RESUMEN
Comprehensive cancer genome studies have revealed genetically-defined subtypes of prostate cancer with distinct truncal driver mutations. Because prostate cancer has been largely seen as a rather uniform disease, the clinical significance of this discovery remained largely obscure. However, recent findings imply distinct biological features and therapeutic vulnerabilities linked to specific truncal mutations. Here we review our current understanding of prostate cancers harboring recurrent point mutations in the ubiquitin ligase adaptor protein SPOP and discuss opportunities for future clinical translation. More specifically, activation of the androgen receptor (AR) signaling emerges as the key oncogenic pathway. SPOP-mutant prostate cancer patients respond to AR inhibition in various clinical settings. Molecular insights on how mutant SPOP promotes tumorigenesis may open more specific therapeutic avenues which, in combination with conventional AR-targeting agents, could improve the outcome of patients with SPOP-mutant prostate cancer.
Asunto(s)
Biomarcadores de Tumor , Mutación , Proteínas Nucleares/genética , Neoplasias de la Próstata/etiología , Proteínas Represoras/genética , Alelos , Toma de Decisiones Clínicas , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Mutación con Pérdida de Función , Masculino , Modelos Biológicos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Atención al Paciente , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/terapia , Conformación Proteica , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.
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Proteínas de Escherichia coli/química , Haptoglobinas/química , Hemoglobinas/química , Hidrocarburos Fluorados/química , Mioglobina/química , Proteínas Represoras/química , Alquilación , Animales , Escherichia coli/química , Caballos , Humanos , Espectrometría de Masas/métodos , Conformación ProteicaRESUMEN
ETV6 is an ETS family transcriptional repressor for which head-to-tail polymerization of its PNT domain facilitates cooperative binding to DNA by its ETS domain. Chromosomal translocations frequently fuse the ETV6 PNT domain to one of several protein tyrosine kinases. The resulting chimeric oncoproteins undergo ligand-independent self-association, autophosphorylation, and aberrant stimulation of downstream signaling pathways, leading to a variety of cancers. Currently, no small-molecule inhibitors of ETV6 PNT domain polymerization are known and no assays targeting PNT domain polymerization have been described. In this study, we developed complementary experimental and computational approaches for identifying such inhibitory compounds. One mammalian cellular approach utilized a mutant PNT domain heterodimer system covalently attached to split Gaussia luciferase fragments. In this protein-fragment complementation assay, inhibition of PNT domain heterodimerization reduces luminescence. A yeast assay took advantage of activation of the reporter HIS3 gene upon heterodimerization of mutant PNT domains fused to DNA-binding and transactivation domains. In this two-hybrid screen, inhibition of PNT domain heterodimerization prevents cell growth in medium lacking histidine. The Bristol University Docking Engine (BUDE) was used to identify virtual ligands from the ZINC8 library predicted to bind the PNT domain polymerization interfaces. More than 75 hits from these three assays were tested by nuclear magnetic resonance spectroscopy for binding to the purified ETV6 PNT domain. Although none were found to bind, the lessons learned from this study may facilitate future approaches for developing therapeutics that act against ETV6 oncoproteins by disrupting PNT domain polymerization.
Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/química , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Bioensayo/métodos , Genes Reporteros , Humanos , Unión Proteica , Relación Estructura-Actividad , Proteína ETS de Variante de Translocación 6RESUMEN
Cleft lip and palate (CL/P) is among the most common congenital malformations and affects 1 in 700 newborns. CL/P is caused by genetic and environmental factors (maternal smoking, alcohol or drug use and others). Many genes and loci were associated with cleft lip/palate but the amount of heterogeneity justifies identifying new causal genes and variants. AHRR (Aryl-Hydrocarbon Receptor Repressor) gene has recently been related to CL/P however, few functional studies analyze the genotypephenotype interaction of AHRR with CL/P. Several studies associate the molecular pathway of AHRR to CL/P which indicates this gene as a functional candidate in CL/P etiology. METHODS: Systematic Literature Review was performed using PUBMED database with the keywords cleft lip, cleft palate, orofacial cleft, AHRR and synonyms. SLR resulted in 37 included articles. RESULTS: AHRR is a positional and functional candidate gene for CL/P. In silico analysis detected interactions with other genes previously associated to CL/P like ARNT and CYP1A1. AHRR protein regulates cellular toxicity through TCDD mediated AHR pathway. Exposure to TCDD in animal embryos is AHR mediated and lead to cleft palate due to palate fusion failure and post fusion rupture. AHRR regulates cellular growth and differentiation, fundamental to lip and palatogenesis. AHRR decreases carcinogenesis and recently a higher tumor risk has been described in CL/P patients and families. AHRR is also a smoking biomarker due to changed methylation sites found in smokers DNA although folate intake may partially revert these methylation alterations. This corroborates the role of maternal smoking and lack of folate supplementation as risk factors for CL/P. CONCLUSION: This research identified the importance of AHRR in dioxin response and demonstrated an example of genetic and environmental interaction, indispensable in the development of many complex diseases.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Labio Leporino/genética , Fisura del Paladar/genética , Proteínas Represoras/genética , Fumar/efectos adversos , Secuencias de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores/metabolismo , Metilación de ADN , Suplementos Dietéticos , Femenino , Ácido Fólico/metabolismo , Estudios de Asociación Genética , Humanos , Recién Nacido , Masculino , Modelos Moleculares , Dominios Proteicos , Isoformas de ARN/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de RiesgoRESUMEN
The phytohormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile) causes protein-protein interactions (PPI) between F-box Protein CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor. A total of 13 JAZ subtypes are encoded in the genome of Arabidopsis thaliana; however, their genetic redundancy obfuscates the individual function of each JAZ. One approach to decipher this redundant signaling network is chemical genetics, using small molecules specific to individual JAZ subtype, for which a reliable and high-throughput screening system of the ligands for all combinations of COI1-JAZs would be indispensable. In this chapter, we describe a fluorescence anisotropy-based quantitative screening system for the ligands of COI1-JAZ co-receptors. Our method is applicable to agonists and antagonists of the COI1-JAZs.
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Descubrimiento de Drogas/métodos , Polarización de Fluorescencia , Proteínas de Plantas/agonistas , Proteínas de Plantas/antagonistas & inhibidores , Proteínas Recombinantes de Fusión , Proteínas Represoras , Factores de Transcripción , Evaluación Preclínica de Medicamentos , Polarización de Fluorescencia/métodos , Ligandos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Represoras/química , Flujo de TrabajoRESUMEN
The NSD family (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1) are histone lysine methyltransferases (HMTases) essential for chromatin regulation. The NSDs are oncoproteins, drivers of a number of tumors and are considered important drug-targets but the lack of potent and selective inhibitors hampers further therapeutic development and limits exploration of their biology. In particular, MMSET/NSD2 selective inhibition is being pursued for therapeutic interventions against multiple myeloma (MM) cases, especially in multiple myeloma t(4;14)(p16.3;q32) translocation that is associated with a significantly worse prognosis than other MM subgroups. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma and remains an incurable malignancy. Here we report the discovery of LEM-14, an NSD2 specific inhibitor with an in vitro IC50 of 132⯵M and that is inactive against the closely related NSD1 and NSD3. LEM-14-1189, a LEM-14 derivative, differentially inhibits the NSDs with in vitro IC50 of 418⯵M (NSD1), IC50 of 111⯵M (NSD2) and IC50 of 60⯵M (NSD3). We propose LEM-14 and derivative LEM-14-1189 as tools for studying the biology of the NSDs and constitute meaningful steps toward potent NSDs therapeutic inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Dominio Catalítico , Diseño de Fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Cinética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Conformación Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Interfaz Usuario-ComputadorRESUMEN
