 1993), and decreased
328
expression has been associated with colon cancer (Oduwole et al., 2003).
329
When first cloned, a putative C-terminal ER retention signal was identified and led to the assumption that
330
the part of the protein containing the active site is luminal (Wu et al., 1993). A subsequent study
331
conducting immunofluorescence experiments demonstrated that 17β-HSD2 co-localized at the ER
332
membrane with the ER-luminal protein BiP, and the presence of 17β-HSD2 in the ER lumen was
333
suggested (Puranen et al., 1999). However, given the size of the ER lipid bilayer, the diffraction limit of
334
the microscope does not allow from double immunofluorescence labeling experiments to distinguish
335
between proteins facing the ER lumen from proteins facing the cytoplasm. To solve this issue, we fused
336
roGFP2 to the C-terminus of 17β-HSD2, and also immediately downstream of the two putative N-terminal
337
transmembrane helices. These experiments revealed a cytosolic localization in both cases based on
338
roGFP2 oxidation. This was additionally supported by selective permeabilization experiments using
339
digitonin (Tsachaki et al., 2015). Taken together, these results strongly support a topology of 17β-HSD2
340
where the substrate and co-factor binding sites are oriented toward the cytosol.
341
Another microsomal enzyme initially suggested to have a luminal orientation is 17β-HSD3, whose
342
principal function is the reduction of A4 to testosterone in the testicular Leydig cells (Geissler et al.,
343
1994). Hu et al. hypothesized a coupling of 17β-HSD3 and 11β-hydroxysteroid dehydrogenase type 1
344
(11β-HSD1) activities, where 17β-HSD3 would utilize ER luminal NADPH to generate testosterone (Hu
345
et al., 2008). The formed NADP+ would then serve as co-factor for 11β-HSD1 to catalyze the inactivation
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of the potent glucocorticoid cortisol to cortisone under stress conditions. However, although an
347
interrelationship between the activity of 11β-HSD1 as an oxoreductase or dehydrogenase and the A4
348
conversion to testosterone was proposed (Latif et al., 2011), a direct functional link between the two
349
enzymes and competition for NADPH co-factor in the ER lumen was not unequivocally shown.
350
Importantly, it was later demonstrated that the active site of 17β-HSD3 protrudes into the cytosol, based
351
on both proteinase K protection and selective permeabilization experiments (Legeza et al., 2013).
352
Moreover, knockdown of glucose 6-phosphate dehydrogenase (G6PD), the enzyme producing NADPH in
353
the cytosol, significantly inhibited 17β-HSD3 activity, while modulation of hexose-6-phosphate
354
dehydrogenase (H6PD), generating NADPH in the ER lumen, had no effect. Conversely and as expected,
355
knockdown of H6PD decreased 11β-HSD1 oxoreduction activity, while knockdown of G6PD had no
356
effect.
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2.5.2 17β-HSD6, 7, 9 and 12: ER proteins with luminal orientation of their active site
359
A common feature of all 17β-HSDs located at the ER (including 17β-HSD11 and 17β-HSD13) is the
360
presence of a hydrophobic domain, predicted to form transmembrane helices, starting at amino acids 1-10
361
(Fig. 2). The co-factor binding site typically follows immediately downstream of this hydrophobic region.
362
An exception to this rule constitutes 17β-HSD7 (Krazeisen et al., 1999), which only contains a highly
363
hydrophobic region closer to the C-terminus (amino acids 238-258 out of 341). Addition of roGFP2 at the
364
C-terminus of 17β-HSD7 led to low oxidation of the sensor indicative of cytosolic localization, which was
365
supported by selective permeabilization experiments of a C-terminally tagged protein (Tsachaki et al.,
366
2015). Furthermore, an N-terminal FLAG-tag on 17β-HSD7 was protected in selective permeabilization
367
experiments, suggesting luminal orientation. The addition of the epitope at the N-terminus did not change
368
ER localization of the enzyme. Taken together, these results indicate that 17β-HSD7 is an ER membrane
369
protein with its substrate and co-factor binding site oriented toward the ER lumen, although the retention
370
signal that keeps it in the ER remains obscure.
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17β-HSD7 is a reductive enzyme able to convert estrone to estradiol similar to 17β-HSD1 albeit with
372
lower efficiency (Laplante et al., 2009). Its relative in vivo contribution to the estradiol pool is unclear, but
373
elevated expression of the enzyme has been shown in breast cancer tissue (Shehu et al., 2011; Wang et al.,
374
2017). The focus on the function of 17β-HSD7 was shifted away from sex steroid metabolism when it was
375
identified as the homolog of the yeast 3-ketosteroid reductase Erg27p, which participates in cholesterol
376
synthesis (Marijanovic et al., 2003). It was shown that 17β-HSD7 is able to convert zymosterone to
377
zymosterol in vitro, and mice deficient of the murine enzyme die at embryonic day 10.5 (Jokela et al.,
378
2010). Analysis of knockout embryos preceding this stage showed severe developmental abnormalities,
379
which were correlated with defects in the cholesterol synthesis pathway. The part of cholesterol
380
biosynthesis in which 17β-HSD7 acts (namely, the post-squalene phase) takes place at the ER. However,
381
the membrane topology of the enzymes participating in this pathway is insufficiently investigated.
382
Intriguingly, according to the hidden Markov algorithm TMHMM and TOPCONS predictions, lanosterol
383
synthase (LSS), which converts squalene epoxide to lanosterol in this series of reactions, is not predicted
384
to contain any transmembrane domains. This implies that it is a soluble enzyme and supports that at least
385
part of the post-squalene cholesterol synthesis takes place in the ER lumen. Further complications come
386
from the fact that the yeast homologue of lanosterol synthase Erg7p was found to localize mainly in lipid
387
droplets, and to be occasionally distributed between the ER and lipid droplets (Milla et al., 2002; Mullner
388
et al., 2004). Thus, further studies are need to shed more light on this aspect of cholesterol metabolism.
389
Another ER enzyme that has been linked with reductive conversion of estrone to estradiol is 17β-HSD12
390
(Luu-The et al., 2006), although with very low efficiency compared to 17β-HSD1 (Laplante et al., 2009)
391
(own observations). Interestingly, it was shown that 17β-HSD12 is the 3-ketoacyl-coA reductase (KAR) in
392
the four-step cascade leading to elongation of long-chain fatty acids in the ER (Moon et al., 2003).
393
Although only one multifunctional enzyme is required for elongation of fatty acids with less than 16
394
carbon atoms in the cytosol, four different enzymatic entities are responsible for elongating longer fatty
395
acids in the ER (Kihara, 2012). Through this process, vital fatty acids are produced, including arachidonic
396
acid, eicosapentaenoic acid and docosahexaenoic acid. Arachidonic acid is the precursor of a variety of
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biologically active lipids, including prostaglandins, thromboxanes, leukotriens, eicosanoids and lipoxins.
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The importance of 17β-HSD12 function was demonstrated in mice, where knockout led to lethality at
399
embryonic day E9.5, with severely impaired organogenesis (Rantakari et al., 2010). Embryonic stem cells
400
heterozygous for 17β-HSD12 showed decreased arachidonic acid levels, suggesting an important role of
401
this enzyme in the synthesis of arachidonic acid and other biologically active lipids and in neuronal
402
development. Additionally, observations from 17β-HSD12 knockout mice revealed a role in female
403
reproduction with impaired meiosis in the ovaries of heterozygous mice and failure in oogenesis and
404
ovulation (Kemilainen et al., 2016). Importantly, arachidonic acid and several important metabolites such
405
as prostaglandins D2, E2 and F2α as well as thromboxane B2 were decreased in the ovaries, while steroid
406
hormones were not altered. High expression of 17β-HSD12 was correlated with poor prognosis in ovarian
407
and breast cancer (Nagasaki et al., 2009; Song et al., 2006; Szajnik et al., 2012) and knockdown of 17β-
408
HSD12 in breast and ovarian carcinoma cell lines led to reduced cell proliferation. This effect could not be
409
rescued by estradiol, but rather by arachidonic acid supplementation (Nagasaki et al., 2009; Szajnik et al.,
410
2012). Recently, 17β-HSD12 expression has been associated with COX-2 expression in ovarian cancer,
411
further emphasizing its function in arachidonic acid production and cell proliferation (Kemilainen et al.,
412
2018). All these findings underline the important role of 17β-HSD12 in long-chain fatty acid elongation.
