RESUMO
ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46 kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.
Assuntos
Cobre/metabolismo , NADH NADPH Oxirredutases/metabolismo , Periodicidade , Sítios de Ligação/genética , Western Blotting , Escherichia coli , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , NAD/metabolismo , Oligonucleotídeos/genética , Oxigênio/metabolismo , Fenantrolinas , Isomerases de Dissulfetos de Proteínas/metabolismo , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Zinco/metabolismoRESUMO
A proteomics approach with detection on western blots using an S-peptide tagged pan-tNOX (ENOX2) recombinant (scFv) antibody followed by alkaline phosphatase-linked anti S has revealed a family of more than 20 ENOX2 isoforms of varying molecular weights (34 to 94 kDa) and mostly of low isoelectric points (4.6 +/- 0.7) based on serum analysis. Different isoforms characterize cancers of different tissue origins indicative of both cancer presence and tissue site of origin. ENOX2 proteins are cancer-associated and differ from constitutive (CNOX or ENOX1) proteins primarily by the absence of a drug binding site to which the cancer-specific scFv is directed. All are located on the cell surface where they function both as terminal oxidases for plasma membrane electron transport and carry out protein disulfide-thiol interchange. These proteins are shed into the blood and can also be found in urine. The tNOX isoform technology is under development as a clinical aid to identify unknown or uncertain primary cancers, evaluation of metastatic spread in post surgery patients, monitoring remission following cessation of therapy and for early diagnosis in at-risk populations.
Assuntos
NADH NADPH Oxirredutases/análise , NADH NADPH Oxirredutases/metabolismo , Neoplasias/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias do Colo/metabolismo , Eletroforese em Gel Bidimensional , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Linfoma não Hodgkin/metabolismo , Masculino , Melanoma/metabolismo , NADH NADPH Oxirredutases/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias da Próstata/metabolismo , Isoformas de Proteínas/genética , Neoplasias do Colo do ÚteroRESUMO
This study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin +DTT + recombinant his-tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2-dependent insulin reduction by high density SDS-PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide-thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX-catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS-PAGE.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Ditiotreitol/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Insulina/metabolismo , NADH NADPH Oxirredutases/genética , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/metabolismoRESUMO
Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.
Assuntos
Isoflavonas/farmacologia , NADH NADPH Oxirredutases/antagonistas & inibidores , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Coenzimas/metabolismo , Dissulfetos/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Isoflavonas/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução/efeitos dos fármacos , Ligação Proteica , RNA Interferente Pequeno/genética , Compostos de Sulfidrila/metabolismo , Transfecção , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface.
Assuntos
Trifosfato de Adenosina/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Lipídeos/síntese química , Microscopia Eletrônica , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.
Assuntos
Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Neoplasias/sangue , Animais , Anticorpos Monoclonais/química , Apoptose , Western Blotting , Dissulfetos/química , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Epitopos , Feminino , Células HeLa , Humanos , Hibridomas/metabolismo , Imuno-Histoquímica , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Oxigênio/metabolismo , Testes de Precipitina , Fatores de TempoRESUMO
Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Membrana Celular/enzimologia , Doxorrubicina/farmacologia , Fígado/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/isolamento & purificação , Animais , Ânions , Western Blotting , Catepsina D/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Células K562 , Microscopia de Fluorescência , NAD/metabolismo , Oxirredução , Testes de Precipitina , Ratos , Células Tumorais CultivadasRESUMO
NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.