Prohibitin (PHB) was originally isolated and characterized as an anti-proliferative gene in rat liver. The evolutionarily conserved PHB gene encodes two human protein isoforms with molecular weights of ~33 kDa, PHB1 and PHB2. PHB1 and PHB2 belong to the prohibitin domain family, and both are widely distributed in different cellular compartments such as the mitochondria, nucleus, and cell membrane. Most studies have confirmed differential expression of PHB1 and PHB2 in cancers compared to corresponding normal tissues. Furthermore, studies verified that PHB1 and PHB2 are involved in the biological processes of tumorigenesis, including cancer cell proliferation, apoptosis, and metastasis. Two small molecule inhibitors, Rocaglamide (RocA) and fluorizoline, derived from medicinal plants, were demonstrated to interact directly with PHB1 and thus inhibit the interaction of PHB with Raf-1, impeding Raf-1/ERK signaling cascades and significantly suppressing cancer cell metastasis. In addition, a short peptide ERAP and a natural product xanthohumol were shown to target PHB2 directly and prohibit cancer progression in estrogen-dependent cancers. As more efficient biomarkers and targets are urgently needed for cancer diagnosis and treatment, here we summarize the functional role of prohibitin domain family proteins, focusing on PHB1 and PHB2 in tumorigenesis and cancer development, with the expectation that targeting the prohibitin domain family will offer more clues for cancer therapy.
Asunto(s)
Carcinogénesis/patología , Neoplasias/diagnóstico , Neoplasias/terapia , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Animales , Apoptosis , Humanos , Terapia Molecular Dirigida , Prohibitinas , Dominios ProteicosRESUMEN
Three series of novel urushiol derivatives were designed by introducing a hydroxamic acid moiety into the tail of an alkyl side chain and substituents with differing electronic properties or steric bulk onto the benzene ring and alkyl side chain. The compounds' binding affinity toward HDAC8 was screened by Glide docking. The highest-scoring compounds were processed further with molecular docking, MD simulations, and binding free energy studies to analyze the binding modes and mechanisms. Ten compounds had Glide scores of -8.2 to -10.2, which revealed that introducing hydroxy, carbonyl, amino, or methyl ether groups into the alkyl side chain or addition of -F, -Cl, sulfonamide, benzamido, amino, or hydroxy substituents on the benzene ring could significantly increase binding affinity. Molecular docking studies revealed that zinc ion coordination, hydrogen bonding, and hydrophobic interactions contributed to the high calculated binding affinities of these compounds toward HDAC8. MD simulations and binding free energy studies showed that all complexes possessed good stability, as characterized by low RMSDs, low RMSFs of residues, moderate hydrogen bonding and zinc ion coordination and low values of binding free energies. Hie147, Tyr121, Phe175, Hip110, Phe119, Tyr273, Lys21, Gly118, Gln230, Leu122, Gly269, and Gly107 contributed favorably to the binding; and Van der Waals and electrostatic interactions provided major contributions to the stability of these complexes. These results show the potential of urushiol derivatives as HDAC8 binding lead compounds, which have great therapeutic potential in the treatment of various malignancies, neurological disorders, and human parasitic diseases.
Asunto(s)
Catecoles/química , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Represoras/química , Biocatálisis/efectos de los fármacos , Catecoles/metabolismo , Catecoles/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ácidos Hidroxámicos/química , Estructura Molecular , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismo , Electricidad EstáticaRESUMEN
The response regulator PhoP, which is part of the PhoP/PhoQ two-component system, regulates the expression of multiple genes involved in controlling virulence in Salmonella enterica serovar Typhimurium and other species of Gram-negative bacteria. Modulating the phosphorylation-mediated dimerization in the receiver domain may interfere with the transcriptional function of PhoP. In this study, we analyzed the therapeutic potential of the PhoP receiver domain by exploring it as a potential target for drug design. The structural information was then applied to identify the first hit compounds from commercial chemical libraries by combining pharmacophore modelling and docking methods with a GFP (Green Fluorescent Protein)-based promoter-fusion bioassay. In total, one hundred and forty compounds were selected, purchased, and tested for biological activity. Several novel scaffolds showed acceptable potency to modulate the transcriptional function of PhoP, either by enhancing or inhibiting the expression of PhoP-dependent genes. These compounds may be used as the starting point for developing modulators that target the protein-protein interface of the PhoP protein as an alternative strategy against antibiotic resistance.