413
In an early study, protection of activity after protease treatment of microsomes was used in order to assess
414
the membrane topology of the enzymes involved in rat long-chain fatty acid elongation (Osei et al.,
415
1989). Based on the results, the first condensing enzyme in this cascade as well as the last trans-2-enoyl-
416
CoA reductase of the elongation are facing the cytosolic part of the ER membrane, whereas the 3-
417
hydroxyacyl-CoA dehydratase had an activity embedded in the ER membrane. The yeast 3-hydroxyacyl-
418
CoA dehydratase (Phs1) was later determined to be a protein that transverses the ER membrane six times,
419
and the critical residues for its enzymatic activity were found to be located within its transmembrane
420
domains, which has not been reported for any other hydratase (Kihara et al., 2008). 17β-HSD12 is
421
predicted to possess up to two transmembrane domains, one close to the N- and one upstream of the C-
422
terminus. Fusion of its C-terminus to roGFP2 showed that this part of the protein is cytosolic, whereas
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addition of roGFP2 upstream of its substrate and co-factor binding site, suggested that this part is luminal
424
(Tsachaki et al., 2015). The above results suggest a complicated mechanism for long-chain fatty acid
425
elongation, with enzymatic catalysis taking place on two different sides of the ER membrane, as well as
426
within the membrane.
427
The membrane topology of 17β-HSD9 (Su et al., 1999) has been elaborately investigated using the
428
proteinase K protection assay and deletion constructs (Simon et al., 1999). It was demonstrated that 17β-
429
HSD9 contains a transmembrane domain starting at the beginning of the protein, followed by the main
430
luminal part and a C-terminal transmembrane domain that leaves six amino acids in the cytosolic
431
compartment. Although the native protein does not seem to be glycosylated, introduction of an N-
432
glycosylation site at the proposed luminal region induced glycosylation, further supporting the luminal
433
localization of the main part of the enzyme. A similar topology was demonstrated for 17β-HSD6 using
434
roGFP2 fusions at the C-terminus, revealing cytoplasmic orientation of the very C-terminus and the part
435
following the first transmembrane region, which indicates luminal orientation of the main part of the
436
enzyme (Tsachaki et al., 2015).
437
The two enzymes also share functional similarity. Both enzymes are involved in the metabolism of
438
retinoids, hence their alternative names retinol dehydrogenase (RODH) for 17β-HSD6 and 11-cis retinol
439
dehydrogenase for 17β-HSD9. Their substrate preference differs though, with 17β-HSD6 accepting
440
mostly all-trans retinol and 17β-HSD9 9-cis retinol or 11-cis retinol (Napoli, 2001; Wang et al., 1999).
441
The membrane topology of these enzymes indicates that at least a part of the complex retinoid metabolism
442
takes place in the lumen of the ER and implies the existence of an unknown transport mechanism of
443
retinoids from the cytosol to the ER. Besides their role in retinoid metabolism, both enzymes are able to
444
oxidize estradiol to estrone, as well as androgens at the 3α or 17β position. Both 17β-HSD6 and 17β-
445
HSD9 can catalyze the conversion of 3α-adiol to DHT, although based on their tissue distribution 17β-
446
HSD6 is considered the biological relevant enzyme for the oxidation of 3a-adiol (Bauman et al., 2006;
447
Biswas et al., 1997; Napoli, 2001; Su et al., 1999; Wang et al., 1999). These reactions are considered to be
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of paramount importance for DHT synthesis/inactivation through the alternative or ‘backdoor’ pathway,
449
bypassing testosterone production (Biason-Lauber et al., 2013; Fukami et al., 2013). This mechanism of
450
regulating local DHT levels seems to be central in the pathology of prostate cancer. Given the topology of
451
17β-HSD6 it remains unclear why DHT should be formed within the ER since unliganded AR resides in
452
the cytoplasm. Conversely, the enzymes that reduce androsterone to 3a-adiol (17β-HSD3) and DHT to 3α-
453
adiol (AKR1C2) are active in the cytosolic compartment, which may allow intracellular control of DHT
454
concentrations through subcellular compartmentalization. Further research is needed to understand the
455
combined action of several enzymes participating in the same metabolic pathway and located in different
456
compartments.
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3. Conclusion remarks
The subcellular localization of all 17β-HSD enzymes has now been determined. This effort required in
460
most cases confirmation by both indirect immunofluorescence and biochemical methods, such as
461
subcellular fractionation. Erroneous intracellular distribution has been initially assumed, for example in
462
case of 17β-HSD10, mainly due to misinterpretation of experimental data in the absence of suitable
463
positive and negative controls or appropriate markers for each cell compartment. For instance, it was
464
noted that cytochrome C oxidase rather than cytochrome C should be used as mitochondrial marker, since
465
upon mitochondrial damage cytochrome C is released into the cytosol, thus not representing an ideal
466
mitochondria-specific marker (Yang et al., 2014).
467
Since the 17β-HSDs that localize to the ER and/or lipid droplets are integral membrane proteins, unveiling
468
their membrane topology has been the focus of several studies. Topology determination is a laborious
469
process often requiring mutagenesis, protein tagging or the use of fusion proteins. These manipulations
470
may disturb proper topology, making the application of different methods indispensable. The most
471
commonly used method for membrane topology determination involves the proteinase K protection assay
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using isolated microsomes, which reveals whether a part of the protein is protected from digestion, i.e.
473
luminal. This is a multi-step, indirect method, which can provide reliable results if appropriate controls are
474
employed. Another approach involves the selective permeabilization of the plasma membrane with
475
detergents such as digitonin or the use of pore-forming agents like streptolysin-O, and subsequent
476
immunofluorescence detection. This method requires extensive optimization of experimental conditions,
477
as well as the use of suitable luminal and cytosolic control proteins. Another recently added technique
478
includes the fusion of the protein of interest with roGFP2 and ratiometric calculation of the sensor
479
oxidation status, indicative of luminal (more oxidized) or cytosolic localization (Tsachaki et al., 2015).
480
Ideally, at least two independent methods are applied for membrane topology determination.
481
Subcellular localization and membrane topology dictates the compartment in which an enzyme is active,
482
and may provide insight into its biological function. This is of particularly interest for 17β-HSDs, as many
483
of these enzymes accept several common substrates in vitro that often do not represent genuine in vivo
484
substrates. A typical example is the interconversion of estrone and estradiol, where 17β-HSD1, 7 and 12
485
can perform the reductive and 17β-HSD2, 4, 6, 8, 9 and 10 the oxidative reaction. The relative
486
contribution of these enzymes in estrogen metabolism is far from being fully understood and may depend
487
on specific cellular conditions. However, given the cytoplasmic/nuclear localization of estrogen receptors
488
one might anticipate that mitochondrial or peroxisomal localization of a given 17β-HSD indicates a
489
different major physiological role for these enzymes. Determination of the subcellular localization
490
supports the hypothesis generation process when limited information exists regarding the substrates or
491
function of an enzyme. Therefore, resolving the subcellular localization and membrane topology of the
492
17β-HSD enzymes paved the way for understanding their biological roles and will continue to be
493
important for future functional studies.
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Acknowledgements
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This work was supported by the Swiss National Science Foundation No 31003A-179400. 20
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Figure Legends
501
Figure 1. Schematic representation of the subcellular localization of the 17β-HSD enzymes. 17β-
502
HSD1, 5 and 14 are cytosolic. According to one study, 17β-HSD5 translocates into the nucleus in complex
503
with the androgen receptor. 17β-HSD11 and 17β-HSD13 have been reported to localize to both the lipid
504
droplets and the ER. 17β-HSD2, 17β-HSD3, 17β-HSD6, 17β-HSD7, 17β-HSD9 and 17β-HSD12 localize
505
at the ER. 17β-HSD8 and 10 are present in mitochondria, and 17β-HSD4 in peroxisomes.
