RESUMO
In human adrenarche during childhood, the secretion of dehydroepiandrosterone (DHEA) from the adrenal gland increases due to its increased synthesis and/or decreased metabolism. DHEA is synthesized by 17α-hydroxylase/17,20-lyase, and is metabolized by 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2). In this study, the inhibition of purified human 3ßHSD2 by the adrenal steroids, androstenedione, cortisone, and cortisol, was investigated and related to changes in secondary enzyme structure. Solubilized, purified 3ßHSD2 was inhibited competitively by androstenedione with high affinity, by cortisone at lower affinity, and by cortisol only at very high, nonphysiologic levels. When purified 3ßHSD2 was bound to lipid vesicles, the competitive Ki values for androstenedione and cortisone were slightly decreased, and the Ki value of cortisol was decreased 2.5-fold, although still at a nonphysiologic level. The circular dichroism spectrum that measured 3ßHSD2 secondary structure was significantly altered by the binding of cortisol, but not by androstenedione and cortisone. Our import studies show that 3ßHSD2 binds in the intermitochondrial space as a membrane-associated protein. Androstenedione inhibits purified 3ßHSD2 at physiologic levels, but similar actions for cortisol and cortisone are not supported. In summary, our results have clarified the mechanisms for limiting the metabolism of DHEA during human adrenarche.
Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Adrenarca/efeitos dos fármacos , Adrenarca/fisiologia , Androstenodiona/farmacologia , Inibidores Enzimáticos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Adrenarca/metabolismo , Androstenodiona/metabolismo , Linhagem Celular , Cortisona/metabolismo , Cortisona/farmacologia , Inibidores Enzimáticos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hidrocortisona/farmacologia , Lipossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , SolubilidadeRESUMO
The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building 'next generation' genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness.
RESUMO
The lack of routine viral genomic surveillance delayed the initial detection of SARS-CoV-2, allowing the virus to spread unfettered at the outset of the U.S. epidemic. Over subsequent months, poor surveillance enabled variants to emerge unnoticed. Against this backdrop, long-standing social and racial inequities have contributed to a greater burden of cases and deaths among minority groups. To begin to address these problems, we developed a new variant surveillance model geared toward building microbial genome sequencing capacity at universities in or near rural areas and engaging the participation of their local communities. The resulting genomic surveillance network has generated more than 1,000 SARS-CoV-2 genomes to date, including the first confirmed case in northeast Louisiana of Omicron, and the first and sixth confirmed cases in Georgia of the emergent BA.2.75 and BQ.1.1 variants, respectively. In agreement with other studies, significantly higher viral gene copy numbers were observed in Delta variant samples compared to those from Omicron BA.1 variant infections, and lower copy numbers were seen in asymptomatic infections relative to symptomatic ones. Collectively, the results and outcomes from our collaborative work demonstrate that establishing genomic surveillance capacity at smaller academic institutions in rural areas and fostering relationships between academic teams and local health clinics represent a robust pathway to improve pandemic readiness. Author summary: Genomic surveillance involves decoding a pathogenâ™s genetic code to track its spread and evolution. During the pandemic, genomic surveillance programs around the world provided valuable data to scientists, doctors, and public health officials. Knowing the complete SARS-CoV-2 genome has helped detect the emergence of new variants, including ones that are more transmissible or cause more severe disease, and has supported the development of diagnostics, vaccines, and therapeutics. The impact of genomic surveillance on public health depends on representative sampling that accurately reflects the diversity and distribution of populations, as well as rapid turnaround time from sampling to data sharing. After a slow start, SARS-CoV-2 genomic surveillance in the United States grew exponentially. Despite this, many rural regions and ethnic minorities remain poorly represented, leaving significant gaps in the data that informs public health responses. To address this problem, we formed a network of universities and clinics in Louisiana, Georgia, and Mississippi with the goal of increasing SARS-CoV-2 sequencing volume, representation, and equity. Our results demonstrate the advantages of rapidly sequencing pathogens in the same communities where the cases occur and present a model that leverages existing academic and clinical infrastructure for a powerful decentralized genomic surveillance system.