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Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Diseño de Fármacos , Simulación del Acoplamiento Molecular , Péptidos/química , Proteínas Represoras/química , Activación Transcripcional , Sitios de Unión , Evaluación Preclínica de Medicamentos , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Proteínas Represoras/ultraestructuraRESUMEN
MicroRNAs (miRNAs) are highly conserved â¼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover â¼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.
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Carbazoles/química , MicroARNs/metabolismo , Nitrilos/química , ARN sin Sentido/genética , Compuestos de Vinilo/química , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Proteínas Argonautas/química , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/química , Adhesiones Focales/metabolismo , GTP Fosfohidrolasas/química , Proteínas Homeobox A10 , Proteínas de Homeodominio/química , Humanos , Proteínas de la Membrana/química , Ratones , MicroARNs/química , Proteínas Represoras/químicaRESUMEN
ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC50 values of 2 µg/ml and 1 µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-[(2-hydroxy-1-naphthalenyl) azo] scaffold with 4 molecules exhibiting IC50 ≤ 10 µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC50 of 60 µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC50 values also inhibited M. tuberculosis growth in broth culture with MIC90 ranging from 17.5 µg/ml to 100 µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/química , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Represoras/química , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon, and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.
Asunto(s)
Genes Dominantes , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Mutación/genética , Obesidad/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Mapeo Cromosómico , Dieta , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Hipotálamo/metabolismo , Luciferasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Neuropéptidos/genética , Neuropéptidos/metabolismo , Obesidad/metabolismo , Obesidad/patología , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Receptor de Melanocortina Tipo 4/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Secuenciación del ExomaRESUMEN
AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas de Escherichia coli/metabolismo , Ácidos Nucleicos/farmacología , Oligonucleótidos/farmacología , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Proteínas Represoras/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Escherichia coli/química , Ácidos Nucleicos/síntesis química , Ácidos Nucleicos/química , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Unión Proteica/efectos de los fármacos , ARN/química , Proteínas de Unión al ARN/química , Proteínas Represoras/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PRDM14 is a PR (PRDI-BF1-RIZ1 homologous) domain protein with six zinc fingers and essential roles in genome-wide epigenetic reprogramming. This protein is required for the establishment of germ cells and the maintenance of the embryonic stem cell ground state. In this study, we cloned the full-length cDNA and genomic DNA of the Paralichthys olivaceus prdm14 (Po-prdm14) gene and isolated the 5' regulatory region of Po-prdm14 by whole-genome sequencing. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM14 was homologous to mammalian PRDM14. Results of real-time quantitative polymerase chain reaction amplification (RT-qPCR) and in situ hybridization (ISH) in embryos demonstrated that Po-prdm14 was highly expressed between the morula and late gastrula stages, with its expression peaking in the early gastrula stage. Relatively low expression of Po-prdm14 was observed in the other developmental stages. ISH of gonadal tissues revealed that the transcripts were located in the nucleus of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes. We also presume that the Po-prdm14 transcription factor binding sites and their conserved binding region among vertebrates. The combined results suggest that Po-PRDM14 has a conserved function in teleosts and mammals.