506
Figure 2. Membrane topology of 17β-HSD enzymes of the ER and lipid droplets. For every protein
507
we illustrate the GXXXGXG motif (which is part of the co-factor binding site) and the active site, along
508
with the amino acid number at which these consensus sequences start. At the C-terminus the total amino
509
acid number is shown. 17β-HSD2, 17β-HSD3, 17β-HSD11 and 17β-HSD13 exhibit cytosolic, and 17β-
510
HSD6, 17β-HSD7, 17β-HSD9 and 17β-HSD12 luminal localization of the GXXXGXG motif and active
511
site.
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Highlights •
The 17β-hydroxysteroid dehydrogenases (17β-HSDs) consists of enzymes with diverse functions, spanning from sex steroid and retinoid metabolism to fatty acid synthesis/oxidation. Investigations of subcellular localization have shown that different 17β-HSDs are present in the
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cytosol, endoplasmic reticulum, lipid droplets, peroxisomes or mitochondria. The 17β-HSDs that reside in the endoplasmic reticulum and lipid droplets are integral membrane proteins and their
Subcellular localization and membrane topology of the 17β-HSD enzymes is tightly correlated
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topology has now been resolved.
S0303-7207(18)30217-X
DOI:
10.1016/j.mce.2018.07.003
Reference:
MCE 10270
To appear in:
Molecular and Cellular Endocrinology
Received Date: 22 August 2017 Revised Date:
18 June 2018
Accepted Date: 3 July 2018
Please cite this article as: Tsachaki, M., Odermatt, A., Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases, Molecular and Cellular Endocrinology (2018), doi: 10.1016/ j.mce.2018.07.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Subcellular localization and membrane topology of 17β-hydroxysteroid dehydrogenases
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Maria Tsachakia and Alex Odermatta*
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of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland
Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University
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* Corresponding author: Prof. Alex Odermatt, Division of Molecular and Systems Toxicology,
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Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel,
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Switzerland. [email protected]
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Abstract
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The 17β-hydroxysteroid dehydrogenases (17β-HSDs) comprise enzymes initially identified by their ability
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to interconvert active and inactive forms of sex steroids, a vital process for the tissue-specific control of
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estrogen and androgen balance. However, most 17β-HSDs have now been shown to accept substrates
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other than sex steroids, including bile acids, retinoids and fatty acids, thereby playing unanticipated roles
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in cell physiology. This functional divergence is often reflected by their different subcellular localization,
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with 17β-HSDs found in the cytosol, peroxisome, mitochondria, endoplasmic reticulum and in lipid
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droplets. Moreover, a subset of 17β-HSDs are integral membrane proteins, with their specific topology
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dictating the cellular compartment in which they exert their enzymatic activity. Here, we summarize the
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present knowledge on the subcellular localization and membrane topology of the 17β-HSD enzymes and
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discuss the correlation with their biological functions.
34 Keywords:
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metabolism; steroid
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subcellular
localization;
membrane
topology;
17β-hydroxysteroid
dehydrogenases;
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Abbreviations: 3α-adiol, 5α-androstan-3α,17β-diol; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type
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1; 17β-HSD, 17β-hydroxysteroid dehydrogenase; A4, ∆4-androsten-3,17-dione; Aβ, amyloid peptide β;
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ACS3, acyl-CoA synthase 3; ADRP, adipose differentiation-related protein; AKR, aldo-keto reductase;
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AR, androgen receptor; CBR4, carbonyl reductase type 4; DHEA, dehydroepiandrosterone, DHT, 5α-
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dihydrotestosterone; ER, endoplasmic reticulum; ERAB, ER-associated Aβ peptide binding protein; FAS,
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fatty acid synthesis; G6PD, glucose 6-phosphate dehydrogenase; KAR, 3-ketoacyl-acyl carrier protein
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(ACP) reductase; LSS, lanosterol synthase; NAD, nicotinamide adenine dinucleotide; PAT, perlipin,
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ADRP and tail-interacting protein; PPAR, peroxisome proliferator-activated receptor; PSA, prostate-
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specific antigen; Ribonuclease P, RNase P; RODH, retinol dehydrogenase; roGFP, reduction-oxidation
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sensitive green fluorescent protein; SDR, short-chain dehydrogenase/reductase.
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1. Introduction The 17β-hydroxysteroid dehydrogenases (17β-HSDs) form an enzyme subfamily of the superfamily of
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short-chain dehydrogenases-reductases (SDRs), with the exception of 17β-HSD5 that belongs to the aldo-
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keto reductase (AKR) family (Moeller et al., 2009). At least 14 different 17β-HSDs have been described
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and they were named after their capacity to interconvert 17-oxo and 17-hydroxy sex steroids. They are
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numbered according to the chronological order of their discovery and the first three members (17β-HSD1,
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2 and 3) were recognized for their ability to interconvert the weaker 17-ketosteroid and the more potent
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17β-hydroxy forms of estrogens and/or androgens (Gast et al., 1989; Geissler et al., 1994; Wu et al.,
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1993). Although these enzymes act bi-directionally in vitro, they predominantly function as either
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oxoreductases or dehydrogenases in intact cells as well as in vivo (Khan et al., 2004), depending on the
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availability of a co-factor, which is preferably either nicotinamide adenine dinucleotide NADP(H) or
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NAD(H). Because of the intracellular abundance of these reduced and oxidized co-factors, typically,
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reductive 17β-HSDs use NADPH and oxidative enzymes NAD+ as co-factor (Hedeskov et al., 1987). The
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co-factor binding site is formed by a conserved structural motif called Rossmann fold (Buehner et al.,
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1973), encompassing the conserved sequence GxGxxxG. The Rossmann fold is a common co-factor
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binding element of SDRs. Notably, the AKR 17β-HSD5 does not contain a GxGxxxG motif in its
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sequence.
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That 17β-HSD enzymes are not exclusively dedicated to sex steroid metabolism was realized after the
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discovery of 17β-HSD4, which was shown to participate in peroxisomal β-oxidation of fatty acids
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(Breitling et al., 2001; Leenders et al., 1996; Markus et al., 1996; Seedorf et al., 1995). Since then, the
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notion of a multifunctional 17β-HSD family was gradually established and experimentally supported. The
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diversity in substrate specificity and in vivo function is also mirrored in the subcellular localization of the
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different 17β-HSD enzymes. However, the presence of an enzyme in a specific organelle cannot always be
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deduced through the knowledge of its substrate. Although signal peptides and targeting sequences are
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present in some 17β-HSDs, definitive proof of the presence in a cell compartment requires the
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employment of biochemical methods (such as subcellular fractionation and immunoblotting) or indirect
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immunofluorescence. A critical issue in these studies is the utilization in each method of proper markers,
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in order to undoubtedly designate a certain protein to a cell organelle or the cytosol.
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Since all organelles are encircled by lipid mono- or bilayers, integral membrane proteins can either
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function in the lumen of an organelle or at its cytosolic side. Therefore, the topology (the orientation of
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different parts of a protein in relation to the membrane) is a fundamental feature of membrane proteins.
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All 17β-HSDs residing in the endoplasmic reticulum (ER) or lipid droplets are membrane-bound (17β-
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HSD2, 3, 6, 7, 11, 12 and 13). Different methods have been employed for topology determination of
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membrane-anchored 17β-HSDs, including selective permeabilization of cell membranes with digitonin
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followed by immunofluorescence detection, proteinase K protection assay followed by western blotting
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and, recently, live-cell measurements using reduction-oxidation sensitive green-fluorescent protein
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(roGFP) fusions with the respective 17β-HSD enzyme (Tsachaki et al., 2015).
