RESUMO
BACKGROUND: The cardiac glycoside digitoxin preferentially inhibits the growth of breast cancer cells and targets the Erk pathway. Digitoxin alters the expression of genes that mediate calcium metabolism and IAP genes. PURPOSE: Since the optimal treatment for cancer involves the use of agents in combination, we assessed the growth inhibitory effects of digitoxin combined with agents that alter calcium metabolism, thapsigargin, a sarcoplasmic/ER Ca(2+)-ATPase inhibitor, and the statin simvastatin, as well as digitoxin's effect on the IAP pathway of apoptosis. METHODS: To reveal signaling pathways, we treated human cancer cells with digitoxin, alone or combined with thapsigargin or simvastatin, and measured cell growth using the MTT and colony formation assays. We used histology and Western blot analysis of HEK293 cells to assay effects on IAPs. RESULTS: Digitoxin inhibited the growth of breast, colon and ovarian cancer cells. Consistent with an effect on calcium metabolism, digitoxin exhibited synergy with thapsigargin and simvastatin on ER-negative breast cancer cells. Digitoxin activates expression of Erk pathway genes and suppresses expression of IAP genes. The growth inhibitory effects on HEK293 cells are not blocked by the pancaspase inhibitor zVAD-FMK, indicating that digitoxin may act by a caspase independent pathway of apoptosis. Furthermore, digitoxin does not have an effect on XIAP protein, a major anti-apoptotic protein. CONCLUSION: Digitoxin appears to act through the Erk and stress response pathways and is worthwhile to study to prevent and treat cancer. Our findings warn of possible safety issues for cardiac patients who take a combination of digitoxin and statins.
Assuntos
Apoptose/efeitos dos fármacos , Digitoxina/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Sinvastatina/farmacologia , Tapsigargina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Humanos , Transdução de Sinais/efeitos dos fármacosRESUMO
Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin-proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Tioestreptona/farmacologia , Genes Reporter , Células HEK293 , Humanos , Modelos Moleculares , Inibidores de Proteassoma/farmacologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacosRESUMO
Thiostrepton is a natural antibiotic produced by bacteria of Streptomyces genus. We identified Thiostrepton as a strong hit in a cell-based small molecule screen for DIAP1 stability modulators. It was shown previously that Thiostrepton induces upregulation of several gene products in Streptomyces lividans, including the TipAS and TipAL isoforms, and that it can induce apoptotic cell death in human cancer cells. Furthermore, it was suggested that thiostrepton induces oxidative and proteotoxic stress, as inferred from the transcriptional upregulation of stress-related genes and endoplasmic reticulum (ER) stress genes. We used a combination of biochemical and proteomics approaches to investigate the effect of Thiostrepton and other compounds in human cells. Our mass-spectrometry data and subsequent biochemical validation shows that Thiostrepton (and MG-132 proteasome inhibitor) trigger upregulation of heat shock proteins HspA1A, Hsp70, Hsp90α, or Hsp105 in various human cancer cells. We propose a model where Thiostrepton-induced proteasome inhibition leads to accumulation of protein aggregates that trigger a heat shock response and apoptosis in human cancer cells.
Assuntos
Antibacterianos , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Resposta ao Choque Térmico/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Neoplasias , Proteômica/métodos , Streptomyces lividans/química , Tioestreptona , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tioestreptona/química , Tioestreptona/farmacocinética , Tioestreptona/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Identification of central nervous system (CNS) molecules elucidates normal and pathological brain function. Tumor differentiation factor (TDF) is a recently-found protein secreted by the pituitary into the blood. TDF mRNA was detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. However, TDF has an unclear function. It is not known whether TDF is expressed only by pituitary or by other brain regions. It is also not known precisely where TDF is expressed in the brain or which cells produce TDF. Database searching revealed that this molecule shares no homology with any known protein. Therefore, we investigated the distribution of TDF in the rat brain using immunohistochemistry (IHC) and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions. Double-staining for TDF and glial fibrillary acidic protein (GFAP), an astrocyte marker, showed no co-localization. Double-staining for TDF with NeuN, a neuronal marker, showed co-localization. Not all NeuN positive cells were positive for TDF. Western blotting (WB) using NG108 neuroblastoma and GS9L astrocytoma cell lysate revealed TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized in neurons, not in astrocytes. This is the first report of any cellular localization of TDF. TDF may have specific roles as a pituitary-derived hormone and in the CNS, and appears to be produced by distinct CNS neurons, not astroglia.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Hipófise/metabolismo , Animais , Astrócitos/metabolismo , Linhagem Celular Tumoral , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Neurônios/citologia , RatosRESUMO
The structure of FADD has been solved in solution, revealing that the death effector domain (DED) and death domain (DD) are aligned with one another in an orthogonal, tail-to-tail fashion. Mutagenesis of FADD and functional reconstitution with its binding partners define the interaction with the intracellular domain of CD95 and the prodomain of procaspase-8 and reveal a self-association surface necessary to form a productive complex with an activated "death receptor." The identification of a procaspase-specific binding surface on the FADD DED suggests a preferential interaction with one, but not both, of the DEDs of procaspase-8 in a perpendicular arrangement. FADD self-association is mediated by a "hydrophobic patch" in the vicinity of F25 in the DED. The structure of FADD and its functional characterization, therefore, illustrate the architecture of key components in the death-inducing signaling complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Caspases/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Caspase 8 , Caspases/metabolismo , Proteína de Domínio de Morte Associada a Fas , Humanos , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transdução de Sinais , Transfecção , Receptor fas/metabolismoRESUMO
The first inducible Arthrobacter overexpression system, based on the promoter/operator and the repressor of the 6-D-hydroxynicotine oxidase gene of Arthrobacter nicotinovorans, is described here. Nicotine-dependent overproduction and affinity purification of recombinant proteins are presented. The system will allow the production of complex enzymes and genetic complementation studies in Arthrobacter species.
