17-hydroxysteroid dehydrogenase ( 17-hsd ) enzymes are involved in the interconversion of inactive and active sex - steroid hormones , thereby playing an essential role in the intracrine regulation of estrogen- , androgen- , and progesterone - dependent physiological functions .
17-hsd1 is responsible for the conversion of estrone to estradiol , the most potent natural estrogen .
importantly , 17-hsd1 expression showed a negative correlation with breast cancer progression and was identified as an independent prognostic marker for the disease - free and overall survival of patients with breast cancer [ 2 , 3 ] .
elevated expression of 17-hsd1 was observed in endometrial cancer   and nonsmall cell lung cancer .
furthermore , the expression ratio of 17-hsd1 and 17-hsd2 was found to be a predictor of the response to tamoxifen in postmenopausal breast cancer patients .  
the administration of specific 17-hsd1 inhibitors led to significantly decreased tumor growth in breast cancer cell xenograft tumor mouse models [ 7 , 8 ] and reversed estrogen - dependent endometrial hyperplasia in transgenic mice , indicating that this enzyme is a promising drug target against estrogen - dependent diseases such as endometriosis and endometrial cancer as well as breast and ovarian tumors .
several classes of chemicals inhibiting 17-hsd1 were tested in vitro , including steroid - like molecules , nonsteroidal compounds , and chimeric molecules , acting on both the active center and the cofactor binding site of the enzyme [ 8 , 1016 ] .
however , only a limited number of inhibitors have been tested in vivo so far and further research is needed . in order to develop potent and selective 17-hsd1 inhibitors , a profound understanding of the structure - function relationships of the enzyme is essential .  
17-hsd1 belongs to the short - chain dehydrogenase / reductase ( sdr ) family and contains the conserved rossmann - fold for nucleotide binding , the catalytic triad with residues ser , tyr , and lys   and a dimerization region .
lys is a critical residue for the discrimination between c-18 and c-19 steroid substrates .  
targeting of functionally essential cysteine residues in proteins is suggested as a promising approach for the design of new types of pharmaceutical agents .
, we demonstrated that the structurally related enzyme 11-hydroxysteroid dehydrogenase type 2 ( 11-hsd2 ) contains a cysteine residue at position 90 , analogous to cys on 17-hsd1 .
therefore , we hypothesized that cys on 17-hsd1 may represent a target site for novel inhibitors . in the present study , we applied 3d structure modeling and enzyme activity measurements to investigate the biochemical properties of cysser mutant 17-hsd1 and to compare its sensitivity towards sulfhydryl modifying agents with that of wild - type 17-hsd1 .
cell culture media were purchased from invitrogen ( carlsbad , ca ) and [ 2,4,6,7-h]-estrone from amersham pharmacia ( piscataway , nj , usa ) .
all other chemicals were from fluka ag ( buchs , switzerland ) and of the highest grade available . in order to facilitate detection ,
an octahistidine tag was added to the c - terminus of human 17-hsd1 by pcr amplification using oligonucleotide primers containing the tag - coding sequence ( forward primer 5-taaaccctgaggaggtggcggaggtcttc-3 , reverse primer 5-tgctctagaagcttaatgatgatgatgatgatgatgatgctgcggggcggccggaggatcg-3 ) .
the substitution of cys to serine was introduced into the c - terminal histidine - tagged 17-hsd1 cdna in bluescript vector by site - directed mutagenesis using the forward primer 5-tcatcaccggctcttcctcg-3 and the reverse primer 5-gaggaagagccggtgatgag-3 according to the quick change mutagenesis kit ( stratagene , amsterdam , the netherlands ) , followed by subsequent recloning into pcdna3.1 expression plasmid .  
hek-293 cells were grown to 6070% confluence in dulbecco 's modified eagles medium ( dmem ) supplemented with 10% fetal bovine serum , 4.5  g / l glucose , 50 units / ml penicillin / streptomycin , and 2 mm glutamine , followed by transfection using the calcium phosphate precipitation method .