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For subcellular localization and membrane topology determination, peptide tags, fusion or truncated
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proteins are frequently used. This raises the question of how such interventions could affect intracellular
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targeting. Given the complex nature of the above studies, erroneous initial allocation in a cell organelle or
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membrane topology have been reported, which has led to misconceptions regarding the physiological role
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of certain 17β-HSDs. In this review, we elaborate on current understanding of 17β-HSD subcellular
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localization and membrane topology (summarized in Fig. 1 and 2, respectively), based on previous
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investigations that include both in silico studies and experimental evidence. In addition, we elaborate on
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how this aspect of 17β-HSD protein physiology is potentially linked to their enzymatic function and the
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metabolic pathways in which they are involved.
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2. Subcellular localization of 17β-HSDs 2.1 Cytosolic 17β-HSD1, 5 and 14 4
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17β-HSD1 was the first enzyme identified in this family and its main function is to reduce the weakly
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active 17-keto estrogen estrone to the potent 17β-hydroxy estrogen estradiol and thereby stimulating the
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activity of estrogen receptors, a process particularly important in tissues such as the placenta, the breast
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and the ovaries (Luu The et al., 1989). An elevated expression of 17β-HSD1 has been associated with sex-
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specific malignancies, including breast and endometrial carcinomas (Gunnarsson et al., 2001; Oduwole et
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al., 2004), and inhibitors of this enzyme are considered for potential therapeutic applications to treat
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hormone-dependent cancer (Brozic et al., 2008; Cornel et al., 2012; Hilborn et al., 2017; Zhang et al.,
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2012). Although other 17β-HSDs (mainly 17β-HSD7 and 17β-HSD12) have been reported to be able to
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reduce estrone to estradiol, 17β-HSD1 clearly has the highest affinity, and combined with its expression
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pattern it is considered to be the main enzyme exerting this function (Laplante et al., 2009). It was
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suggested that 17β-HSD1 dwells in the cytosol, since this enzyme has no predicted membrane helices.
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Subsequently, immunofluorescence experiments using a specific antibody against human 17β-HSD1
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confirmed the cytosolic distribution (Tsachaki et al., 2015).
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The inactivation of estradiol to estrone can be performed by several 17β-HSDs (17β-HSD2, 4, 6, 8, 9, 10
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and 14), although the relative in vivo contribution of each of these enzymes to estradiol inactivation
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remains unclear. Among these enzymes, 17β-HSD14 is the only one residing in the cytosol, as shown by
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co-localization with the cytoplasmic protein phalloidin in immunofluorescence labelling experiments
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(Lukacik et al., 2007). In the same study, no co-localization with nuclei or mitochondria could be
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observed. 17β-HSD14 likely plays an important role in the local inactivation of estradiol in tissues where
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it is highly expressed, such as the breast, placenta and brain (Jansson et al., 2006; Lukacik et al., 2007;
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Sivik et al., 2012). Importantly, low expression of 17β-HSD14 was correlated with better prognosis, and
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its expression was shown to be predictive of response to tamoxifen treatment in estrogen-receptor positive
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breast cancer patients (Jansson et al., 2006; Sivik et al., 2012). In this case, the presence of 17β-HSD1 and
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17β-HSD14 in the cytosol could provide an efficient system for the intracellular regulation and
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maintenance of high estradiol concentrations. 17β-HSD14 is also able to oxidize other substrates, at least
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in vitro, including 5-androstene-3β,17β-diol and testosterone (Lukacik et al., 2007). Resolving the crystal
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structure of 17β-HSD14 revealed that the substrate binding pocket is wide enough to accommodate
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substrates other than steroids, raising the possibility that new roles for 17β-HSD14 will be uncovered in
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the future (Bertoletti et al., 2016; Lukacik et al., 2007).
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As mentioned above, all 17β-HSDs belong to the SDR superfamily with the exception of 17β-HSD5 (also
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known as AKR1C3) that is a member of the AKR superfamily (Penning, 2015). AKRs are dedicated in the
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reduction of carbonyl-containing substrates, including sugar aldehydes, keto-prostaglandins and keto-
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steroids. 17β-HSD5 catalyzes the reduction of the prostaglandins PGH2 and PGD2 to PGF2a and 9α,11β-
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PGF2, respectively, both products shown to promote cancer cell proliferation (Matsuura et al., 1998;
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Suzuki-Yamamoto et al., 1999). Importantly, 17β-HSD5 participates in metabolic cascades through which
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the most potent human androgen 5α-dihydrotestosterone (DHT) is synthesized, and has been considered as
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a potential target against prostate cancer (Adeniji et al., 2013). Upon binding of DHT to its cognate
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androgen receptor (AR) in the cytosol, it translocates into the nucleus where it regulates the expression of
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a plethora of target genes. Immunofluorescence experiments showed that in the absence of ligand
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activation 17β-HSD5 co-localizes with the AR in the cytosol, whereas treatment with the 17β-HSD5
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substrate ∆4-androsten-3,17-dione (A4) or the AR agonist R1881 led to translocation of both AR and 17β-
139
HSD5 to the nucleus (Yepuru et al., 2013). Intriguingly, chromatin immunoprecipitation experiments
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demonstrated the binding of 17β-HSD5 to the promoter of the AR-target gene prostate specific antigen
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(PSA), when it was suggested to act as a coactivator of the AR. Nuclear localization has not been shown
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for any other 17β-HSD enzyme, and the specific function of a reductase binding to a gene regulatory
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region remains enigmatic.
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2.2 Peroxisomal 17β-HSD4
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The only enzyme of the 17β-HSDs localized in the peroxisomes is 17β-HSD4 (Adamski et al., 1995).
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Evidence for peroxisomal localization of 17β-HSD4 was provided by immunogold electron microscopy,
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where 17β-HSD4 co-localized with the peroxisomal-resident enzymes catalase and acyl-CoA oxidase
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(Markus et al., 1995). Later, the subcellular localization of 17β-HSD4 was also deduced from the presence
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at its C-terminus of the peroxisomal targeting tripeptide Ala-Lys-Leu (AKL) (Moller et al., 1999), which
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represents an alternate version of the classical Ser-Lys-Leu (SKL) peroxisomal targeting signal. In the
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porcine enzyme this sequence is varied as Ala-Lys-Ile, and its deletion from the protein causes cytosolic
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mislocalization (Moller et al., 1999).
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Although the N-terminal part of the protein is reminiscent of other SDRs, the amino acid sequence of 17β-
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HSD4 points towards a multi-functional enzyme. The diverse roles of 17β-HSD4 in peroxisomes have
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been well-documented and are attributed to distinct protein domains (Breitling et al., 2001). A sterol and
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phospholipid transfer domain present at the C-terminus of 17β-HSD4 is related to sterol carrier protein 2
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(SCP2), and such activity has been clearly demonstrated for the enzyme (Leenders et al., 1996; Markus et
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al., 1996; Seedorf et al., 1995). Additionally, based on sequence similarity to the yeast FOX2 proteins,
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17β-HSD4 was suggested to participate in peroxisomal β-oxidation of fatty acids and bile acids (Leenders
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et al., 1996). Indeed, acyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities of 17β-HSD4 are
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supported by strong experimental evidence (Caira et al., 1998; Leenders et al., 1996; Markus et al., 1996;
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Qin et al., 1997). Knockout of 17β-HSD4 in mice showed severe impairments in β-oxidation of 2-methyl-
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branched fatty acids, the bile acid intermediates di- and trihydroxycoprostanic acid, and very long-chain
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fatty acids (Baes et al., 2000). The importance of 17β-HSD4 for peroxisomal function is further
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underlined by reports for mutations of the HSD17B4 gene, leading to conditions reminiscent of the
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Zellweger syndrome (Suzuki et al., 1997; van Grunsven et al., 1998; van Grunsven et al., 1999).