Asunto(s)
Lenguado/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Lenguado/clasificación , Regulación de la Expresión Génica , Orden Génico , Sitios Genéticos , Gónadas/metabolismo , Datos de Secuencia Molecular , Motivos de Nucleótidos , Filogenia , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Alineación de Secuencia , Homología de Secuencia , Sitio de Iniciación de la TranscripciónRESUMEN
By integrating the microarray expression data and a global E. faecalis transcriptional network we identified a sub-network activated by zinc and copper. Our analyses indicated that the transcriptional response of the bacterium to copper and zinc exposure involved the activation of two modules, module I that contains genes implicated in zinc homeostasis, including the Zur transcriptional repressor, and module II containing a set of genes associated with general stress response and basal metabolism. Bacterial exposure to zinc and copper led to the repression of the zinc uptake systems of module I. Upon deletion of Zur, exposure to different zinc and copper conditions induced complementary homeostatic mechanisms (ATPase efflux proteins) to control the intracellular concentrations of zinc. The transcriptional activation of zinc homeostasis genes by zinc and copper reveals a functional interplay between these two metals, in which exposure to copper also impacts on the zinc homeostasis. Finally, we present a new zinc homeostasis model in E. faecalis, positioning this bacterium as one of the most complete systems biology model in metals described to date.
Asunto(s)
Proteínas Bacterianas/genética , Cobre/metabolismo , Enterococcus faecalis/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/genética , Zinc/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/metabolismo , Infecciones por Bacterias Grampositivas/microbiología , Homeostasis , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Alineación de SecuenciaRESUMEN
As key regulators of epigenetic regulation, human histone deacetylases (HDACs) have been identified as drug targets for the treatment of several cancers. The proper recognition of zinc-binding groups (ZBGs) will help improve the accuracy of virtual screening for novel HDAC inhibitors. Here, we developed a high-specificity ZBG-based pharmacophore model for HDAC8 inhibitors by incorporating customized ZBG features. Subsequently, pharmacophore-based virtual screening led to the discovery of three novel HDAC8 inhibitors with low micromole IC50 values (1.8-1.9 µM). Further studies demonstrated that compound H8-A5 was selective for HDAC8 over HDAC 1/4 and showed antiproliferation activity in MDA-MB-231 cancer cells. Molecular docking and molecular dynamic studies suggested a possible binding mode for H8-A5, which provides a good starting point for the development of HDAC8 inhibitors in cancer treatment.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Zinc/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Histona Desacetilasas/química , Humanos , Conformación Proteica , Proteínas Represoras/química , Interfaz Usuario-ComputadorRESUMEN
Protein arginine methyltransferases (PRMTs) are a family of enzymes that can methylate protein arginine residues. PRMTs' substrates include histones and a variety of non-histone proteins. Previous studies have shown that yeast Hmt1 is a type I PRMT and methylates histone H4 arginine 3 and several mRNA-binding proteins. Hmt1 forms dimers or oligomers, but how dimerization or oligomerization affects its activity remains largely unknown. We now report that Hmt1 can methylate histone H3 arginine 2 (H3R2) in vitro. The dimerization but not hexamerization is essential for Hmt1's activity. Interestingly, the methyltransferase activity of Hmt1 on histone H3R2 requires reciprocal contributions from two Hmt1 molecules. Our results suggest an intermolecular trans-complementary mechanism by which Hmt1 dimer methylates its substrates.
