RESUMO
Fibroblasts within the mammary tumor microenvironment are active participants in carcinogenesis mediating both tumor initiation and progression. Our group has previously demonstrated that genetic loss of phosphatase and tensin homolog (PTEN) in mammary fibroblasts induces an oncogenic secretome that remodels the extracellular milieu accelerating ErbB2-driven mammary tumor progression. While these prior studies highlighted a tumor suppressive role for stromal PTEN, how the adjacent normal epithelium transforms in response to PTEN loss was not previously addressed. To identify these early events, we have evaluated both phenotypic and genetic changes within the pre-neoplastic mammary epithelium of mice with and without stromal PTEN expression. We report that fibroblast-specific PTEN deletion greatly restricts mammary ductal elongation and induces aberrant alveolar side-branching. These mice concomitantly exhibit an expansion of the mammary epithelial stem cell (MaSC) enriched basal/myoepithelial population and an increase in in vitro stem cell activity. Further analysis revealed that NOTCH signaling, specifically through NOTCH3, is diminished in these cells. Mechanistically, JAGGED-1, a transmembrane ligand for the NOTCH receptor, is downregulated in the PTEN-null fibroblasts leading to a loss in the paracrine activation of NOTCH signaling from the surrounding stroma. Reintroduction of JAGGED-1 expression within the PTEN-null fibroblasts was sufficient to abrogate the observed increase in colony forming activity implying a direct role for stromal JAGGED-1 in regulation of MaSC properties. Importantly, breast cancer patients whose tumors express both low stromal JAG1 and low stromal PTEN exhibit a shorter time to recurrence than those whose tumors express low levels of either alone suggesting similar stromal signaling in advanced disease. Combined, these results unveil a novel stromal PTEN-to-JAGGED-1 axis in maintaining the MaSC niche, and subsequently inhibiting breast cancer initiation and disease progression.
Assuntos
Células Epiteliais/citologia , Proteína Jagged-1/metabolismo , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Animais/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Células-Tronco/citologia , Células 3T3 , Animais , Fibroblastos Associados a Câncer/metabolismo , Proliferação de Células , Células Epiteliais/patologia , Feminino , Humanos , Proteína Jagged-1/deficiência , Proteína Jagged-1/genética , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/metabolismo , Receptor Notch3/metabolismo , Transdução de Sinais , Células Estromais/citologia , Microambiente TumoralRESUMO
This corrects the article DOI: 10.1038/onc.2016.383.
RESUMO
Recent studies have demonstrated that arachidonic acid (AA) may serve as an important signal that blocks cell proliferation of certain neoplastic cells. The current study was conducted to determine whether disruption of AA homeostasis influences breast cancer cell proliferation and death. Initial experiments revealed that inhibition of AA remodeling through membrane phospholipids by inhibitors of the enzyme, coenzyme A-independent transacylase (CoA-IT), attenuates the proliferation of the estrogen receptor-negative, MDA-MB-231, and estrogen receptor-positive, MCF-7 breast cancer cell lines. This growth inhibition was accompanied by a marked accumulation of AA in both free fatty acid and triglyceride forms, a marker of intracellular AA stress within mammalian cells. Cell cycle synchronization experiments revealed that the CoA-IT inhibitor, SB-98625, blocked MDA-MB-231 cell replication in early to mid G1 phase. Time-lapse video microscopy, used to observe the changes in cell morphology associated with apoptosis, indicated that SB-98625 treatment induced early rounding and occasional blebbing but not late apoptotic events, blistering, and lysis. The cyclooxygenase inhibitors, NS-398 and indomethacin, were found to be less potent blockers of cell proliferation and poor inducers of cellular AA accumulation than CoA-IT inhibitors in these breast cancer cell lines. Finally, AA provided exogenously blocked the proliferation of MCF-7 cells, and this effect could be attenuated in MCF-7 cells overexpressing the glutathione peroxidase gene, GSHPx-1. Taken together, these experiments suggest that disruption of AA remodeling in a manner that increases intracellular AA may represent a novel therapeutic strategy to reduce cancer cell proliferation and that an oxidized AA metabolite is likely to mediate this effect.