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Despite the above functions attributed to 17β-HSD4, it has also been considered important for estrogen
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metabolism, since it was first described as an estradiol-inactivating enzyme in porcine uterus (Adamski et
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al., 1992). Luteneization of equine ovarian follicles, a process accompanied by a decrease in estradiol and
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increase in progesterone levels, was linked to increase in the expression of 17β-HSD4 (Brown et al.,
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2004). Additionally, single nucleotide polymorphisms in the HSD17B4 gene were well-correlated with
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endometrial cancer risk (Karageorgi et al., 2011). According to its amino acid sequence, 17β-HSD4 is
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unlikely to contain any transmembrane helices and is thus expected to be a soluble protein within the
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peroxisomal matrix. Since estrogen metabolism in this compartment has not been demonstrated, and the
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presence of 17β-HSD4 in the cytosol has never been shown, the actual involvement of 17β-HSD4 in
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estrogen metabolism remains obscure.
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2.3 Mitochondrial 17β-HSD8 and 17β-HSD10
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Fatty acid synthesis (FAS) in mitochondria is responsible for producing a number of long-chain fatty
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acids, including the respiratory complex I component 3-hydroxymyristoyl-ACP and lipoic acid (Hiltunen
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et al., 2010). The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) has been recently
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discovered to be 17β-HSD8. The presence of 17β-HSD8 in mitochondria was demonstrated by co-
184
localization with the MitoTracker Red dye in immunofluorescence labeling experiments (Chen et al.,
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2009). The primary sequence of 17β-HSD8 indicates that it is a soluble enzyme and should thus reside in
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the mitochondrial matrix. Moreover, it has been shown to form a heteroterameric complex with carbonyl
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reductase type 4 (HsCBR4), where it acts as a scaffold to the functional subunit CBR4 (Venkatesan et al.,
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2014). Based on these findings and the fact that it likely acts as an oxidase in vivo, it was suggested that
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17β-HSD8 could function in channeling 3R-hydroxyacyl-CoA intermediates to β-oxidation in
190
mitochondria. Three-dimensional models of 17β-HSD8 in complex with NAD+ showed that it accepts
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substrates similar to those of the β-ketoacyl-ACP reductases of Brassica napus, further supporting a role
192
in fatty acid metabolism (Pletnev et al., 2005).
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The gene encoding 17β-HSD10 is located on the X chromosome and several mutations of the enzyme are
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linked with mental retardation (Yang et al., 2011). The function of this enzyme remains to be further
195
investigated. Elevated 17β-HSD10 levels have been reported in Alzheimer’s patients (He et al., 2002). It
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has been shown to bind the amyloid β (Aβ) peptide, which accumulates in the brain of Alzheimer’s
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patients and is thought to play a major role in the disease progression (Kissinger et al., 2004). 17β-HSD10
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is also important for isoleucine metabolism and oxidative inactivation of the potent neurosteroid
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allopregnanolone (He et al., 2005; Ofman et al., 2003). Besides, 17β-HSD10 was shown to catalyze the
200
conversion of the inactive androgen 5α-androstan-3α,17β-diol (3α-adiol) to the potent androgen DHT,
201
with a potential role in the backdoor pathway of androgen generation that is thought to promote prostate
202
cancer (Auchus, 2004; Mohler, 2014; Penning, 2010).
203
Initially, 17β-HSD10 was reported to reside in the ER in HeLa cells (giving rise to the suggested name
204
ER-associated Aβ peptide binding protein-ERAB), and addition of Aβ peptide led to its re-distribution to
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the plasma membrane in the human neuroblastoma cell line SK-N-SH (Yan et al., 1997), an observation
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that was supported by confocal microscopy and subcellular fractionation studies. However, in the
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immunofluorescence experiments presented in this work no plasma membrane marker was used to
208
confirm such localization. Additionally, no compartment-specific marker was demonstrated to validate
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proper separation of all cell fractions. In a follow-up work by the same group, this localization could be
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reproduced, however the additional presence of 17β-HSD10 in the mitochondria of the same cells was
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shown (Yan et al., 1999). Although it cannot be excluded that 17β-HSD10 also localizes in the ER or at
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mitochondria/ER contact sites in the cell lines tested above, all subsequent studies using specific markers
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for both immunofluorescence labeling and subcellular fractionation experiments have reported exclusively
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mitochondrial localization (He et al., 2001; He et al., 1999; He et al., 2000). Interestingly, an early study
215
identified bovine 17β-HSD10 as the 3-hydroxyacyl-CoA dehydrogenase of the mitochondrial fatty acid β-
216
oxidation (Furuta et al., 1997). It was also demonstrated that its protein sequence contains a 16-amino acid
217
N-terminal non-cleaved mitochondrial signal peptide, similar to that of human and rat thiolases.
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Nevertheless, fusion of the 15 first amino acids of human 17β-HSD10 to GFP failed to result in
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mitochondrial localization of the fusion protein, whereas fusion of the first 34 amino acids was sufficient
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(Shafqat et al., 2003). This implies that additional elements following this signal peptide are necessary for
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mitochondrial targeting.
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An important role of 17β-HSD10, directly linked to its subcellular localization, is in mitochondrial tRNA
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processing, as an essential component of Ribonuclease P (RNase P) (Holzmann et al., 2008; Vilardo et al.,
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2012). The role of 17β-HSD10 in mitochondrial tRNA processing is believed to underlie the etiology of
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the HSD10 disease, which is characterized by severe neurological defects and cardiomyopathy
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(Deutschmann et al., 2014; Falk et al., 2016; Vilardo et al., 2015). The plethora of substrates and functions
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attributed to 17β-HSD10 points towards a multifunctional enzyme (Yang et al., 2014; Yang et al., 2005),
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and the precise mechanisms by which its malfunction or absence leads to disease remain to be more
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clearly determined.
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2.4 Lipid droplet-associated 17β-HSD11 and 17β-HSD13
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Lipid droplets are cytoplasmic organelles dedicated to lipid storage and are regarded to be formed by
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budding off from ER membranes (Pol et al., 2014). They contain a lipid monolayer and they are mostly
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found in adipocytes, although they are also present in metabolically active tissues, such as the liver and the
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muscle. 17β-HSD11 was the first enzyme in the 17β-HSD family shown to be located in lipid droplets. In
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the first study describing its subcellular localization, N-terminally myc-tagged 17β-HSD11 transiently
237
transfected into the mouse adrenal Y1 cell line was regarded to be in the cytoplasm based on
238
immunofluorescence labeling (Chai et al., 2003). However, no organelle or cytoplasmic markers were
239
employed in this study, and the microscopic image was likely misinterpreted. Also, the N-terminal tag
240
might have affected the localization of the protein due to its close proximity to the membrane spanning
241
helix. Later, it was shown that 17β-HSD11 is one of the most abundant proteins in the lipid droplet
242
fraction of the human hepatoma cell line HuH7, together with Adipose differentiation-related protein
243
(ADRP) and acyl-CoA synthase 3 (ACS3). This was demonstrated by mass spectrometric analysis and
244
confirmed by immunoblotting using a specific antibody (Fujimoto et al., 2004). When cells were treated
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with oleic acid, which promotes lipid droplet formation, the lipid droplet localization became more
246
evident. In this study, 17β-HSD11 exhibited an exclusive lipid droplet localization; it could not be
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detected by immunoblotting in the ER cellular protein fraction nor did it show any ER labeling in
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fluorescence labeling experiments. Interestingly, 17β-HSD11 expression was found to be upregulated in
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the mouse intestine upon activation of peroxisome proliferator-activated receptor alpha (PPARα), which
250
has major roles in lipid metabolism (Motojima, 2004). In subcellular fractionation studies 17β-HSD11 was
251
present in both the ER and the lipid droplet fraction when mice were fed normal diet, and specifically
252
localized in the lipid droplet fraction when they were treated with a PPARα agonist (Yokoi et al., 2007).
253
Moreover, in the Chinese hamster ovary cell line 17β-HSD11 was found to localize in the ER under
254
normal conditions and to be transferred from the ER to the lipid droplets after oleic acid treatment.
