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1.
Artigo em Inglês | MEDLINE | ID: mdl-30218714

RESUMO

Androgens are a recognized class of endocrine disrupting compounds with the ability to impact reproductive status in aquatic organisms. The current study utilized in vitro exposure of mummichog (Fundulus heteroclitus) testis tissue to either the aromatizable androgen 17α-methyltestosterone (MT) or the non-aromatizable androgen 5α-dihydrotestosterone (DHT) over the course of 24 h to determine if there were differential effects on steroidogenic gene expression. Testis tissue was exposed to androgen concentrations of 10-12 M, 10-9 M and 10-6 M for 6, 12, 18 or 24 h, after which a suite of steroidogenic genes, including steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase (3ßhsd) and cytochrome P450 17A1 (cyp17a1), were quantified using real-time polymerase chain reaction. Both androgens affected steroidogenic gene expression, with most alterations occurring at the 24-hour time point. The gene with the highest fold-change, and shortest interval to expression alteration, was 3ßhsd for both androgens. Potential differences between the two model androgens were observed in increased expression of cyp17a1 and 11ß-hydroxysteroid dehydrogenase (11ßhsd), which were only altered after exposure to DHT and in expression levels of cytochrome P450 11A1 (cyp11a1), which was upregulated by MT but not altered by DHT. Results from this study show both androgens interact at the gonadal level of the hypothalamus-pituitary-gonadal axis and may possess some distinct gene expression impacts. These data strengthen the current research initiatives of establishing in vitro test systems that allow toxic potential of untested chemicals to be predicted from molecular perturbations.


Assuntos
Androgênios/toxicidade , Disruptores Endócrinos/toxicidade , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peixes Listrados/fisiologia , Testículo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Oceano Atlântico , Di-Hidrotestosterona/toxicidade , Estuários , Proteínas de Peixes/genética , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/crescimento & desenvolvimento , Sistema Hipotálamo-Hipofisário/metabolismo , Peixes Listrados/crescimento & desenvolvimento , Cinética , Masculino , Metiltestosterona/toxicidade , Novo Brunswick , Especificidade de Órgãos , Fosfoproteínas/metabolismo , Esteroide 17-alfa-Hidroxilase/química , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
2.
Int J Biol Macromol ; 120(Pt A): 673-682, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30170058

RESUMO

Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3ß-Hydroxysteroid dehydrogenase (3ßHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3ßHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3ßHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3ßHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3ßHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3ßHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3ßHSD was highly homologous to the 3ßHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3ßHSD and 3ßHSD from Rana rugosa. The optimized expression of recombinant Bbg-3ßHSD were achieved by inducing with 0.1 mmol L-1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3ß-oxidized by Bbg-3ßHSD when NAD+ was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3ßHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L-1.


Assuntos
3-Hidroxiesteroide Desidrogenases/química , Proteínas de Anfíbios/química , Bufo bufo/metabolismo , Desidroepiandrosterona/química , NAD/química , Pregnenolona/química , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Clonagem Molecular , Desidroepiandrosterona/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ponto Isoelétrico , Cinética , Peso Molecular , NAD/metabolismo , Fases de Leitura Aberta , Filogenia , Pregnenolona/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
3.
Biosci Biotechnol Biochem ; 82(10): 1702-1707, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29912635

RESUMO

We overexpressed and purified 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD) and its mutants where the active site residues known as the SYK triad, Ser114, Tyr153, and Lys157, were mutated. Ps3αHSD catalyzes the reaction by using a nucleotide cofactor. The NADH binding affinity of K157A mutant was much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity would result in decreased kcat. Compared to the wild-type, the mutants S114A and Y153F showed higher Km and lower kcat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F resulted in a significant decrease in kcat relative to the single mutant. These results are supported by the notion that Tyr153 is a catalytic base and Ser114 would be a substitute. Loss of hydrogen bonding with NADH upon the Y153F mutation resulted in increased enthalpy change, partially compensated by increased entropy change.


Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Pseudomonas/enzimologia , 3-Hidroxiesteroide Desidrogenases/química , Catálise , Domínio Catalítico , Dicroísmo Circular , Cristalografia por Raios X , Cinética , Mutação , Ligação Proteica , Conformação Proteica , Esteroides/metabolismo , Relação Estrutura-Atividade , Termodinâmica , Tirosina/metabolismo
4.
J Steroid Biochem Mol Biol ; 174: 257-264, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29031687

RESUMO

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-Atividade
5.
J Steroid Biochem Mol Biol ; 171: 270-280, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28457968

RESUMO

17beta-hydroxysteroid dehydrogenase type 5 (17ß-HSD5) is an important enzyme associated with sex steroid metabolism in hormone-dependent cancer. However, reports on its expression and its prognostic value in breast cancer are inconsistent. Here, we demonstrate the impact of 17ß-HSD5 expression modulation on the proteome of estrogen receptor-positive (ER+) breast cancer cells. RNA interference technique (siRNA) was used to knock down 17ß-HSD5 gene expression in the ER+ breast cancer cell line MCF-7 and the proteome of the 17ß-HSD5-knockdown cells was compared to that of MCF-7 cells using two-dimensional (2-D) gel electrophoresis followed by mass spectrometry analysis. Ingenuity pathway analysis (IPA) was additionally used to assess functional enrichment analyses of the proteomic dataset, including protein network and canonical pathways. Our proteomic analysis revealed only four differentially expressed protein spots (fold change > 2, p<0.05) between the two cell lines. The four spots were up-regulated in 17ß-HSD5-knockdown MCF-7 cells, and comprised 21 proteins involved in two networks and in functions that include apoptosis inhibition, regulation of cell growth and differentiation, signal transduction and tumor metastasis. Among the proteins are nucleoside diphosphate kinase A (NME1), 78kDa glucose-regulated protein (GRP78) and phosphoglycerate kinase 1 (PGK1). We also showed that expression of 17ß-HSD5 and that of the apoptosis inhibitor GRP78 are strongly but negatively correlated. Consistent with their opposite regulation, GRP78 knockdown decreased MCF-7 cell viability whereas 17ß-HSD5 knockdown or inhibition increased cell viability and proliferation. Besides, IPA analysis revealed that ubiquitination pathway is significantly affected by 17ß-HSD5 knockdown. Furthermore, IPA predicted the proto-oncogene c-Myc as an upstream regulator linked to the tumor-secreted protein PGK1. The latter is over-expressed in invasive ductal breast carcinoma as compared with normal breast tissue and its expression increased following 17ß-HSD5 knockdown. Our present results indicate a 17ß-HSD5 role in down-regulating breast cancer development. We thus propose that 17ß-HSD5 may not be a potent target for breast cancer treatment but its low expression could represent a poor prognosis factor.


Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoglicerato Quinase/metabolismo , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , Membro C3 da Família 1 de alfa-Ceto Redutase , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática , Feminino , Perfilação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/genética , Processamento de Imagem Assistida por Computador , Células MCF-7 , Nucleosídeo NM23 Difosfato Quinases/química , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/genética , Proteômica/métodos , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Receptores Estrogênicos/metabolismo , Eletroforese em Gel Diferencial Bidimensional
6.
Toxicol Ind Health ; 33(3): 265-276, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27102426

RESUMO

The aim of this study was to investigate the beneficial effects of zinc (Zn) in preventing lead (Pb)-induced reproductive toxicity in Wistar rats. The rats were divided into four groups, namely, control group, Pb group, Zn group, and Pb + Zn group. Animals were exposed to Pb (819 mg of Pb/L) or Zn (71 mg of Zn/L) or both through drinking water for 65 days. Rats exposed to Pb showed decreased weights of testes and accessory sex organs. Significant decrease in the testicular daily sperm production, epididymal sperm count, motility, viability, and number of hypoosmotic tail coiled sperm was observed in Pb-exposed rats. Testicular 3ß- and 17ß-hydroxysteroid dehydrogenase activity levels and circulatory testosterone levels were also decreased significantly in Pb-exposed rats. A significant increase in the lipid peroxidation products with a significant decrease in the activities of catalase and superoxide dismutase were observed in the testes and epididymis of Pb-exposed rats. Moreover, the testicular architecture showed lumens devoid of sperm in Pb-exposed rats. Supplementation of Zn mitigated Pb-induced oxidative stress and restored the spermatogenesis and steroidogenesis in Pb-exposed rats. In conclusion, cotreatment of Zn is effective for recovering suppressed spermatogenesis, steroidogenesis, elevated oxidative status, and histological damage in the testis of rats treated with Pb.