the transfection efficiency was 32  3% . to examine the expression levels of the overexpressed proteins , we performed sds - page ( 30  g of total cellular proteins loaded per lane ) followed by immunoblotting and detection of the histidine - tagged proteins using tetra - his antibody ( qiagen gmbh , hilden , germany ; cat .
was visualized using an anti--actin antibody ( santa cruz biotechnology , santa cruz , ca ; cat .
cells were harvested 48  h after transfection , washed twice with phosphate - buffered saline ( pbs ) , and centrifuged for 4  min at 150  g .
supernatants were removed and the cell pellets were quick - frozen in a dry ice - ethanol bath and stored at 80c .
the reductase activity of 17-hsd1 was measured by incubation of cell lysates for 10  min at 37c in the presence of 200  nm radiolabeled estrone and 400  m nadph in a reaction buffer containing 20% glycerol , 1  mm edta , and 50  mm potassium phosphate , ph 7.4 .
inhibitors were diluted from stock solutions in dimethylsulfoxide ( dmso ) and immediately used in the assays .
the dmso concentration did not exceed 0.1% and had no effect on the enzyme activities .  
for the enzyme activity assay in intact cells , hek-293 cells were grown in 10  cm culture dishes , transfected with plasmids for histidine - tagged wild - type 17-hsd1 or cysser mutant , detached 24  h after transfection and distributed in 96-well plates at a density of 10,000 cells per well .
after 16  h , cells were incubated in serum- and steroid - free medium , and the conversion of radiolabeled estrone to estradiol was determined upon incubation for 30  min at 37c in a total volume of 50  l containing 200  nm estrone .
the reaction was stopped by adding methanol containing 2  mm unlabeled estrone and estradiol , followed by separation of steroids by tlc and scintillation counting . for determination of enzyme kinetics , estrone concentrations ranging from 10 to 800  nm were used .
data ( mean  sd ) were obtained from three independent experiments and were calculated using the data analysis toolbox ( elsevier mdl , allschwil , switzerland ) .  
the stability of wild - type and mutant 17-hsd1 protein was analyzed basically as described previously .
briefly , to investigate the protein half - life of wild - type 17-hsd1 and mutant cysser , hek-293 cells grown in six - well plates were transfected with plasmids for mutant and wild - type enzyme , washed once with pbs 24  h after transfection , and incubated with fresh medium containing 50  g / ml cycloheximide . at 0 , 12 , 24 , and 48  h ,
alternatively , cells transfected with plasmids for mutant and wild - type enzyme were washed once 16  h after transfection , followed by incubation in leucine - free dmem ( mp biomedicals , illkirch , france ) for 45  min to deplete endogenous leucine .
the medium was then replaced by 1  ml of leucine - free dmem supplemented with 20  ci / ml l - leucine-[3,4,5-h(n ) ] , followed by incubation for 3  h. the labeling was terminated by addition of 5  mm unlabeled leucine , washing twice with dmem , and incubation in dmem . at 0 , 12 , 24 , and 48  h , aliquots of cells were snap - frozen prior to purification of histidine - tagged proteins by using a ni - nta agarose kit according to the manufacturer ( qiagen ag , hombrechtikon , switzerland ) .
after elution , proteins were subjected to sds - page , the gels were dried and exposed to tritium - sensitive screens ( tr uncoated bafbr : eu screens ) for 16  h , followed by analysis using a cyclone phosphor - imager ( perkinelmer life and analytical sciences , shelton , ct , usa ) .  
the protein sequences of 17-hsd1 from different species and different human sdrs were compared using the clustalw algorithm , run on http://www.ebi.ac.uk/ , with default program parameters .  
to investigate the effect on the structure of 17-hsd1 of mutating cys to ser , we extracted 1fdt from the protein data bank ( pdb ) .
cys was converted to serine using the biopolymer option in the insight ii software package .
the structure of the ser mutant 17-hsd1 was refined with discover 3 for 10,000 iterations , using a distant dependent dielectric constant of 2 .