255
The N-terminal hydrophobic part of mouse 17β-HSD11 (aminoacids 4-16) seems to be important for
256
correct intracellular targeting, since deletion of this region leads to mitochondrial localization (Horiguchi
257
et al., 2008). Based on in silico predictions this should be part of a transmembrane region, which also
258
contains the lipid droplet-targeting motif of the PAT family (perilipin, ADRP and tail-interacting protein).
259
Deletion of this motif or of two of its conserved amino acids causes cytosolic mislocalization of mouse
260
17β-HSD11 (Horiguchi et al., 2008). Analysis of the membrane topology of 17β-HSD11 suggested that it
261
contains a membrane helix close to the N-terminus, followed by the largest part of the protein, containing
262
the Rossmann fold and active sites, which faces the cytosol (Tsachaki et al., 2015).
263
Although 17β-HSD11 has been shown to be able to convert several substrates, including estradiol and 3α-
264
adiol (Brereton et al., 2001; Li et al., 1998; Lundova et al., 2016), the in vivo enzymatic activity is far from
265
being resolved and has surely not been correlated with its subcellular localization. Even less is known
266
about the other lipid droplet-associated enzyme 17β-HSD13 (Liu et al., 2007), which has a surprisingly
267
similar primary amino acid sequence to 17β-HSD11 (65% identical amino acids). The N-terminus of 17β-
268
HSD13, which is also highly hydrophobic and contains a motif similar to the PAT of 17β-HSD11, has
269
been shown to be sufficient for correct targeting of the protein in lipid droplets (Horiguchi et al., 2008).
270
Topology studies showed similar results than those obtained for 17β-HSD11, suggesting that the enzyme
271
transverses the membrane once and its active site protrudes into the cytosol (Tsachaki et al., 2015). The
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recent DiscovEHR human genetics study revealed an association of a 17β-HSD13 splice variant
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(rs72613567:TA) resulting in an unstable truncated protein with a reduced risk of nonalcoholic and
274
alcoholic liver disease (Abul-Husn et al., 2018). This splice variant was associated with a lower risk of
275
nonalcoholic steatohepatitis, but not with simple steatosis, suggesting that this variant inhibits disease
276
progression. Wild-type 17β-HSD13 and the truncated variant were found to be expressed on the surface of
277
the lipid droplet membrane. This study also provided evidence for several bioactive lipids, including
278
leukotriene B3 and B4, as well as eicosanoids, as potential physiologically relevant substrates.
279
Interestingly, ablation of 17β-HSD13 in mice led to hepatic steatosis and inflammation, with evidence for
280
impaired mitochondrial β-oxidation, without alterations in circulating steroid concentrations or
281
reproductive performance (Adam et al., 2018). The above observations should stimulate further research
282
to uncover the physiologically most relevant substrates of 17β-HSD11 and 17β-HSD13, as well as the role
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of these two enzymes in lipid metabolism, control of lipid storage and energy homeostasis.
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2.5 17β-HSD2, 3, 6, 7, 9 and 12 at the ER membrane
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Besides 17β-HSD11 and 17β-HSD13, both located at the ER and in lipid droplets, six other 17β-HSDs
287
have been shown to localize at the ER. All are transmembrane proteins, with their substrate and co-factor
288
binding site facing either the ER lumen or the cytosol. Until recently, attempts to resolve the membrane
289
topology of these enzymes involved mainly proteinase K digestion of isolated microsomes followed by
290
immunoblotting to determine whether the exposed protein part is protected (indicating luminal
291
orientation) or not (supporting cytosolic orientation). This biochemical method relies on the proper
292
microsomal preparation, yielding >90% inside-out microsomes (ER luminal part protected, cytoplasmic
293
side facing the solution). The activity and purity of the proteinase K batch needs to be carefully evaluated.
294
Proteinase K is still active in the presence of detergent and at 4°C, thus proper inactivation to terminate the
295
reaction is essential. Another method is the selective permeabilization of plasma membranes using
296
detergents such as digitonin, followed by indirect immunofluorescence to determine whether a specific
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epitope of the protein of interest faces the cytosolic or luminal side of the ER. Similarly, the plasma
298
membrane-specific pore forming agent streptolysin-O allows selective access to the cytosolic
299
compartment. Importantly, suitable positive and negative controls must be included in the above
300
mentioned methods.
301
Recently, we employed a method for membrane topology determination of SDRs in living cells, based on
302
fusing part or the SDR sequence with the redox-sensitive roGFP2 (Tsachaki et al., 2015). Depending on
303
the oxidation conditions of the compartment, the emission intensity of roGFP2 when excited at two
304
different wavelengths (405 nm and 480 nm) changes. The intensity ratio at the two different wavelengths
305
allows calculating the degree of roGFP2 oxidation. A high oxidation rate of roGFP2 indicates ER luminal
306
localization and a low oxidation rate a cytoplasmic one. By fusing roGFP2 either to the very C-terminus
307
of the different 17β-HSD enzymes or immediately upstream of the co-factor binding and active sites, we
308
could solve the topology of 17β-HSD2, 17β-HSD6, 17β-HSD7 and 17β-HSD12, along with the ER/lipid
309
droplet-localized 17β-HSD11 and 17β-HSD13. These results were corroborated with selective
310
permeabilization of cell membranes using digitonin.
311
For the above methods, fusion proteins of the respective 17β-HSD enzyme with GFP or other epitope tags
312
(for instance myc or flag) at either the C- or N-terminus can be used. Given that many proteins contain at
313
the N-terminus signal sequences for proper targeting, modifications at the C-terminus are usually
314
preferred. Proper localization of the fusion protein at the expected cellular compartment has to be
315
confirmed to avoid artifacts. Unfortunately, even with careful experimental design and validation,
316
structural changes introduced by protein modifications that could inverse proper membrane insertion can
317
never be fully excluded. Below, we revise our present understanding on the membrane topology of ER-
318
resident 17β-HSDs.
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17β-HSD2 is responsible for oxidizing the active 17β-hydroxy forms of many sex steroids to their inactive
322
or weakly active 11-keto forms, exerting a protective function in peripheral tissues to avoid excessive
323
action of these steroids (Moghrabi et al., 1997). Importantly, it exhibits opposite effects to 17β-HSD1 by
324
inactivating estradiol to estrone (Miettinen et al., 1996), and a decreased expression has been associated
325
with breast cancer (Gunnarsson et al., 2001). Additionally, it has a role in androgen inactivation by
326
mediating the conversion of testosterone to A4, androstenediol to dehydroepiandrosterone (DHEA), DHT
327
to androstanedione and 3α-adiol to androsterone (Elo et al., 1996; Wu et al., 1993), and decreased
328
expression has been associated with colon cancer (Oduwole et al., 2003).
329
When first cloned, a putative C-terminal ER retention signal was identified and led to the assumption that
330
the part of the protein containing the active site is luminal (Wu et al., 1993). A subsequent study
331
conducting immunofluorescence experiments demonstrated that 17β-HSD2 co-localized at the ER
332
membrane with the ER-luminal protein BiP, and the presence of 17β-HSD2 in the ER lumen was
333
suggested (Puranen et al., 1999). However, given the size of the ER lipid bilayer, the diffraction limit of
334
the microscope does not allow from double immunofluorescence labeling experiments to distinguish
335
between proteins facing the ER lumen from proteins facing the cytoplasm. To solve this issue, we fused
336
roGFP2 to the C-terminus of 17β-HSD2, and also immediately downstream of the two putative N-terminal
337
transmembrane helices. These experiments revealed a cytosolic localization in both cases based on
338
roGFP2 oxidation. This was additionally supported by selective permeabilization experiments using
339
digitonin (Tsachaki et al., 2015). Taken together, these results strongly support a topology of 17β-HSD2
340
where the substrate and co-factor binding sites are oriented toward the cytosol.
