RESUMEN
NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Inhibidores Enzimáticos/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colesterol/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib/química , Clorhidrato de Erlotinib/metabolismo , Clorhidrato de Erlotinib/farmacología , Humanos , Cinética , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , NAD/química , NAD/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transducción de SeñalRESUMEN
Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.
Asunto(s)
Metabolismo Energético , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Tuberculosis/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Catálisis , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad , Isoenzimas , Modelos Moleculares , Oxiesteroles/química , Oxiesteroles/metabolismo , Proteínas Recombinantes , Relación Estructura-Actividad , Tuberculosis/microbiologíaRESUMEN
BACKGROUND: Congenital hemidysplasia with ichthyosiform erythroderma and limb defects also known as CHILD syndrome is an X-linked dominant, male lethal genodermatosis with a prevalence of 1 in 100,000 live births. Mutations in NSDHL gene located at Xq28 potentially impair the function of NAD(P) H steroid dehydrogenase-like protein and is responsible for its pathogenesis. CASE PRESENTATION: The proband was a 9-month-old twin (T2) girl with a healthy twin sister (T1) of Sri Lankan origin born to non-consanguineous parents. She presented with right sided continuous icthyosiform erythroderma and ipsilateral limb defects and congenital hemidysplasia since birth. Notably the child had ipsilateral hand hypoplasia and syndactyly. There were other visceral abnormalities. We performed whole exome sequencing and found a novel heterozygous variant (NSDHL, c.713C > A, p.Thr238Asn). CONCLUSION: We report a novel missense variant in the NSDHL gene that resides in a highly-conserved region. This variant affects the NAD(P) H steroid dehydrogenase-like protein function via reduction in the number of active sites resulting in the CHILD syndrome phenotype and syndactyly.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/genética , Anomalías Múltiples/genética , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Predisposición Genética a la Enfermedad , Eritrodermia Ictiosiforme Congénita/genética , Deformidades Congénitas de las Extremidades/genética , Mutación/genética , Sindactilia/genética , 3-Hidroxiesteroide Deshidrogenasas/química , Animales , Dominio Catalítico , Secuencia Conservada , Femenino , Humanos , Lactante , Mutación Missense/genética , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Cholesterol biosynthesis is a multistep process in mammals that includes the aerobic removal of three methyl groups from the intermediate lanosterol, one from position 14 and two from position 4. During the demethylations at position 4, a 3-ketosteroid reductase catalyses the conversion of both 4-methylzymosterone and zymosterone to 4-methylzymosterol and zymosterol, respectively, restoring the alcoholic function of lanosterol, which is also maintained in cholesterol. Unlike other eukaryotes, mammals also use the same enzyme as an estrone reductase that can transform estrone (E1) into estradiol (E2). This enzyme, named 17ß-hydroxysteroid dehydrogenase type 7 (HSD17B7), is therefore a multifunctional protein in mammals, and one that belongs to both the HSD17B family, which is involved in steroid-hormone metabolism, and to the family of post-squalene cholesterol biosynthesis enzymes. In the present study, a series of known inhibitors of human HSD17B7's E1-reductase activity have been assayed for potential inhibition against 3-ketosteroid reductase activity. Surprisingly, the assayed compounds lost their inhibition activity when tested in HepG2 cells that were incubated with radiolabelled acetate and against the recombinant overexpressed human enzyme incubated with 4-methylzymosterone (both radiolabelled and not). Preliminary kinetic analyses suggest a mixed or non-competitive inhibition on the E1-reductase activity, which is in agreement with Molecular Dynamics simulations. These results raise questions about the mechanism(s) of action of these possible inhibitors, the enzyme dynamic regulation and the interplay between the two activities.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Estradiol/metabolismo , Estrona/metabolismo , Proteína A6 de Unión a Calcio de la Familia S100/antagonistas & inhibidores , Proteína A6 de Unión a Calcio de la Familia S100/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Colesterol/metabolismo , Inhibidores Enzimáticos/química , Estrógenos/metabolismo , Células Hep G2 , Humanos , Conformación ProteicaRESUMEN
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 24â¯h to determine if there were differential effects on steroidogenic gene expression. Testis tissue was exposed to androgen concentrations of 10-12â¯M, 10-9â¯M and 10-6â¯M for 6, 12, 18 or 24â¯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.
Asunto(s)
Andrógenos/toxicidad , Disruptores Endocrinos/toxicidad , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Peces Killi/fisiología , Testículo/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Océano Atlántico , Dihidrotestosterona/toxicidad , Estuarios , Proteínas de Peces/genética , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/crecimiento & desarrollo , Sistema Hipotálamo-Hipofisario/metabolismo , Peces Killi/crecimiento & desarrollo , Cinética , Masculino , Metiltestosterona/toxicidad , Nuevo Brunswick , Especificidad de Órganos , Fosfoproteínas/metabolismo , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genética , Esteroide 17-alfa-Hidroxilasa/metabolismo , Testículo/crecimiento & desarrollo , Testículo/metabolismoRESUMEN
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 1134â¯bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8â¯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 0.1â¯mmolâ¯L-1 IPTG at 15⯰C for 20â¯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 1.5â¯mmolâ¯L-1.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , Proteínas Anfibias/química , Bufo bufo/metabolismo , Deshidroepiandrosterona/química , NAD/química , Pregnenolona/química , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Clonación Molecular , Deshidroepiandrosterona/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Punto Isoeléctrico , Cinética , Peso Molecular , NAD/metabolismo , Sistemas de Lectura Abierta , Filogenia , Pregnenolona/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Pseudomonas/enzimología , 3-Hidroxiesteroide Deshidrogenasas/química , Catálisis , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Cinética , Mutación , Unión Proteica , Conformación Proteica , Esteroides/metabolismo , Relación Estructura-Actividad , Termodinámica , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Arginina/metabolismo , Dihidrotestosterona/análogos & derivados , Dihidrotestosterona/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Andrógenos , Unión Competitiva , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoglicerato Quinasa/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Femenino , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/genética , Procesamiento de Imagen Asistido por Computador , Células MCF-7 , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosfoglicerato Quinasa/química , Fosfoglicerato Quinasa/genética , Proteómica/métodos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Electroforesis Bidimensional Diferencial en GelRESUMEN
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.