Asunto(s)
Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Arginina/química , Dominio Catalítico , Eliminación de Gen , Genes Fúngicos , Histonas/química , Histonas/genética , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The main aim of this study was to evaluate the uptake of E6 mRNA antisense into cervical cancer cells, induced by human papilloma virus (HPV). In this study, the carrier of the antisense was tri-calcium phosphate nanoparticles (TCP NPs) conjugated with dioleoyl phosphatidyl ethanolamine (DOPE) and/or anti-E6 antibody. At first, TCP NPs were synthesized, coated with carboxy-polyethylene glycol, and then conjugated with anti-E6 antibody and/or DOPE by carbodiimide cross-linker. Then, a single stranded DNA, which was complementary (antisense) of E6 mRNA, was attached to each one. Finally, the uptake of conjugated and unconjugated TCP NPs into HelaS3 cells was separately evaluated by Fourier transform infrared spectroscopy, optical microscopy, and fluorescent microscopy. Also, the cytotoxicity of these carriers was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Overall, 4 types of TCP NPs were used in this study, including 1) TCP NPs conjugated with DOPE (TCP NPs/DOPE), 2) TCP NPs conjugated with DOPE and antibody (TCP NPs/DOPE/Anti-E6 Ab), 3) TCP NPs conjugated with antibody (TCP NPs/Anti-E6 Ab), and 4) TCP NPs which not conjugated with DOPE and antibody (unconjugated TCP NPs). Uptake tests showed that although all types of TCP NPs could transfer antisense of E6 mRNA into HelaS3 cells, TCP NPs/DOPE and TCP NPs/DOPE/Anti-E6 Ab had more uptake than TCP NPs/Anti-E6 Ab and unconjugated TCP NPs. Moreover, MTT assay showed that TCP NPs/DOPE was more toxic than TCP NPs/DOPE/Anti-E6 Ab, TCP NPs/Anti-E6 Ab, and unconjugated TCP NPs. It can be concluded that TCP NPs/DOPE/Anti-E6 Ab is a good choice for oligonucleotide delivery, because of higher uptake and less toxicity, compared with other formulations.
Asunto(s)
Anticuerpos/química , Fosfatos de Calcio/química , Nanopartículas/química , Proteínas Oncogénicas Virales/química , Fosfatidiletanolaminas/química , ARN sin Sentido/química , ARN Mensajero/química , Proteínas Represoras/química , Células HeLa , Humanos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Nickel is an essential transition metal for the survival of Helicobacter pylori in the acidic human stomach. The nickel-responsive transcriptional regulator HpNikR is important for maintaining healthy cytosolic nickel concentrations through the regulation of multiple genes, but its complete regulon and role in nickel homeostasis are not well understood. To investigate potential gene targets of HpNikR, ChIP sequencing was performed using H. pylori grown at neutral pH in nickel-supplemented media and this experiment identified HPG27_866 (frpB2) and HPG27_1499 (ceuE). These two genes are annotated to encode a putative iron transporter and a nickel-binding, periplasmic component of an ABC transporter, respectively. In vitro DNA-binding assays revealed that HpNikR binds both gene promoter sequences in a nickel-responsive manner with affinities on the order of â¼10(-7) M. The recognition sites of HpNikR were identified and loosely correlate with the HpNikR pseudo-consensus sequence (TATTATT-N11-AATAATA). Quantitative PCR experiments revealed that HPG27_866 and HPG27_1499 are transcriptionally repressed following growth of H. pylori G27 in nickel-supplemented media, and that this response is dependent on HpNikR. In contrast, iron supplementation results in activation of HPG27_1499, but no impact on the expression of HPG27_866 was observed. Metal analysis of the Δ866 strain revealed that HPG27_866 has an impact on nickel accumulation. These studies demonstrate that HPG27_866 and HPG27_1499 are both direct targets of HpNikR and that HPG27_866 influences nickel uptake in H. pylori.
Asunto(s)
Proteínas Bacterianas/química , Helicobacter pylori/metabolismo , Níquel/química , Proteínas Represoras/química , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , Citosol/metabolismo , ADN/química , ADN Bacteriano , Desoxirribonucleasa I/química , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Metales/química , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN/química , Homología de Secuencia de Ácido NucleicoRESUMEN
By combining NMR spectroscopy, transmission electron microscopy, and circular dichroism we have identified the structural determinants involved in the interaction of green tea catechins with Aß1-42, PrP106-126, and ataxin-3 oligomers. The data allow the elucidation of their mechanism of action, showing that the flavan-3-ol unit of catechins is essential for interaction. At the same time, the gallate moiety, when present, seems to increase the affinity for the target proteins. These results provide important information for the rational design of new compounds with anti-amyloidogenic activity and/or molecular tools for the specific targeting of amyloid aggregates in vivo.