RESUMO
Human 3beta-hydroxysteroid dehydrogenase/isomerase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a target enzyme for inhibition in the treatment of breast cancer in postmenopausal women. Human 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland in this population. In our recombinant human breast tumor MCF-7 Tet-off cells that express either 3beta-HSD1 or 3beta-HSD2, trilostane and epostane inhibit the DHEA-induced proliferation of MCF-7 3beta-HSD1 cells with 12- to 16-fold lower IC(50) values compared to the MCF-7 3beta-HSD2 cells. The compounds also competitively inhibit purified human 3beta-HSD1 with 12- to 16-fold lower K(i) values compared to the noncompetitive K(i) values measured for human 3beta-HSD2. Using our structural model of 3beta-HSD1, trilostane or 17beta-acetoxy-trilostane was docked in the active site of 3beta-HSD1, and Arg195 in 3beta-HSD1 or Pro195 in 3beta-HSD2 was identified as a potentially critical residue (one of 23 non-identical residues in the two isoenzymes). The P195R mutant of 3beta-HSD2 were created, expressed and purified. Kinetic analyses of enzyme inhibition suggest that the high affinity, competitive inhibition of 3beta-HSD1 by trilostane and epostane may be related to the presence of Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , Neoplasias da Mama/enzimologia , Inibidores Enzimáticos/farmacologia , Arginina/metabolismo , Inibidores da Aromatase/farmacologia , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Coenzimas/metabolismo , Desidroepiandrosterona/farmacologia , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/farmacologia , Estradiol/farmacologia , Estrona/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Proteínas Mutantes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato/efeitos dos fármacosRESUMO
The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , Di-Hidrotestosterona/análogos & derivados , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/isolamento & purificação , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Di-Hidrotestosterona/farmacologia , Relação Dose-Resposta a Droga , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/genética , Cinética , Metionina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Esteroide Isomerases/antagonistas & inibidores , Esteroide Isomerases/química , Esteroide Isomerases/genética , Relação Estrutura-Atividade , Especificidade por Substrato , Testosterona/análogos & derivados , Testosterona/farmacologia , Treonina/metabolismoRESUMO
The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.
Assuntos
3-Hidroxiesteroide Desidrogenases/química , Coenzimas/metabolismo , Cisteína/metabolismo , Modelos Moleculares , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Sítios de Ligação , Cisteína/genética , Humanos , Mutagênese Sítio-Dirigida , Conformação Proteica , Especificidade por SubstratoRESUMO
3ß-Hydroxysteroid dehydrogenase type 1 (3ß-HSD1) is selectively expressed in human placenta, mammary glands and breast tumors in women. Human 3ß-HSD2 is selectively expressed in adrenal glands and ovaries. Based on AutoDock 3 and 4 results, we have exploited key differences in the amino acid sequences of 3ß-HSD1 (Ser194, Arg195) and 3ß-HSD2 (Gly194, Pro195) by designing a selective inhibitor of 3ß-HSD1. 