the analysis of the peptide sequence of human 17-hsd1 using the clustal w algorithm   revealed that residue cys is highly conserved among species , including rodents and zebrafish ( figure 1 ) , suggesting a role for this residue in the stability and/or function of the protein . cys is located between the first two of three highly conserved glycine residues of the rossmann - fold nucleotide binding domain .
interestingly , the cysteine and the two downstream serine residues also are present in all - trans retinol dehydrogenase rdh8 , which like 17-hsd1 belongs to the sdr28c family . moreover , the cysteine residue at this position is conserved in all members of the sdr9c family , except for 17-hsd2 , which has a cysteine residue downstream by two positions .   to begin to understand the role of cys for 17-hsd1 function
, we analyzed the interactions of cys with adjacent amino acids in 17-hsd1 cocrystalized with estradiol and nadp .
the crystal structure of 1fdt reveals that cys has van der waals contacts with ile , gly , gly , ala , and thr .
gly , gly , and thr directly stabilize the binding of the cofactor nadp ( figure 2(a ) ) .
the numerous close contacts of cys with amino acids in the nadp(h)-binding site indicate that substitution of the sulfhydryl group with a larger chemical would disrupt this site and probably alter nadp(h ) binding and catalytic activity . to begin to study the function of this cysteine residue
as revealed by the 3d model ( figure 2(b ) ) , mutating cys to serine does not lead to significant changes in contacts between ser and ile , gly and ala .
however , all three contacts between ser and gly are shorter than between cys and gly , and gly is more distant from a phosphate oxygen on nadp ( figure 2(b ) ) . also , ser is a little more distant from the backbone oxygen on thr , and thr is 4.7    from the oxygen on the adenosine phosphate .
the backbone nitrogen on gly is 4.5    from the same phosphate oxygen on nadp ( figure 2(b ) ) .   in a previous study , sulfhydryl modifying
chemicals were found to exert potent inhibitory effects on 11-hsd2 and substitution of cys by serine abolished enzymatic activity . to test the hypothesis that cys on 17-hsd1 has similar essential stabilizing interactions in the nadph binding region , we assessed the inhibitory potential of the cysteine modifying agent n - ethylmaleimide ( nem ) and of dithiocarbamate chemicals .
nem appeared to be a weak inhibitor with an ic50 of 22  6  m upon simultaneous incubation of lysates of 17-hsd1 expressing hek-293 cells with 200  nm estrone and increasing concentrations of nem . in line with an irreversible mode of inhibition , preincubation with nem resulted in a more pronounced inhibitory effect in a time - dependent manner .
preincubation of lysates with 20  m nem for 1  h prior to the addition of estrone completely abolished 17-hsd1 activity ( data not shown ) .
in contrast to the previously observed potent inhibition of 11-hsd2 , dithiocarbamates turned out to have rather modest inhibitory effects on 17-hsd1 .
the ic50 values for thiram ( 21  4  m ) , disulfiram ( 8  1  m ) , maneb ( 25  3  m ) , and zineb ( 24  3  m ) upon simultaneous incubation of the 17-hsd1 enzyme preparation with substrate and inhibitor were about two orders of magnitude higher than those obtained for 11-hsd2 , indicating that 17-hsd1 is much less prone to inhibition by sulfhydryl modification .  
importantly , as shown in figure 3 , preincubation with nadph for 15  min protected from inhibition by nem , an effect which was concentration dependent .
preincubation with nadph also protected from inhibition by dithiocarbamates ( data not shown ) , suggesting that binding of nadph prevents the covalent modification of cys in the cofactor binding region . to study the role of cys for 17-hsd1 function , we generated mutant cysser and
compared the expression of histidine - tagged wild - type and mutant enzymes upon transient expression in hek-293 cells , which lack endogenous 17-hsd1 expression . despite comparable transfection efficiency ,
mutant cysser was expressed at approximately twofold lower levels than wild - type 17-hsd1 ( figure 4 ) .