Asunto(s)
Suplementos Dietéticos , Epidídimo/efectos de los fármacos , Infertilidad Masculina/prevención & control , Intoxicación por Plomo/prevención & control , Estrés Oxidativo/efectos de los fármacos , Testículo/efectos de los fármacos , Zinc/uso terapéutico , 17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Suplementos Dietéticos/efectos adversos , Epidídimo/metabolismo , Epidídimo/patología , Infertilidad Masculina/etiología , Intoxicación por Plomo/metabolismo , Intoxicación por Plomo/patología , Intoxicación por Plomo/fisiopatología , Peroxidación de Lípido/efectos de los fármacos , Masculino , Tamaño de los Órganos/efectos de los fármacos , Compuestos Organometálicos/antagonistas & inhibidores , Compuestos Organometálicos/toxicidad , Sustancias Protectoras/efectos adversos , Sustancias Protectoras/uso terapéutico , Distribución Aleatoria , Ratas Wistar , Espermatogénesis/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Testículo/metabolismo , Testículo/patología , Testosterona/sangre , Enfermedades Transmitidas por el Agua/metabolismo , Enfermedades Transmitidas por el Agua/patología , Enfermedades Transmitidas por el Agua/fisiopatología , Enfermedades Transmitidas por el Agua/prevención & control , Zinc/efectos adversosRESUMEN
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.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Estrés del Retículo Endoplásmico , Gónadas/metabolismo , Mitocondrias/metabolismo , Fosfatos/metabolismo , Estrés Fisiológico , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Citoplasma/metabolismo , Masculino , Mamíferos/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Fosfoproteínas/metabolismo , Factor de Transcripción CHOP/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
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.
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3-Hidroxiesteroide Deshidrogenasas/metabolismo , Antineoplásicos Alquilantes/uso terapéutico , Benzamidas/uso terapéutico , Carcinoma/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Diseño de Fármacos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Mesilatos/uso terapéutico , Modelos Moleculares , Organofosfonatos/uso terapéutico , Profármacos/uso terapéutico , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Activación Metabólica/efectos de los fármacos , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacología , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Carcinoma/metabolismo , Carcinoma/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Células HCT116 , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/genética , Mesilatos/química , Mesilatos/metabolismo , Mesilatos/farmacología , Ratones Desnudos , Simulación del Acoplamiento Molecular , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Organofosfonatos/farmacología , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacología , Distribución Aleatoria , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Organismos Libres de Patógenos Específicos , Especificidad por Sustrato , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Andrógenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidorreductasas/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/química , Andrógenos/química , Animales , Sistema Enzimático del Citocromo P-450/química , Humanos , Redes y Vías Metabólicas , Modelos Moleculares , Oxidorreductasas/químicaRESUMEN
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.
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3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/química , Conformación Proteica/efectos de los fármacos , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Cristalografía por Rayos X , Humanos , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Modelos Moleculares , NADP/metabolismoRESUMEN
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 (IC50â¼100nM) 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.
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3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Dominio Catalítico , Técnicas de Química Sintética , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Enlace de Hidrógeno , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Concentración 50 Inhibidora , Modelos Moleculares , Estructura Molecular , Morfolinas/química , Relación Estructura-ActividadRESUMEN
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.
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Adiponectina/metabolismo , Citocinas/antagonistas & inhibidores , Regulación hacia Abajo , Células Intersticiales del Testículo/metabolismo , FN-kappa B/antagonistas & inhibidores , Receptores de Adiponectina/metabolismo , Transducción de Señal , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Línea Celular , Ciclooxigenasa 2/química , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Silenciador del Gen , Células Intersticiales del Testículo/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/química , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Receptores de Adiponectina/antagonistas & inhibidores , Receptores de Adiponectina/genética , Regulación hacia ArribaRESUMEN
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.
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3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Colesterol/metabolismo , Dicroismo Circular , Detergentes/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Glucósidos/metabolismo , Glucósidos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Células Intersticiales del Testículo/efectos de los fármacos , Células Intersticiales del Testículo/metabolismo , Masculino , Espectrometría de Masas/métodos , Ratones , Modelos Moleculares , Chaperonas Moleculares/farmacología , Peso Molecular , Pregnenolona/metabolismo , Progesterona/metabolismo , Conformación Proteica , Pliegue de ProteínaRESUMEN
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.
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3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Indometacina/análogos & derivados , Indometacina/farmacología , Orquiectomía , Neoplasias de la Próstata/enzimología , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Dominio Catalítico , Genes Reporteros/genética , Células HeLa , Humanos , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Masculino , Modelos Moleculares , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/cirugía , Receptores Androgénicos/genética , Especificidad por SustratoRESUMEN
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.
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Dietilhexil Ftalato/toxicidad , Células Intersticiales del Testículo/efectos de los fármacos , Testículo/efectos de los fármacos , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Insulina/química , Insulina/genética , Células Intersticiales del Testículo/citología , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/sangre , Masculino , Proteínas/química , Proteínas/genética , ARN Mensajero/química , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Long-Evans , Reacción en Cadena en Tiempo Real de la Polimerasa , Testículo/citología , Testículo/metabolismo , Testosterona/sangreRESUMEN
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.