Assuntos
Aciltransferases/antagonistas & inibidores , Apoptose , Ácido Araquidônico/metabolismo , Neoplasias da Mama/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Aciltransferases/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ácidos Graxos não Esterificados/farmacologia , Humanos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has become a potential target for the prevention and treatment of breast cancer. However, recent in vitro and in vivo studies have raised the question of whether activation of PPARgamma leads to the promotion or reduction of tumor formation. Studies using several cancer cell lines, animal models, and a variety of PPARgamma agonists have shown discordant results, including changes in cellular proliferation, differentiation, and apoptosis of cancer cells and tumors. METHODS: We studied the effects of low-, moderate-, and high-dose treatment of the PPARgamma ligands 15-deoxy-delta1214 prostaglandin J2 (15dPGJ2) and troglitazone (TGZ) on parameters of cell growth, differentiation, and apoptosis in the epithelial breast cancer cell line MDA-MB-231. RESULTS: The biologic effects of these compounds depend largely on ligand concentration and the degree of PPARgamma activation. For example, low concentrations of 15dPGJ2 (<2.5 microM) and TGZ (<5 microM) increased cellular proliferation, but concentrations of 15dPGJ2 > or = 10 microM and of TGZ at 100 microM blocked cell growth. TGZ (100 microM) slowed cell cycle progression, and 15dPGJ2 (10 microM) caused an S-phase arrest in the cell cycle and induced morphological characteristics consistent with apoptosis. Expression of CD36, a marker of differentiation in these cells, was induced by 2.5 microM 15dPGJ2 or 5 to 100 microM TGZ. However, higher concentrations of 15dPGJ2 did not alter CD36 expression. Transcriptional activation studies demonstrated that 15dPGJ2 is a more potent PPARgamma ligand than TGZ. Regardless of the ligand used, though, low transcriptional activation correlated with an increased cellular proliferation, whereas higher levels of activation correlated with cell cycle arrest and apoptosis. CONCLUSIONS: PPARgamma activation induces several important and seemingly opposite changes in neoplastic cells, depending on the magnitude of PPARgamma activation. These data may explain, at least in part, some of the discordant results previously reported.
Assuntos
Neoplasias da Mama/patologia , Cromanos/farmacologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Ativação Transcricional/efeitos dos fármacos , Troglitazona , Células Tumorais CultivadasRESUMO
PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten-miR-320-Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Microambiente Tumoral/genética , Animais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Fibroblastos/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/metabolismo , Proteína Proto-Oncogênica c-ets-2/genética , Proteína Proto-Oncogênica c-ets-2/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismoRESUMO
Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.
Assuntos
Centrossomo/metabolismo , Ciclina D1/fisiologia , Quinase 4 Dependente de Ciclina/fisiologia , Genes ras/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Apoptose/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Centrossomo/patologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Doença da Mama Fibrocística/genética , Doença da Mama Fibrocística/metabolismo , Genes ras/genética , Humanos , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Quinases Relacionadas a NIMA , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the rate-limiting step, the conversion of nitrate to nitrite, in nitrate assimilation. Initial rate studies of nitrate reductase activity, performed under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substrate inhibition at NADH concentrations below 300 microM and NO3- concentrations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (Vmax = 9.2 mumol NADH/min/nmol heme and Km = 2.3 microM) as the physiological electron donor but could also utilize alpha-NADH (Vmax = 5.6 mumol NADH/min/nmol heme and Km = 131 microM) and NADPH (Vmax = 0.6 mumol NADPH/min/nmol heme and Km = 910 microM) though with significantly decreased efficiency. Examination of various NADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleotide (NHDH) was used most efficiently (Vmax = 9.3 mumol NHDH/min/nmol heme and Km = 7.9 microM), while reduced nicotinamide mononucleotide (NMNH) was utilized least efficiently (Vmax = 0.07 mumol NMNH/min/nmol heme and Km = 676 microM). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result in the most significant decreases in Vmax, indicating poor reducing substrates. Product inhibition studies indicated both NAD+ (Ki = 2.2 mM) and NADP+ (Ki = 10.5 mM) to be competitive inhibitors of Chlorella NR. A variety of NAD+ analogs were also determined to act as competitive inhibitors with varying degrees of efficiency. 3-Pyridinealdehyde adenine dinucleotide was the most efficient inhibitor (Ki = 0.74 mM) while nicotinamide was the least efficient (Ki = 18.1 mM). Overall, changing substituents on the nicotinamide ring or its complete deletion produced the most effective inhibitors compared to NAD+. In contrast, changes in the adenine or ribose moieties produced less effective inhibitors when compared to NAD+. These results represent the most comprehensive analysis of the effect of modifications of the physiological reductant (NADH) and product (NAD+) on nitrate reductase activity.
Assuntos
Chlorella/enzimologia , NAD/análogos & derivados , Nitrato Redutases/antagonistas & inibidores , Nitrato Redutases/metabolismo , Ligação Competitiva , Cinética , NADP/análogos & derivados , Oxirredução , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The low molecular weight "blue" copper protein, azurin, has been purified from Pseudomonas putida (NCIB 9869) to homogeneity using various chromatographic techniques including reverse-phase HPLC. The amino acid sequence of the N-terminus of the reduced and carboxymethylated protein yielded a single sequence corresponding to AECKV. The complete sequence, comprising 128 amino acid residues with a C-terminal sequence corresponding to TVTLK, was determined from the peptides obtained from a Staphylococcus aureus V8 digest of the protein and confirmed using peptides obtained following cyanogen bromide and endoprotease Asp-N digests. The amino acid sequence contained three cysteine residues at positions 3, 26, and 112, was devoid of tryptophan, and showed closest similarity (90% identical residues) to the previously determined sequence of azurin isolated from Pseudomonas fluorescens biotype B [Ambler, R.P. (1971) in Developpements Recents Dans L'Etude Chimique De La Structures Des proteins (Preverio, A., Pechere, J.-F., and Coletti-Preverio, M.-A., Eds.), pp. 289-305, INSERM, Paris]. Examination of the complete sequence indicated P. putida azurin contained unique Asp and Ala residues at positions 19 and 21, respectively, that have not been found in any other azurin sequence.