moreover , several low - molecular weight bands were detected , indicating a lower stability of the cysser mutant protein .
next , we performed experiments in transfected hek-293 cells using cycloheximide in order to block de novo protein synthesis and estimate protein half - life .
the signal detected for histidine - tagged 17-hsd1 protein was not significantly decreased 48  h after blocking translation with cycloheximide ( figure 5(a ) ) , indicating a protein half - life greater than 48  h. longer incubations are not appropriate due to the cytotoxicity of cycloheximide .
in contrast , mutant cysser was less stable and the protein half - life of the mutant enzyme was estimated to be 26  7  h ( mean  sd ) .
a pulse - chase experiment using h - leucine labeling confirmed the decreased stability of the mutant protein ( estimated protein half - life of 15  6  h ) compared with the wild - type 17-hsd1 ( estimated protein half - life of 36  10  h ) ( figure 5(b ) ) .
the differences in the protein half - life estimation by the pulse - chase and cycloheximide methods may be explained by the incomplete inhibition of protein synthesis in the latter approach .   a comparison of the enzyme activities of lysates expressing wild - type 17-hsd1 or mutant cysser showed approximately 50% lower activity for the mutant ( data not shown ) .
however , after correction for protein expression using anti - histidine - tag antibody there was no significant difference between wild - type and mutant enzymes .
the analysis of enzyme kinetics revealed a slightly higher apparent km but no significant change in the maximal velocity ( vmax ) for estrone reduction ( table 1 ) .
in intact cells , the activity of mutant cysser was indistinguishable from that of the wild - type enzyme ( data not shown ) .  
next , we compared the effect of different dithiocarbamate chemicals on the activity of wild - type 17-hsd1 and mutant cysser .
mutant cysser was protected from inhibition by all of the dithiocarbamates tested ( figure 6 ) .
similarly , the cysteine - modifying agent nem inhibited the activity of the wild - type enzyme approximately two times stronger than that of mutant cysser , confirming the role of this cysteine for 17-hsd1 function ( data not shown ) .
analysis of the 17-hsd1 protein sequences from different species revealed that the cysteine at position 10 in the human enzyme is highly conserved .
protein sequence alignment further revealed that a cysteine at this position in the rossmann - fold nucleotide binding region is conserved in the sdr subfamilies sdr28c and sdr9c .
the 3d structure of 17-hsd1 predicts several van der waals contacts between cys and residues involved directly in the binding of nadph .
the 3d model of ser mutant 17-hsd1 finds that although most contacts are conserved there are changes that may be significant . for example , ser is closer to gly , and gly is more distant from a phosphate oxygen on nadp .
also , in the ser mutant 17-hsd1 , the backbone oxygen on thr and backbone nitrogen on gly move to 4.7    and 4.5    , respectively , from the oxygen on the adenosine phosphate . in wild - type 17-hsd1 ,
the backbone oxygen on thr and backbone nitrogen on gly are 3.3    and 3.8    , respectively , from this oxygen on nadp .
the loss of these two stabilizing contacts could reduce the affinity for nadp(h ) in the ser mutant 17-hsd1 .
another possible contribution to lower stability of nadp(h ) in the ser mutant may be the different chemical properties of the thiol group on cys and alcohol on ser .
for instance , only the thiol group is predicted to be partially deprotonated at neutral ph , which could better stabilize the site around cys .
km values for nadph will need to be determined in follow - on studies to address this question .
nevertheless , our prediction is supported by results from huang et al . who demonstrated that insertion of a positively charged lysine residue in the neighborhood of cys and ser led to a more than 20-fold increase in the preference of 17-hsd1 for nadp(h ) against nad(h ) .
in a previous study , we found that the related sdr enzyme 11-hsd2 also contains a cysteine residue at the position corresponding to cys on 17-hsd1 in the cofactor binding region .
the 11-hsd2 cysser mutant almost completely lost its enzymatic activity , due to impaired protein folding and mislocalization of the mutant protein .  