341
Another microsomal enzyme initially suggested to have a luminal orientation is 17β-HSD3, whose
342
principal function is the reduction of A4 to testosterone in the testicular Leydig cells (Geissler et al.,
343
1994). Hu et al. hypothesized a coupling of 17β-HSD3 and 11β-hydroxysteroid dehydrogenase type 1
344
(11β-HSD1) activities, where 17β-HSD3 would utilize ER luminal NADPH to generate testosterone (Hu
345
et al., 2008). The formed NADP+ would then serve as co-factor for 11β-HSD1 to catalyze the inactivation
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of the potent glucocorticoid cortisol to cortisone under stress conditions. However, although an
347
interrelationship between the activity of 11β-HSD1 as an oxoreductase or dehydrogenase and the A4
348
conversion to testosterone was proposed (Latif et al., 2011), a direct functional link between the two
349
enzymes and competition for NADPH co-factor in the ER lumen was not unequivocally shown.
350
Importantly, it was later demonstrated that the active site of 17β-HSD3 protrudes into the cytosol, based
351
on both proteinase K protection and selective permeabilization experiments (Legeza et al., 2013).
352
Moreover, knockdown of glucose 6-phosphate dehydrogenase (G6PD), the enzyme producing NADPH in
353
the cytosol, significantly inhibited 17β-HSD3 activity, while modulation of hexose-6-phosphate
354
dehydrogenase (H6PD), generating NADPH in the ER lumen, had no effect. Conversely and as expected,
355
knockdown of H6PD decreased 11β-HSD1 oxoreduction activity, while knockdown of G6PD had no
356
effect.
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2.5.2 17β-HSD6, 7, 9 and 12: ER proteins with luminal orientation of their active site
359
A common feature of all 17β-HSDs located at the ER (including 17β-HSD11 and 17β-HSD13) is the
360
presence of a hydrophobic domain, predicted to form transmembrane helices, starting at amino acids 1-10
361
(Fig. 2). The co-factor binding site typically follows immediately downstream of this hydrophobic region.
362
An exception to this rule constitutes 17β-HSD7 (Krazeisen et al., 1999), which only contains a highly
363
hydrophobic region closer to the C-terminus (amino acids 238-258 out of 341). Addition of roGFP2 at the
364
C-terminus of 17β-HSD7 led to low oxidation of the sensor indicative of cytosolic localization, which was
365
supported by selective permeabilization experiments of a C-terminally tagged protein (Tsachaki et al.,
366
2015). Furthermore, an N-terminal FLAG-tag on 17β-HSD7 was protected in selective permeabilization
367
experiments, suggesting luminal orientation. The addition of the epitope at the N-terminus did not change
368
ER localization of the enzyme. Taken together, these results indicate that 17β-HSD7 is an ER membrane
369
protein with its substrate and co-factor binding site oriented toward the ER lumen, although the retention
370
signal that keeps it in the ER remains obscure.
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17β-HSD7 is a reductive enzyme able to convert estrone to estradiol similar to 17β-HSD1 albeit with
372
lower efficiency (Laplante et al., 2009). Its relative in vivo contribution to the estradiol pool is unclear, but
373
elevated expression of the enzyme has been shown in breast cancer tissue (Shehu et al., 2011; Wang et al.,
374
2017). The focus on the function of 17β-HSD7 was shifted away from sex steroid metabolism when it was
375
identified as the homolog of the yeast 3-ketosteroid reductase Erg27p, which participates in cholesterol
376
synthesis (Marijanovic et al., 2003). It was shown that 17β-HSD7 is able to convert zymosterone to
377
zymosterol in vitro, and mice deficient of the murine enzyme die at embryonic day 10.5 (Jokela et al.,
378
2010). Analysis of knockout embryos preceding this stage showed severe developmental abnormalities,
379
which were correlated with defects in the cholesterol synthesis pathway. The part of cholesterol
380
biosynthesis in which 17β-HSD7 acts (namely, the post-squalene phase) takes place at the ER. However,
381
the membrane topology of the enzymes participating in this pathway is insufficiently investigated.
382
Intriguingly, according to the hidden Markov algorithm TMHMM and TOPCONS predictions, lanosterol
383
synthase (LSS), which converts squalene epoxide to lanosterol in this series of reactions, is not predicted
384
to contain any transmembrane domains. This implies that it is a soluble enzyme and supports that at least
385
part of the post-squalene cholesterol synthesis takes place in the ER lumen. Further complications come
386
from the fact that the yeast homologue of lanosterol synthase Erg7p was found to localize mainly in lipid
387
droplets, and to be occasionally distributed between the ER and lipid droplets (Milla et al., 2002; Mullner
388
et al., 2004). Thus, further studies are need to shed more light on this aspect of cholesterol metabolism.
389
Another ER enzyme that has been linked with reductive conversion of estrone to estradiol is 17β-HSD12
390
(Luu-The et al., 2006), although with very low efficiency compared to 17β-HSD1 (Laplante et al., 2009)
391
(own observations). Interestingly, it was shown that 17β-HSD12 is the 3-ketoacyl-coA reductase (KAR) in
392
the four-step cascade leading to elongation of long-chain fatty acids in the ER (Moon et al., 2003).
393
Although only one multifunctional enzyme is required for elongation of fatty acids with less than 16
394
carbon atoms in the cytosol, four different enzymatic entities are responsible for elongating longer fatty
395
acids in the ER (Kihara, 2012). Through this process, vital fatty acids are produced, including arachidonic
396
acid, eicosapentaenoic acid and docosahexaenoic acid. Arachidonic acid is the precursor of a variety of
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biologically active lipids, including prostaglandins, thromboxanes, leukotriens, eicosanoids and lipoxins.
398
The importance of 17β-HSD12 function was demonstrated in mice, where knockout led to lethality at
399
embryonic day E9.5, with severely impaired organogenesis (Rantakari et al., 2010). Embryonic stem cells
400
heterozygous for 17β-HSD12 showed decreased arachidonic acid levels, suggesting an important role of
401
this enzyme in the synthesis of arachidonic acid and other biologically active lipids and in neuronal
402
development. Additionally, observations from 17β-HSD12 knockout mice revealed a role in female
403
reproduction with impaired meiosis in the ovaries of heterozygous mice and failure in oogenesis and
404
ovulation (Kemilainen et al., 2016). Importantly, arachidonic acid and several important metabolites such
405
as prostaglandins D2, E2 and F2α as well as thromboxane B2 were decreased in the ovaries, while steroid
406
hormones were not altered. High expression of 17β-HSD12 was correlated with poor prognosis in ovarian
407
and breast cancer (Nagasaki et al., 2009; Song et al., 2006; Szajnik et al., 2012) and knockdown of 17β-
408
HSD12 in breast and ovarian carcinoma cell lines led to reduced cell proliferation. This effect could not be
409
rescued by estradiol, but rather by arachidonic acid supplementation (Nagasaki et al., 2009; Szajnik et al.,
410
2012). Recently, 17β-HSD12 expression has been associated with COX-2 expression in ovarian cancer,
411
further emphasizing its function in arachidonic acid production and cell proliferation (Kemilainen et al.,
412
2018). All these findings underline the important role of 17β-HSD12 in long-chain fatty acid elongation.
413
In an early study, protection of activity after protease treatment of microsomes was used in order to assess
414
the membrane topology of the enzymes involved in rat long-chain fatty acid elongation (Osei et al.,
415
1989). Based on the results, the first condensing enzyme in this cascade as well as the last trans-2-enoyl-
416
CoA reductase of the elongation are facing the cytosolic part of the ER membrane, whereas the 3-
417
hydroxyacyl-CoA dehydratase had an activity embedded in the ER membrane. The yeast 3-hydroxyacyl-
418
CoA dehydratase (Phs1) was later determined to be a protein that transverses the ER membrane six times,
419
and the critical residues for its enzymatic activity were found to be located within its transmembrane
420
domains, which has not been reported for any other hydratase (Kihara et al., 2008). 17β-HSD12 is
421
predicted to possess up to two transmembrane domains, one close to the N- and one upstream of the C-
422
terminus. Fusion of its C-terminus to roGFP2 showed that this part of the protein is cytosolic, whereas
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addition of roGFP2 upstream of its substrate and co-factor binding site, suggested that this part is luminal
424
(Tsachaki et al., 2015). The above results suggest a complicated mechanism for long-chain fatty acid
425
elongation, with enzymatic catalysis taking place on two different sides of the ER membrane, as well as
426
within the membrane.
