RESUMO
Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fator de Crescimento de Hepatócito/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Proto-Oncogênicas c-met/genética , Serina Endopeptidases/biossíntese , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Doxiciclina/administração & dosagem , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Animais , Camundongos , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Serina Endopeptidases/genética , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures. Recently, it has been discovered that they act as sensors in the hypoxic response in humans and other animals. Substrate oxidation is coupled to conversion of 2OG to succinate and carbon dioxide. Kinetic, spectroscopic and structural studies are consistent with a consensus mechanism in which ordered binding of (co)substrates enables control of reactive intermediates. Binding of the substrate to the active site triggers the enzyme for ligation of dioxygen to the metal. Oxidative decarboxylation of 2OG then generates the ferryl species thought to mediate substrate oxidation. Structural studies reveal a conserved double-stranded beta-helix core responsible for binding the iron, via a 2His-1carboxylate motif and the 2OG side chain. The rigidity of this core contrasts with the conformational flexibility of surrounding regions that are involved in binding the substrate. Here we discuss the roles of 2OG oxygenases in terms of the generic structural and mechanistic features that render the 2OG oxygenases suited for their functions.
Assuntos
Hipóxia Celular/fisiologia , Ferro/química , Ferro/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Modelos Biológicos , Modelos Químicos , Transdução de Sinais/fisiologia , Animais , Catálise , Humanos , Modelos Moleculares , OxirreduçãoRESUMO
A widely used generic assay for 2-oxoglutarate-dependent oxygenases relies upon monitoring the release of 14CO2 from labeled [1-14C]-2-oxoglutarate. We report an alternative assay in which depletion of 2-oxoglutarate is monitored by its postincubation derivatization with o-phenylenediamine to form a product amenable to fluorescence analysis. The utility of the procedure is demonstrated by assays with hypoxia-inducible factor hydroxylases where it was shown to give results similar to those reported with the radioactive assay, but it is more efficient and readily adapted to a multiwell format. The process should be amenable to the assay of other 2-oxoglutarate-consuming enzymes and to the discovery of inhibitors.
Assuntos
Ácidos Cetoglutáricos/metabolismo , Oxigenases/análise , Ácidos Cetoglutáricos/química , Cinética , Oxigenases de Função Mista , Fenilenodiaminas/química , Pró-Colágeno-Prolina Dioxigenase/análise , Proteínas Repressoras/metabolismo , Espectrometria de Fluorescência , Fatores de Transcrição/metabolismoRESUMO
Humans, like other complex aerobic organisms, possess highly evolved systems for the delivery of dioxygen to all the cells of the body. These systems are regulated since excessive levels of dioxygen are toxic. In animals hypoxia causes an increase in the transcription levels of specific genes, including those encoding for vascular endothelial growth factor and erythropoietin. At the transcriptional level, the hypoxic response is mediated by hypoxia-inducible factor (HIF), an alpha,beta-heterodimeric protein. HIF-beta is constitutively present, but HIF-alpha levels are regulated by dioxygen. Under hypoxic conditions, levels of HIF-alpha rise, allowing its dimerization with HIF-beta and enabling transcriptional activation. Under normoxic conditions both the level of HIF-alpha and its ability to enable transcription are directly controlled by its post-translational oxidation by oxygenases. Hydroxylation of HIF-alpha at either of two conserved prolyl residues enables its recognition by the von Hippel-Lindau tumour suppressor protein which targets it for proteasomal degradation. Hydroxylation of an asparaginyl residue in the C-terminal transactivation domain of HIF-alpha directly prevents its interaction with the coactivator p300 from the transcription complex. Hydroxylation of HIF-alpha is catalysed by members of the iron (II) and 2-oxoglutarate dependent oxygenase family. In humans, three prolyl-hydroxylase isozymes (PHD1-3, for prolyl hydroxylase domain enzymes) and an asparaginyl hydroxylase (FIH, for factor inhibiting HIF) have been identified. Recent studies have identified additional post-translational modifications of HIF-alpha including acetylation and phosphorylation. Modulation of the HIF mediated hypoxic response is of potential use in a wide range of disease states including cardiovascular disease and cancer. Here we review current knowledge of the HIF pathway focusing on its regulation by dioxygen and discussion of potential targets and challenges in attempts to modulate the pathway for medicinal application.
Assuntos
Doenças Cardiovasculares/metabolismo , Proteínas de Ligação a DNA/fisiologia , Neoplasias/metabolismo , Receptores de Hidrocarboneto Arílico/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Doenças Cardiovasculares/terapia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Terapia Genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias/terapia , Oxigênio/fisiologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sensing of ambient dioxygen levels and appropriate feedback mechanisms are essential processes for all multicellular organisms. In animals, moderate hypoxia causes an increase in the transcription levels of specific genes, including those encoding vascular endothelial growth factor and erythropoietin. The hypoxic response is mediated by hypoxia-inducible factor (HIF), an alphabeta heterodimeric transcription factor in which both the HIF subunits are members of the basic helix-loop-helix PAS (PER-ARNT-SIM) domain family. Under hypoxic conditions, levels of HIFalpha rise, allowing dimerization with HIFbeta and initiating transcriptional activation. Two types of dioxygen-dependent modification to HIFalpha have been identified, both of which inhibit the transcriptional response. Firstly, HIFalpha undergoes trans -4-hydroxylation at two conserved proline residues that enable its recognition by the von Hippel-Lindau tumour-suppressor protein. Subsequent ubiquitinylation, mediated by an ubiquitin ligase complex, targets HIFalpha for degradation. Secondly, hydroxylation of an asparagine residue in the C-terminal transactivation domain of HIFalpha directly prevents its interaction with the co-activator p300. Hydroxylation of HIFalpha is catalysed by enzymes of the iron(II)- and 2-oxoglutarate-dependent dioxygenase family. In humans, three prolyl hydroxylase isoenzymes (PHD1-3) and an asparagine hydroxylase [factor inhibiting HIF (FIH)] have been identified. The role of 2-oxoglutarate oxygenases in the hypoxic and other signalling pathways is discussed.
Assuntos
Ferro/fisiologia , Oxigenases de Função Mista/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Drosophila/enzimologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Oxigenases de Função Mista/química , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Helminto/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , 2,2'-Dipiridil/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Células HeLa , Proteínas de Helminto/química , Proteínas de Helminto/genética , Homeostase , Humanos , Hidroxilação , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Indicadores e Reagentes , Ligases/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Isoformas de Proteínas , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed. The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer. The masses of the monomeric species were consistent with the presence of a [2Fe-2S] cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer. Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact. Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment. A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+.
Assuntos
Escherichia coli/enzimologia , Ferro/química , Enxofre/química , Sulfurtransferases/química , Soluções Tampão , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Mononucleotídeo de Flavina/metabolismo , Flavoproteínas , Sulfurtransferases/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biotina/análogos & derivados , Biotina/metabolismo , Clonagem Molecular , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Flavodoxina/química , Flavodoxina/metabolismo , Peso Molecular , Ligação Proteica , Análise de Sequência de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report Mössbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer. The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states. The Mössbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (2Fe-2S)(2+) center.
Assuntos
Proteínas de Bactérias/química , Sulfurtransferases/química , Sítios de Ligação , Escherichia coli , Ferro , Especificidade por Substrato , EnxofreRESUMO
Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.