Assuntos
Suplementos Nutricionais , Epididimo/efeitos dos fármacos , Infertilidade Masculina/prevenção & controle , Intoxicação por Chumbo/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Testículo/efeitos dos fármacos , Zinco/uso terapêutico , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Suplementos Nutricionais/efeitos adversos , Epididimo/metabolismo , Epididimo/patologia , Infertilidade Masculina/etiologia , Intoxicação por Chumbo/metabolismo , Intoxicação por Chumbo/patologia , Intoxicação por Chumbo/fisiopatologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Compostos Organometálicos/antagonistas & inibidores , Compostos Organometálicos/toxicidade , Substâncias Protetoras/efeitos adversos , Substâncias Protetoras/uso terapêutico , Distribuição Aleatória , Ratos Wistar , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Testículo/metabolismo , Testículo/patologia , Testosterona/sangue , Doenças Transmitidas pela Água/metabolismo , Doenças Transmitidas pela Água/patologia , Doenças Transmitidas pela Água/fisiopatologia , Doenças Transmitidas pela Água/prevenção & controle , Zinco/efeitos adversos
7.
Mol Cell Biol ; 36(24): 3058-3074, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27697863

RESUMO

The acute response to stress consists of a series of physiological programs to promote survival by generating glucocorticoids and activating stress response genes that increase the synthesis of many chaperone proteins specific to individual organelles. In the endoplasmic reticulum (ER), short-term stress triggers activation of the unfolded protein response (UPR) module that either leads to neutralization of the initial stress or adaptation to it; chronic stress favors cell death. UPR induces expression of the transcription factor, C/EBP homology protein (CHOP), and its deletion protects against the lethal consequences of prolonged UPR. Here, we show that stress-induced CHOP expression coincides with increased metabolic activity. During stress, the ER and mitochondria come close to each other, resulting in the formation of a complex consisting of the mitochondrial translocase, translocase of outer mitochondrial membrane 22 (Tom22), steroidogenic acute regulatory protein (StAR), and 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2) via its intermembrane space (IMS)-exposed charged unstructured loop region. Stress increased the circulation of phosphates, which elevated pregnenolone synthesis by 2-fold by increasing the stability of 3ßHSD2 and its association with the mitochondrion-associated ER membrane (MAM) and mitochondrial proteins. In summary, cytoplasmic CHOP plays a central role in coordinating the interaction of MAM proteins with the outer mitochondrial membrane translocase, Tom22, to activate metabolic activity in the IMS by enhanced phosphate circulation.


Assuntos
Glândulas Suprarrenais/metabolismo , Estresse do Retículo Endoplasmático , Gônadas/metabolismo , Mitocôndrias/metabolismo , Fosfatos/metabolismo , Estresse Fisiológico , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Citoplasma/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfoproteínas/metabolismo , Fator de Transcrição CHOP/metabolismo , Resposta a Proteínas não Dobradas
8.
Biochem Pharmacol ; 116: 176-87, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27453434

RESUMO

The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.


Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Antineoplásicos Alquilantes/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Desenho de Drogas , Hidroxiprostaglandina Desidrogenases/metabolismo , Mesilatos/uso terapêutico , Modelos Moleculares , Organofosfonatos/uso terapêutico , Pró-Fármacos/uso terapêutico , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , Ativação Metabólica/efeitos dos fármacos , Membro C3 da Família 1 de alfa-Ceto Redutase , Animais , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacologia , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Carcinoma/metabolismo , Carcinoma/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HCT116 , Humanos , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/genética , Mesilatos/química , Mesilatos/metabolismo , Mesilatos/farmacologia , Camundongos Nus , Simulação de Acoplamento Molecular , Nitrorredutases/genética , Nitrorredutases/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Livres de Patógenos Específicos , Especificidade por Substrato , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Steroid Biochem Mol Biol ; 161: 54-72, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26924584

RESUMO

Androgen-metabolizing enzymes convert cholesterol, a relatively inert molecule, into some of the most potent chemical messengers in vertebrates. This conversion involves thermodynamically challenging reactions catalyzed by P450 enzymes and redox reactions catalyzed by Aldo-Keto Reductases (AKRs). This review covers the structures of these enzymes with a focus on active site interactions and proposed mechanisms. Due to their role in a number of diseases, particularly in cancer, androgen-metabolizing enzymes have been targets of drug design. Hence we will also highlight how existing knowledge of structure is being used to this end.


Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiesteroide Desidrogenases/metabolismo , Androgênios/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Oxirredutases/metabolismo , 17-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/química , Androgênios/química , Animais , Sistema Enzimático do Citocromo P-450/química , Humanos , Redes e Vias Metabólicas , Modelos Moleculares , Oxirredutases/química
10.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 4): 918-27, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25849402

RESUMO

Type 5 17ß-hydroxysteroid dehydrogenase (17ß-HSD5) is an aldo-keto reductase expressed in the human prostate which catalyzes the conversion of androstenedione to testosterone. Testosterone is converted to 5α-dihydrotestosterone, which is present at high concentrations in patients with castration-resistant prostate cancer (CRPC). Inhibition of 17ß-HSD5 is therefore considered to be a promising therapy for treating CRPC. In the present study, crystal structures of complexes of 17ß-HSD5 with structurally diverse inhibitors derived from high-throughput screening were determined. In the structures of the complexes, various functional groups, including amide, nitro, pyrazole and hydroxyl groups, form hydrogen bonds to the catalytic residues His117 and Tyr55. In addition, major conformational changes of 17ß-HSD5 were observed following the binding of the structurally diverse inhibitors. These results demonstrate interactions between 17ß-HSD5 and inhibitors at the atomic level and enable structure-based drug design for anti-CRPC therapy.


Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Conformação Proteica/efeitos dos fármacos , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Cristalografia por Raios X , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Modelos Moleculares , NADP/metabolismo
11.
Bioorg Med Chem ; 22(3): 967-77, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24411201

RESUMO

Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50∼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure-activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the 'oxyanion hole' of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.


Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Domínio Catalítico , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Ligações de Hidrogênio , Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/metabolismo , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Morfolinas/química , Relação Estrutura-Atividade
12.
FEBS J ; 280(16): 3920-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23773620

RESUMO

Adiponectin is an adipocyte hormone that is predominantly secreted by adipocytes, and has important roles in glucose and lipid homeostasis. Recent studies have shown that adiponectin is also involved in the regulation of many endocrine organs, such as the ovary, adrenal gland, and pituitary. However, its biological role in male testes is largely unexplored. The present findings demonstrate the presence of adeponectin receptors (adiponectin receptor 1 and adiponectin receptor 2) in TM3 cells derived from mouse Leydig cells. Proinflammatory cytokine treatment significantly downregulated mRNA and protein levels of adiponectin receptor 1 and adiponectin receptor 2. However, adiponectin pretreatment successfully inhibited the signaling pathway mediated by proinflammatory cytokines. At the molecular level, we provide compelling evidence that adeponectin achieves this by suppressing nuclear factor-κB activation through promotion of AMP-activated protein kinase phosphorylation. Thus, our data clearly indicate that adiponectin plays a protective role in Leydig cells through its anti-inflammatory actions.


Assuntos
Adiponectina/metabolismo , Citocinas/antagonistas & inibidores , Regulação para Baixo , Células Intersticiais do Testículo/metabolismo , NF-kappa B/antagonistas & inibidores , Receptores de Adiponectina/metabolismo , Transdução de Sinais , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Inativação Gênica , Células Intersticiais do Testículo/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Adiponectina/antagonistas & inibidores , Receptores de Adiponectina/genética , Regulação para Cima
13.
ACS Chem Biol ; 8(5): 1000-8, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23485106