Assuntos
Azurina/química , Pseudomonas putida/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dados de Sequência MolecularRESUMO
A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus. The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis. Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups. The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains. NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence. The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively. This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein. The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components.
Assuntos
Citocromos b5/genética , Nitrato Redutases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia em Gel , Clonagem Molecular , Citocromos b5/isolamento & purificação , Citocromos b5/metabolismo , DNA Recombinante , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Imunoquímica , Cinética , Fígado/enzimologia , Dados de Sequência Molecular , Nitrato Redutase (NADH) , Nitrato Redutases/isolamento & purificação , Nitrato Redutases/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise Espectral , Verduras/enzimologiaRESUMO
Cytochrome c has been purified to homogeneity from alligator liver (Alligator mississipiensis) using aluminum sulfate precipitation, CM-cellulose and gel-filtration chromatography, and reverse-phase HPLC. The protein exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an approximate molecular weight of 12,000 Da. Oxidized and reduced visible spectra yielded maxima at 408 (tau) nm and 315 (delta), 415 (tau), 520 (beta), and 550 (alpha) nm, respectively, while potentiometric titrations in the presence of dye-mediators yielded an Eo of +265 mV. N-terminal analysis of the protein yielded no sequence which indicates a blocked residue. A combination of amino acid sequencing, using peptides obtained from Staphylococcus aureus V8 protease, endoproteinase Lys-C, and CNBr digests of the protein and total amino acid analyses, using equine and avian cytochromes c as standards yielded the primary sequence GDVEKGKKIFVQKCAQCHTVEKGGKHKTGPNLHGLIGRKTGQAPGFSYTEANKNKGITWGEETLMEYLE NPKKYIPGTKMIFAGIKKKPERADLIAYLKEATSN. Comparison with sequences of other cytochromes c indicated the closest similarity to cytochrome c from snapping turtle (Chelydra serpentina) with substitutions at five positions corresponding to residues 32 (His-->Asn), 44 (Glu-->Pro), 89 (Ala-->Pro), 100 (Asp-->Glu), and 104 (Lys-->Asn), respectively. The presence of Pro and Asn residues at positions 89 and 104, respectively, are unique to alligator cytochrome c.
Assuntos
Jacarés e Crocodilos , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Grupo dos Citocromos c/isolamento & purificação , Heme/metabolismo , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Análise de Sequência , Homologia de Sequência de Aminoácidos , Espectrofotometria , Tartarugas/genéticaRESUMO
Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.
Assuntos
Flavinas/química , NAD/farmacologia , Nitrato Redutases/química , Difosfato de Adenosina/farmacologia , Eletroquímica , Eletrodos , Flavina-Adenina Dinucleotídeo/química , Grafite , Cinética , Cloreto de Magnésio/farmacologia , Mutagênese Sítio-Dirigida , NAD/análogos & derivados , Nitrato Redutase , Nitrato Redutases/genética , Oxirredução , Proteínas Recombinantes/química , Spinacia oleracea/enzimologia , Relação Estrutura-AtividadeRESUMO
Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.
Assuntos
Cisteína/química , Flavinas/química , Mutagênese Sítio-Dirigida , Nitrato Redutases/antagonistas & inibidores , Nitrato Redutases/química , Spinacia oleracea , Reagentes de Sulfidrila/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Ferricianetos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , Dados de Sequência Molecular , NAD/metabolismo , NAD/farmacologia , Nitrato Redutase (NADH) , Nitrato Redutases/genética , Oxirredução , Espectrofotometria , Relação Estrutura-AtividadeRESUMO
The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.
Assuntos
Flavina-Adenina Dinucleotídeo/análise , Nitrato Redutases/metabolismo , Spinacia oleracea/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cromatografia de Afinidade , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Brometo de Cianogênio , Primers do DNA , Escherichia coli , Cinética , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Nitrato Redutase (NADH) , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Plasmídeos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Serina Endopeptidases , Espectrofotometria , Especificidade por SubstratoRESUMO
The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the protein's composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.
Assuntos
Proteínas Ferro-Enxofre , Oxirredutases/química , Rhodobacter sphaeroides/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.