Assuntos
Arthrobacter/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Nicotina/farmacologia , Plasmídeos/genética , Arthrobacter/classificação , Sequência de Bases , Genes Reporter , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Plasmídeos/efeitos dos fármacos , Mapeamento por RestriçãoRESUMO
The death domain and death effector domain are two common motifs that mediate protein-protein interactions between components of cell death signaling complexes. The mechanism by which these domains engage their binding partners has been explored by extensive mutagenesis of two death adaptors, FADD and TRADD, suggesting that a death adaptor can discriminate its intended binding partners from other proteins harboring similar motifs. Death adaptors are found to utilize one of two topologically conserved surfaces for protein-protein interaction, whether that partner is another adaptor or its cognate receptor. These surfaces are topologically related to the interaction between death domains observed in the x-ray crystal structure of the Drosophila adaptor Tube bound to Pelle kinase. Comparing the topology of protein-protein interactions for FADD complexes to TRADD complexes reveals that FADD uses a Tube-like surface in each of its death motifs to engage either CD95 or TRADD. TRADD reverses these roles, employing a Pelle-like surface to interact with either receptor TNFR1 or adaptor FADD. Since death adaptors display a Tube-like or Pelle-like preference for engaging their binding partners, Tube/Pelle-like pairing provides a mechanism for death adaptor discrimination of death receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoptose/fisiologia , Linhagem Celular , Proteína de Domínio de Morte Associada a Fas , Humanos , Células Jurkat , Dados de Sequência Molecular , Mutação , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Receptor fas/metabolismoRESUMO
Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation gamma-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating gamma-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between -48 and -88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the -35 promoter region of the purU-mabO-folD operon.
Assuntos
Arthrobacter/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Óperon/genética , Plasmídeos , Transativadores/genética , Transativadores/metabolismo , Sequência de Aminoácidos , Arthrobacter/fisiologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Mutagênese Insercional , Nicotina/metabolismo , Óperon/fisiologia , Regiões Promotoras Genéticas , Ligação Proteica , RNA Bacteriano/análise , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência , Transativadores/isolamento & purificação , Sítio de Iniciação de Transcrição/fisiologia , Transcrição GênicaRESUMO
Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A Km of 140 microm and a kcat of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation.
Assuntos
Arthrobacter/enzimologia , Nicotina/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Arthrobacter/metabolismo , Sequência de Bases , Western Blotting , Primers do DNA , Flavinas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
Utilization of L-nicotine as growth substrate by Arthrobacter nicotinovorans pAO1 starts with hydroxylation of the pyridine ring at C6. Next, the pyrrolidine ring is oxidized by 6-hydroxy-L-nicotine oxidase, which acts strictly stereo-specific on the L-enantiomer. Surprisingly, L-nicotine also induces the synthesis of a 6-hydroxy-d-nicotine-specific oxidase in the bacteria. Genes of nicotine-degrading enzymes are located on the catabolic plasmid pAO1. The pAO1 sequence revealed that the 6-hydroxy-D-nicotine oxidase gene is flanked by two open reading frames with a similarity to amino acid permeases and a divergently transcribed open reading frame with a similarity to proteins of the tetracycline repressor TetR family. Reverse transcription PCR and primer extension analysis of RNA transcripts isolated from A. nicotinovorans pAO1 indicated that the 6-hydroxy-D-nicotine oxidase gene represents a transcriptional unit. DNA electromobility shift assays established that the purified TetR-similar protein represents the 6-hydroxy-D-nicotine oxidase gene repressor HdnoR and binds to the 6-hydroxy-D-nicotine oxidase gene operator with a Kd of 21 nM. The enantiomers 6-hydroxy-D- and 6-hydroxy-L-nicotine acted in vitro as inducers. In vivo analysis of 6-hydroxy-D-nicotine oxidase gene transcripts from bacteria grown with L- and D-nicotine confirmed this conclusion. The poor discrimination by HdnoR between the 6-hydroxy-L- and 6-hydroxy-D-nicotine enantiomers explains the presence of the 6-hydroxy-D-nicotine-specific enzyme in bacteria grown on L-nicotine.
Assuntos
Arthrobacter/genética , Nicotina/farmacologia , Oxirredutases/genética , Proteínas Repressoras , Transcrição Gênica/efeitos dos fármacos , Sequência de Aminoácidos , Clonagem Molecular , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Família Multigênica , Nicotina/análogos & derivados , Ligação Proteica/efeitos dos fármacos , EstereoisomerismoRESUMO
The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His(6)-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.