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Cysteine-10 on 17 ? -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals.

ABSTRACT: 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, Cys(10), 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 Cys(10) 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. Cys(10)Ser mutant 17 ? -HSD1 was partially protected from inhibition by NEM and dithiocarbamates, emphasizing the importance of Cys(10) in the cofactor binding region. Substitution of Cys(10) with serine resulted in a decreased protein half-life, without significantly altering kinetic properties. Despite the fact that Cys(10) 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.

SUBMITTER: Nashev LG

PROVIDER: S-EPMC3855964 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Cysteine-10 on 17 β -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals.

Nashev Lyubomir G LG Atanasov Atanas G AG Baker Michael E ME Odermatt Alex A

International journal of cell biology 20131117

17 β -Hydroxysteroid dehydrogenase type 1 (17 β -HSD1) catalyzes the conversion of estrone to the potent estrogen estradiol. 17 β -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 stru ...[more]

PMID: 24348564

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Project description:Background and aims17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model.Approach and resultsSingle-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets.ConclusionsExtensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.

Project description:17?-Hydroxysteroid dehydrogenase type 1 (17?-HSD1) catalyzes the reduction of estrone to estradiol, which is the most potent estrogen in humans. Inhibition of 17?-HSD1 and thereby reducing the intracellular estradiol concentration is thus a promising approach for the treatment of estrogen dependent diseases. In the past, several steroidal and non-steroidal inhibitors of 17?-HSD1 have been described but so far there is no cocrystal structure of the latter in complex with 17?-HSD1. However, a distinct knowledge of active site topologies and protein-ligand interactions is a prerequisite for structure-based drug design and optimization. An elegant strategy to enhance this knowledge is to compare inhibition values obtained for one compound toward ortholog proteins from various species, which are highly conserved in sequence and differ only in few residues. In this study the inhibitory potencies of selected members of different non-steroidal inhibitor classes toward marmoset 17?-HSD1 were determined and the data were compared with the values obtained for the human enzyme. A species specific inhibition profile was observed in the class of the (hydroxyphenyl)naphthols. Using a combination of computational methods, including homology modelling, molecular docking, MD simulation, and binding energy calculation, a reasonable model of the three-dimensional structure of marmoset 17?-HSD1 was developed and inhibition data were rationalized on the structural basis. In marmoset 17?-HSD1, residues 190 to 196 form a small ?-helix, which induces conformational changes compared to the human enzyme. The docking poses suggest these conformational changes as determinants for species specificity and energy decomposition analysis highlighted the outstanding role of Asn152 as interaction partner for inhibitor binding. In summary, this strategy of comparing the biological activities of inhibitors toward highly conserved ortholog proteins might be an alternative to laborious x-ray or site-directed mutagenesis experiments in certain cases. Additionally, it facilitates inhibitor design and optimization by offering new information on protein-ligand interactions.

Dataset Information

Cysteine-10 on 17 ? -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals.

Publications

Cysteine-10 on 17 β -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals.

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