2,16-Dicyano-4,5-epoxy-androstane-3,17-dione (16-cyano-17-keto-trilostane or DiCN-AND) was synthesized in a 4-step procedure from androstenedione. In purified 3ß-HSD inhibition studies, DiCN-AND competitively inhibited 3ß- HSD1 with Ki=4.7µM and noncompetitively inhibited 3ß-HSD2 with a 6.5-fold higher Ki=30.7µM. We previously reported similar isoenzyme-specific inhibition profiles for trilostane. Based on our docking results, we created, expressed and purified the chimeric S194G-1 mutant of 3ß-HSD1. Trilostane inhibited S194G-1 (Ki=0.67µM) with a noncompetitive mode compared to its 6.7-fold higher affinity, competitive inhibition of 3ß-HSD1 (Ki=0.10µM). DiCN-AND inhibited S194G-1 with a 6.3-fold higher Ki (29.5µM) than measured for 3ß-HSD1 (Ki=4.7µM) but with the same competitive mode for both enzyme species. Since DiCN-AND noncompetitively inhibits 3ß-HSD2, which has the Gly194 and Pro195 of 3ß-HSD2 in place of the Ser194 and Arg195 in 3ß-HSD1, this suggests that Arg195 alone in 3ß-HSD1 or S194G-1 is required to bind DiCN-AND in the substrate binding site (competitive inhibition). However, both Ser194 and Arg195 are required to bind trilostane in the 3ß-HSD1 substrate site based on its noncompetitive inhibition of S194G-1 and 3ß-HSD2. In support of this hypothesis, DiCN-AND inhibited our chimeric R195P-1 mutant noncompetitively with a Ki=41.3µM (similar to the 3ß-HSD2 inhibition profile). Since DiCN-AND competitively inhibited S194G-1 that still contains R195 but noncompetitively inhibited R195P-1 that still contains S194, our data provides strong evidence that the Arg195 being mutated to Pro195 (as present in 3ß-HSD2) shifts the inhibition mode from competitive to noncompetitive in 3ß-HSD1. This supports the key role of Arg195 in 3ß-HSD1 for the high affinity, competitive binding of the trilostane analogs. Our new structure/function information for the design of targeted 3ß-HSD1 inhibitors may lead to important new treatments for the prevention of spontaneous premature birth.
Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/metabolismo , Arginina/metabolismo , Di-Hidrotestosterona/análogos & derivados , Di-Hidrotestosterona/metabolismo , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , Androgênios , Ligação Competitiva , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Relação Estrutura-AtividadeRESUMO
In postmenopausal women, human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) is a critical enzyme in the conversion of DHEA to estradiol in breast tumors, while 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to determine if Arg195 in 3beta-HSD1 vs. Pro195 in 3beta-HSD2 in the substrate/inhibitor binding site is a critical structural difference responsible for the higher affinity of 3beta-HSD1 for inhibitor and substrate steroids compared to 3beta-HSD2 and whether Asp61, Glu192 and Thr8 are fingerprint residues for cofactor and substrate binding using site-directed mutagenesis. The R195P-1 mutant of 3beta-HSD1 and the P195R-2 mutant of 3beta-HSD2 have been created, expressed, purified and characterized kinetically. Dixon analyses of the inhibition of the R195P-1 mutant, P195R-2 mutant, wild-type 3beta-HSD1 and wild-type 3beta-HSD2 by trilostane has produced kinetic profiles that show inhibition of 3beta-HSD1 by trilostane (K(i)=0.10microM, competitive) with a 16-fold lower K(i) and different mode than measured for 3beta-HSD2 (K(i)=1.60microM, noncompetitive). The R195P-1 mutation shifts the high-affinity, competitive inhibition profile of 3beta-HSD1 to a low-affinity (trilostane K(i)=2.56microM), noncompetitive inhibition profile similar to that of 3beta-HSD2 containing Pro195. The P195R-2 mutation shifts the low-affinity, noncompetitive inhibition profile of 3beta-HSD2 to a high-affinity (trilostane K(i)=0.19microM), competitive inhibition profile similar to that of 3beta-HSD1 containing Arg195. Michaelis-Menten kinetics for DHEA, 16beta-hydroxy-DHEA and 16alpha-hydroxy-DHEA substrate utilization by the R195P-1 and P195R-2 enzymes provide further validation for higher affinity binding due to Arg195 in 3beta-HSD1. Comparisons of the Michaelis-Menten values of cofactor and substrate for the targeted mutants of 3beta-HSD1 (D61N, D61V, E192A, T8A) clarify the functions of these residues as well.