RESUMO

The steroidogenic enzyme 3-ß hydroxysteroid dehydrogenase 2 (3ßHSD2) mediates the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione through both its dehydrogenase and isomerase activities, making it necessary for the protein to undergo a reversible conformational change. We hypothesized that chaperones assist 3ßHSD2 in switching between the conformations to initiate, enhance, and maintain activity. In the presence of the chaperone lauryl maltoside (LM), 3ßHSD2 immediately converted pregnenolone to progesterone, with a 6.4-fold increase in synthesis. Using far-UV circular dichroism (CD), we found that addition of LM increased 3ßHSD2's α-helical content, which over time reverted to control levels, suggesting the formation of a stable but reversible conformation possibly due to hydrophobic interactions of the protein with LM micelles. We also found that LM increased fluorescence resonance energy transfer (FRET) about 11-fold between 3ßHSD2 and fluorescing ANS molecules. This observation supports the idea that detergent(s) act as chaperones to assist 3ßHSD2 in forming stable complexes, which in turn promotes proper folding. Mass spectrometric fingerprinting illustrated that LM incubation resulted in an ordered fragmentation of molecular mass from 39 to 13 kDa, as compared to limited or no proteolysis in the absence of LM. In addition, space-filling modeling demonstrated that 3ßHSD2 association with detergents likely exposed the hydrophobic region, leading to its proteolysis. We conclude that detergents help 3ßHSD2 to refold in order to rejuvenate, contributing to the ability of cells to rapidly produce steroids when needed.


Assuntos
3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Colesterol/metabolismo , Dicroísmo Circular , Detergentes/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glucosídeos/metabolismo , Glucosídeos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Espectrometria de Massas/métodos , Camundongos , Modelos Moleculares , Chaperonas Moleculares/farmacologia , Peso Molecular , Pregnenolona/metabolismo , Progesterona/metabolismo , Conformação Proteica , Dobramento de Proteína
14.
J Med Chem ; 56(6): 2429-46, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23432095

RESUMO

Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP(+)·2'-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.


Assuntos
3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Indometacina/análogos & derivados , Indometacina/farmacologia , Orquiectomia , Neoplasias da Próstata/enzimologia , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Domínio Catalítico , Genes Reporter/genética , Células HeLa , Humanos , Hidroxiprostaglandina Desidrogenases/química , Hidroxiprostaglandina Desidrogenases/metabolismo , Masculino , Modelos Moleculares , Metástase Neoplásica , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Receptores Androgênicos/genética , Especificidade por Substrato
15.
Toxicol Lett ; 215(2): 84-91, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23064086

RESUMO

The objective of the present study is to determine whether di-(2-ethylhexyl) phthalate (DEHP) exposure at adulthood affects regeneration of rat Leydig cells. 90-day-old Long-Evans rats received intraperitoneal injection of 75 mg/kg ethane dimethanesulfonate (EDS) to eliminate mature Leydig cells, and then were randomly divided into 3 groups, in which rats were gavaged with the corn oil (control) or 10 or 750 mg/kg DEHP daily for 35 days. Serum testosterone and luteinizing hormone levels were assessed by RIA, Leydig cell numbers and proliferation rate were evaluated, and the mRNA levels of Leydig cell specific genes were measured by qPCR. Both 10 and 750 mg/kg DEHP treatments increased Leydig cell numbers on day 14, 21 and 35 post-EDS, due to significant increase of the number of Leydig cell precursors from day 14 to 21 post-EDS. However, serum testosterone levels were halved in 10 and 750 mg/kg DEHP groups compared to control on day 35 post-EDS despite the increased Leydig cell numbers. Quantitative PCR showed that Leydig cell specific genes including Lhcgr, Cyp11a1, Hsd3b1, and Insl3 were significantly down-regulated in 750 mg/kg DEHP-treated testes on post-EDS day 21 and beyond. The present study suggests that DEHP increases Leydig cell proliferation but inhibits differentiation during the regeneration of Leydig cells.


Assuntos
Dietilexilftalato/toxicidade , Células Intersticiais do Testículo/efeitos dos fármacos , Testículo/efeitos dos fármacos , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Insulina/química , Insulina/genética , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Hormônio Luteinizante/sangue , Masculino , Proteínas/química , Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Long-Evans , Reação em Cadeia da Polimerase em Tempo Real , Testículo/citologia , Testículo/metabolismo , Testosterona/sangue
16.
PLoS One ; 7(8): e43965, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22937138

RESUMO

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.