interestingly , mutations of the analogous residue cys in the human ( r)-3-hydroxybutyrate dehydrogenase led to a slight increase in the apparent km for both nadp(h ) and nad(h ) .
the cysser mutant of the ( r)-3-hydroxybutyrate dehydrogenase , analogous to 17-hsd1 cysser , showed a twofold lower apparent vmax compared with the wild - type enzyme .
the slight increase in the apparent km of 17-hsd1 mutant cysser for estrone , observed in our measurements in cell lysates , indicates a decreased affinity for the substrate as a result of disturbed interactions with the cofactor nadph . a careful structural comparison between the interactions of residues with the cofactor and substrate in 17-hsd1 , 11-hsd2 , and ( r)-3-hydroxybutyrate dehydrogenase should offer an explanation for the different effects of the modification of the analogous cysteine residues of the three sdrs . in human retinol dehydrogenase
, the function of the analogous cys is not studied in detail ; however , boerman and napoli showed that the protein contains cysteine residues in close proximity , which are essential for the catalytic activity . in our experiments ,
the preincubation with increasing concentrations of nadph was able to protect 17-hsd1 from inhibition by the sulfhydryl modifying agents nem and dithiocarbamates , suggesting an indirect role of cys in stabilizing the binding of the cofactor .
dithiocarbamates and nem were able to inhibit the activity of 17-hsd1 , although at concentrations significantly higher than those needed for inhibition of 11-hsd2 .
the high sensitivity of 11-hsd2 toward sulfhydryl modifying chemicals was recently shown to be dependent on the presence of a cysteine residue in the substrate binding region .
our western blot experiments detected low - molecular bands for the 17-hsd1 cysser mutant , suggesting increased degradation of the mutant enzyme .
the more rapid degradation is likely due to changes in the protein conformation and thus exposure of amino acid residues normally buried inside the protein , followed by activation of the proteasome .
a limitation of the present study includes that wild - type and mutant enzymes were overexpressed and expression levels are higher than endogenous levels .
thus , the estimated half - life of the proteins may be different in an endogenous situation .
nevertheless , the results demonstrate a reduced stability of the mutant compared with the wild - type enzyme .  
the present work provides novel information on the structure - activity relationship of 17-hsd1 and reveals that cys is involved in essential stabilizing interactions in the cofactor binding region .
future studies using protease digestion of purified 17-hsd1 wild - type and cysser mutant proteins should provide further mechanistic insight into the stabilizing interactions of this residue .
 17-hydroxysteroid dehydrogenase type 1 ( 17-hsd1 ) catalyzes the conversion of estrone to the potent estrogen estradiol . 
 17-hsd1 is highly expressed in breast and ovary tissues and represents a prognostic marker for the tumor progression and survival of patients with breast cancer and other estrogen - dependent tumors . 
 therefore , the enzyme is considered a promising drug target against estrogen - dependent cancers . for the development of novel inhibitors , 
 an improved understanding of the structure - function relationships is essential . in the present study 
 , we examined the role of a cysteine residue , cys10 , in the rossmann - fold nadph binding region , for 17-hsd1 function and tested the sensitivity towards sulfhydryl modifying chemicals . 
 3d structure modeling revealed important interactions of cys10 with residues involved in the stabilization of amino acids of the nadph binding pocket . 
 analysis of enzyme activity revealed that 17-hsd1 was irreversibly inhibited by the sulfhydryl modifying agents n - ethylmaleimide ( nem ) and dithiocarbamates . 
 preincubation with increasing concentrations of nadph protected 17-hsd1 from inhibition by these chemicals . 
 cys10ser mutant 17-hsd1 was partially protected from inhibition by nem and dithiocarbamates , emphasizing the importance of cys10 in the cofactor binding region . 
 substitution of cys10 with serine resulted in a decreased protein half - life , without significantly altering kinetic properties . despite the fact that cys10 on 17-hsd1 seems to have limited potential as a target for new enzyme inhibitors , the present study provides new insight into the structure - function relationships of this enzyme . 