427
The membrane topology of 17β-HSD9 (Su et al., 1999) has been elaborately investigated using the
428
proteinase K protection assay and deletion constructs (Simon et al., 1999). It was demonstrated that 17β-
429
HSD9 contains a transmembrane domain starting at the beginning of the protein, followed by the main
430
luminal part and a C-terminal transmembrane domain that leaves six amino acids in the cytosolic
431
compartment. Although the native protein does not seem to be glycosylated, introduction of an N-
432
glycosylation site at the proposed luminal region induced glycosylation, further supporting the luminal
433
localization of the main part of the enzyme. A similar topology was demonstrated for 17β-HSD6 using
434
roGFP2 fusions at the C-terminus, revealing cytoplasmic orientation of the very C-terminus and the part
435
following the first transmembrane region, which indicates luminal orientation of the main part of the
436
enzyme (Tsachaki et al., 2015).
437
The two enzymes also share functional similarity. Both enzymes are involved in the metabolism of
438
retinoids, hence their alternative names retinol dehydrogenase (RODH) for 17β-HSD6 and 11-cis retinol
439
dehydrogenase for 17β-HSD9. Their substrate preference differs though, with 17β-HSD6 accepting
440
mostly all-trans retinol and 17β-HSD9 9-cis retinol or 11-cis retinol (Napoli, 2001; Wang et al., 1999).
441
The membrane topology of these enzymes indicates that at least a part of the complex retinoid metabolism
442
takes place in the lumen of the ER and implies the existence of an unknown transport mechanism of
443
retinoids from the cytosol to the ER. Besides their role in retinoid metabolism, both enzymes are able to
444
oxidize estradiol to estrone, as well as androgens at the 3α or 17β position. Both 17β-HSD6 and 17β-
445
HSD9 can catalyze the conversion of 3α-adiol to DHT, although based on their tissue distribution 17β-
446
HSD6 is considered the biological relevant enzyme for the oxidation of 3a-adiol (Bauman et al., 2006;
447
Biswas et al., 1997; Napoli, 2001; Su et al., 1999; Wang et al., 1999). These reactions are considered to be
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of paramount importance for DHT synthesis/inactivation through the alternative or ‘backdoor’ pathway,
449
bypassing testosterone production (Biason-Lauber et al., 2013; Fukami et al., 2013). This mechanism of
450
regulating local DHT levels seems to be central in the pathology of prostate cancer. Given the topology of
451
17β-HSD6 it remains unclear why DHT should be formed within the ER since unliganded AR resides in
452
the cytoplasm. Conversely, the enzymes that reduce androsterone to 3a-adiol (17β-HSD3) and DHT to 3α-
453
adiol (AKR1C2) are active in the cytosolic compartment, which may allow intracellular control of DHT
454
concentrations through subcellular compartmentalization. Further research is needed to understand the
455
combined action of several enzymes participating in the same metabolic pathway and located in different
456
compartments.
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3. Conclusion remarks
The subcellular localization of all 17β-HSD enzymes has now been determined. This effort required in
460
most cases confirmation by both indirect immunofluorescence and biochemical methods, such as
461
subcellular fractionation. Erroneous intracellular distribution has been initially assumed, for example in
462
case of 17β-HSD10, mainly due to misinterpretation of experimental data in the absence of suitable
463
positive and negative controls or appropriate markers for each cell compartment. For instance, it was
464
noted that cytochrome C oxidase rather than cytochrome C should be used as mitochondrial marker, since
465
upon mitochondrial damage cytochrome C is released into the cytosol, thus not representing an ideal
466
mitochondria-specific marker (Yang et al., 2014).
467
Since the 17β-HSDs that localize to the ER and/or lipid droplets are integral membrane proteins, unveiling
468
their membrane topology has been the focus of several studies. Topology determination is a laborious
469
process often requiring mutagenesis, protein tagging or the use of fusion proteins. These manipulations
470
may disturb proper topology, making the application of different methods indispensable. The most
471
commonly used method for membrane topology determination involves the proteinase K protection assay
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using isolated microsomes, which reveals whether a part of the protein is protected from digestion, i.e.
473
luminal. This is a multi-step, indirect method, which can provide reliable results if appropriate controls are
474
employed. Another approach involves the selective permeabilization of the plasma membrane with
475
detergents such as digitonin or the use of pore-forming agents like streptolysin-O, and subsequent
476
immunofluorescence detection. This method requires extensive optimization of experimental conditions,
477
as well as the use of suitable luminal and cytosolic control proteins. Another recently added technique
478
includes the fusion of the protein of interest with roGFP2 and ratiometric calculation of the sensor
479
oxidation status, indicative of luminal (more oxidized) or cytosolic localization (Tsachaki et al., 2015).
480
Ideally, at least two independent methods are applied for membrane topology determination.
481
Subcellular localization and membrane topology dictates the compartment in which an enzyme is active,
482
and may provide insight into its biological function. This is of particularly interest for 17β-HSDs, as many
483
of these enzymes accept several common substrates in vitro that often do not represent genuine in vivo
484
substrates. A typical example is the interconversion of estrone and estradiol, where 17β-HSD1, 7 and 12
485
can perform the reductive and 17β-HSD2, 4, 6, 8, 9 and 10 the oxidative reaction. The relative
486
contribution of these enzymes in estrogen metabolism is far from being fully understood and may depend
487
on specific cellular conditions. However, given the cytoplasmic/nuclear localization of estrogen receptors
488
one might anticipate that mitochondrial or peroxisomal localization of a given 17β-HSD indicates a
489
different major physiological role for these enzymes. Determination of the subcellular localization
490
supports the hypothesis generation process when limited information exists regarding the substrates or
491
function of an enzyme. Therefore, resolving the subcellular localization and membrane topology of the
492
17β-HSD enzymes paved the way for understanding their biological roles and will continue to be
493
important for future functional studies.
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Acknowledgements
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This work was supported by the Swiss National Science Foundation No 31003A-179400. 20
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Figure Legends
501
Figure 1. Schematic representation of the subcellular localization of the 17β-HSD enzymes. 17β-
502
HSD1, 5 and 14 are cytosolic. According to one study, 17β-HSD5 translocates into the nucleus in complex
503
with the androgen receptor. 17β-HSD11 and 17β-HSD13 have been reported to localize to both the lipid
504
droplets and the ER. 17β-HSD2, 17β-HSD3, 17β-HSD6, 17β-HSD7, 17β-HSD9 and 17β-HSD12 localize
505
at the ER. 17β-HSD8 and 10 are present in mitochondria, and 17β-HSD4 in peroxisomes.
506
Figure 2. Membrane topology of 17β-HSD enzymes of the ER and lipid droplets. For every protein
507
we illustrate the GXXXGXG motif (which is part of the co-factor binding site) and the active site, along
508
with the amino acid number at which these consensus sequences start. At the C-terminus the total amino
509
acid number is shown. 17β-HSD2, 17β-HSD3, 17β-HSD11 and 17β-HSD13 exhibit cytosolic, and 17β-
510
HSD6, 17β-HSD7, 17β-HSD9 and 17β-HSD12 luminal localization of the GXXXGXG motif and active
511
site.
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Highlights •
The 17β-hydroxysteroid dehydrogenases (17β-HSDs) consists of enzymes with diverse functions, spanning from sex steroid and retinoid metabolism to fatty acid synthesis/oxidation. Investigations of subcellular localization have shown that different 17β-HSDs are present in the
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cytosol, endoplasmic reticulum, lipid droplets, peroxisomes or mitochondria. The 17β-HSDs that reside in the endoplasmic reticulum and lipid droplets are integral membrane proteins and their
Subcellular localization and membrane topology of the 17β-HSD enzymes is tightly correlated
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with their biological function.
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topology has now been resolved.