Assuntos
3-Hidroxiesteroide Desidrogenases/química , Anti-Inflamatórios não Esteroides/química , Hidroxiprostaglandina Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Anti-Inflamatórios não Esteroides/metabolismo , Ácido Flufenâmico/química , Ácido Flufenâmico/metabolismo , Flurbiprofeno/química , Flurbiprofeno/metabolismo , Hidroxiprostaglandina Desidrogenases/metabolismo , Ibuprofeno/química , Ibuprofeno/metabolismo , Indometacina/química , Indometacina/metabolismo , Ácido Meclofenâmico/química , Ácido Meclofenâmico/metabolismo , Ácido Mefenâmico/química , Ácido Mefenâmico/metabolismo , Naproxeno/química , Naproxeno/metabolismo , Sulindaco/química , Sulindaco/metabolismo , Tolmetino/análogos & derivados , Tolmetino/química , Tolmetino/metabolismo
17.
J Med Chem ; 55(17): 7417-24, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22881866

RESUMO

Human aldo-keto reductases 1C1-1C4 (AKR1C1-AKR1C4) function in vivo as 3-keto-, 17-keto-, and 20-ketosteroid reductases and regulate the activity of androgens, estrogens, and progesterone and the occupancy and transactivation of their corresponding receptors. Aberrant expression and action of AKR1C enzymes can lead to different pathophysiological conditions. AKR1C enzymes thus represent important targets for development of new drugs. We performed a virtual high-throughput screen of a fragment library that was followed by biochemical evaluation on AKR1C1-AKR1C4 enzymes. Twenty-four structurally diverse compounds were discovered with low µM K(i) values for AKR1C1, AKR1C3, or both. Two structural series included the salicylates and the N-phenylanthranilic acids, and additionally a series of inhibitors with completely novel scaffolds was discovered. Two of the best selective AKR1C3 inhibitors had K(i) values of 0.1 and 2.7 µM, exceeding expected activity for fragments. The compounds identified represent an excellent starting point for further hit-to-lead development.


Assuntos
20-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , 20-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/química , Membro C3 da Família 1 de alfa-Ceto Redutase , Hidroxiprostaglandina Desidrogenases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ácido Salicílico/química
18.
Artigo em Inglês | MEDLINE | ID: mdl-22505408

RESUMO

Aldo-keto reductase 1C3 (AKR1C3) is a human enzyme that catalyzes the NADPH-dependent reduction of steroids and prostaglandins. AKR1C3 overexpression is associated with the proliferation of hormone-dependent cancers, most notably breast and prostate cancers. Nonsteroidal anti-inflammatory drugs (NSAIDs) and their analogues are well characterized inhibitors of AKR1C3. Here, the X-ray crystal structure of 3-phenoxybenzoic acid in complex with AKR1C3 is presented. This structure provides useful information for the future development of new anticancer agents by structure-guided drug design.


Assuntos
3-Hidroxiesteroide Desidrogenases/química , Benzoatos/química , Inibidores Enzimáticos/química , Hidroxiprostaglandina Desidrogenases/química , Domínios e Motivos de Interação entre Proteínas , 3-Hidroxiesteroide Desidrogenases/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase , Benzoatos/metabolismo , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica
19.
J Biol Chem ; 287(20): 16609-22, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22437839

RESUMO

Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5ß-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5ß-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3ß-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3ß-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5ß-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3ß-HSD activity on the 5ß-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.


Assuntos
3-Hidroxiesteroide Desidrogenases/química , Substituição de Aminoácidos , Mutação de Sentido Incorreto , NADP/química , Oxirredutases/química , Mutação Puntual , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Humanos , NADP/genética , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , Especificidade por Substrato/genética
20.
Int J Biol Sci ; 8(1): 125-38, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22215981

RESUMO

Molting hormone (ecdysteroid) is one of the most important hormones in insects. The synthesis and inactivation of the ecdysteroid regulate the developmental process of insects. A major pathway of ecdysone inactivation is that ecdysone is converted to 3-dehydroecdysone, and then further to 3-epiecdysone in insects. Two enzymes (ecdysone oxidase: EO and 3DE-3α-reductase) participate in this pathway. In this study, based on the previously characterized cDNAs in Spodoptera littoralis, we cloned and characterized EO and 3DE-3α-reductase genes in the silkworm, Bombyx mori. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and 3DE-3α-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3α-reductase gene, RT-PCR and western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized 3DE-3α-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the 5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer. The functional characterization of the enzymes participating in ecdysone inactivation in the silkworm provides hints for the artificial regulation of the silkworm development and biological control of pests.


Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Bombyx/enzimologia , 3-Hidroxiesteroide Desidrogenases/análise , 3-Hidroxiesteroide Desidrogenases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Clonagem Molecular , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Análise de Sequência de Proteína
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