RESUMO
Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados , Polissacarídeos/química , Cromatografia Líquida , Neoplasias/terapia , Mapeamento de Peptídeos , Espectrometria de Massas em TandemRESUMO
Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.
Assuntos
Antígeno CA-19-9/imunologia , Imunoconjugados/imunologia , Imagem Multimodal/métodos , Neoplasias Pancreáticas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Desferroxamina/química , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes/química , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos Knockout , Camundongos Nus , Microscopia de Fluorescência , Estrutura Molecular , Neoplasias Pancreáticas/diagnóstico , Radioisótopos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Distribuição Tecidual , Transplante Heterólogo , Zircônio/químicaRESUMO
The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.
Assuntos
Imunoconjugados/química , Imagem Óptica/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Neoplasias Colorretais/diagnóstico , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/química , Galactose/química , Humanos , Imunoconjugados/farmacocinética , Camundongos , Imagem Multimodal , Neoplasias Experimentais/diagnóstico , Polissacarídeos/química , Polissacarídeos/imunologia , Radioisótopos , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , ZircônioRESUMO
An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.
Assuntos
Anticorpos/metabolismo , Desferroxamina/metabolismo , Marcação por Isótopo , Compostos Organometálicos/metabolismo , Zircônio/metabolismo , beta-Galactosidase/metabolismo , Animais , Anticorpos/química , Sítios de Ligação , Química Click , Desferroxamina/química , Humanos , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Compostos Organometálicos/química , Compostos Organometálicos/farmacocinética , Polissacarídeos/química , Polissacarídeos/metabolismo , Células Tumorais Cultivadas , Zircônio/química , beta-Galactosidase/químicaRESUMO
The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Translocadoras de Prótons/fisiologia , Bactérias/enzimologia , Potenciais da Membrana , Modelos Moleculares , Proteínas Motores Moleculares , Conformação Proteica , Dobramento de Proteína , ATPases Translocadoras de Prótons/química , RotaçãoRESUMO
High content screening (HCS)-based multiparametric measurements are very useful in early toxicity testing and safety assessment during drug development, and useful in evaluating the impact from new food supplements and environmental toxicants. Mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phosphoplipidosis, and steatosis are a few of the important markers routinely studied for the assessment of drug-induced liver injury and toxicity. Mitochondrial dysfunction leads to oxidative stress and cell death. Liver injury from drug-induced phospholipidosis and steatosis is routinely studied in hepatotoxicity investigations to determine the risk factors and fate of drugs or chemical compounds as some drugs can lead to defects in lipid metabolism and accumulation of lipids in lysosomes. In this chapter, we describe fluorescent reagents and the protocols for the measurement of various parameters such as mitochondrial membrane potential, plasma membrane permeability, oxidative stress, phospholipidosis, and steatosis using high content imaging-based methodologies and instrumentation.
Assuntos
Ensaios de Triagem em Larga Escala , Processamento de Imagem Assistida por Computador , Imagem Molecular , Animais , Linhagem Celular , Sobrevivência Celular , Descoberta de Drogas , Humanos , Metabolismo dos Lipídeos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Imagem Molecular/métodos , Estresse Oxidativo , Fosfolipídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Testes de ToxicidadeRESUMO
Mitochondrial dysfunction has been shown to be a pharmacotoxicological response to a variety of currently-marketed drugs. In order to reduce attrition due to mitochondrial toxicity, high throughput-applicable screens are needed for early stage drug discovery. We describe, here, a set of immunocapture based assays to identify compounds that directly inhibit four of the oxidative phosphorylation (OXPHOS) complexes: I, II, IV, and V. Intra- and inter-assay variation were determined and specificity tested by using classical mitochondrial inhibitors. Twenty drugs, some with known mitochondrial toxicity and others with no known mitochondrial liability, were studied. Direct inhibition of one or more of the OXPHOS complexes was identified for many of the drugs. Novel information was obtained for several drugs including ones with previously unknown effects on oxidative phosphorylation. A major advantage of the immunocapture approach is that it can be used throughout drug screening from early compound evaluation to clinical trials.
Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Desacopladores/toxicidade , Animais , Anticorpos Monoclonais , Bovinos , Avaliação Pré-Clínica de Medicamentos/métodos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/toxicidade , Imunoquímica , Técnicas In Vitro , Oligomicinas/toxicidade , Cianeto de Potássio/toxicidade , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Rotenona/toxicidade , Succinato Citocromo c Oxirredutase/antagonistas & inibidores , Succinato Citocromo c Oxirredutase/metabolismo , Tenoiltrifluoracetona/toxicidadeRESUMO
Comparative analysis of cytoplasmic organelles in a variety of tumors relative to normal tissues generally reveals a strong diminution in mitochondrial content and in oxidative phosphorylation capacity. However, little is known about what triggers these modifications and whether or not they are physiologically reversible. We hypothesized that energy substrate availability could play an important role in this phenomenon. The physiological effects of a change in substrate availability were examined on a human cancer cell line (HeLa), focusing specifically on its ability to use glycolysis versus oxidative phosphorylation, and the effect that energy substrate type has on mitochondrial composition, structure, and function. Changes in oxidative phosphorylation were measured in vivo by a variety of techniques, including the use of two novel ratiometric green fluorescent protein biosensors, the expression level of oxidative phosphorylation and some glycolytic enzymes were determined by Western blot, mitochondrial DNA content was measured by real-time PCR, and mitochondrial morphology was monitored by both confocal and electron microscopy. Our data show that the defective mitochondrial system described in cancer cells can be dramatically improved by solely changing substrate availability and that HeLa cells can adapt their mitochondrial network structurally and functionally to derive energy by glutaminolysis only. This could also provide an explanation for the enhancement of oxidative phosphorylation capacity observed after tumor regression or removal. Our work demonstrates that the pleomorphic, highly dynamic structure of the mitochondrion can be remodeled to accommodate a change in oxidative phosphorylation activity. We compared our finding on HeLa cells with those for nontransformed fibroblasts to help distinguish the regulatory pathways.
Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neoplasias/metabolismo , Neoplasias/ultraestrutura , Técnicas Biossensoriais , Divisão Celular/fisiologia , Meios de Cultura , DNA Mitocondrial/metabolismo , Metabolismo Energético , Galactose/administração & dosagem , Galactose/metabolismo , Glucose/deficiência , Glucose/metabolismo , Glicólise , Células HeLa , Hexoquinase/biossíntese , Hexoquinase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/metabolismo , Oxirredução , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologiaRESUMO
A series of novel water-soluble PEGylated dibenzosilole-based conjugated polymers were prepared as ultra-bright fluorescent labels for biomolecules. Due to their superior solubility and brightness, antibody conjugates labeled with functionalized polymers showed significantly enhanced signal and sensitivity relative to traditional fluorophores in functional flow cytometry applications.
Assuntos
Anticorpos/química , Corantes Fluorescentes/química , Compostos de Organossilício/química , Polietilenoglicóis/química , Polímeros/química , Água/química , Técnicas Biossensoriais , Citometria de Fluxo , SolubilidadeRESUMO
The availability of monoclonal antibodies (mAbs) against the proteins of the oxidative phosphorylation chain (OXPHOS) and other mitochondrial components facilitates the analysis and ultimately the diagnosis of mitochondrially related diseases. mAbs against each of the five complexes and pyruvate dehydrogenase (PDH) are the basis of a rapid and simple immunocytochemical approach [Hanson, B.J., Capaldi, R.A., Marusich, M.F. and Sherwood, S.W., J. Histochem. Cytochem. 50 (2002) 1281-1288]. This approach can be used to detect if complexes have altered assembly in mitochondrial disease due to mutations in nuclear encoded genes, such as in Leigh's disease, or in mitochondrially encoded genes, e.g., MELAS. Other mAbs have recently been obtained that can immunocapture each of the five OXPHOS complexes, PDH and the adenine nucleotide translocase (ANT) from very small amounts of tissue such as that obtained from cell culture or needle biopsies from patients. When adapted to a 96-well plate format, these mAbs allow measurement of the specific activity of each of the mitochondrial components individually and analysis of their subunit composition and state of posttranslational modification. The immunocapture protocol should be useful not only in the analysis of genetic mitochondrial diseases but also in evaluating and ultimately diagnosing late-onset mitochondrial disorders including Parkinson's disease, Alzheimer's disease, and late-onset diabetes, which are thought to result from accumulated oxidative damage to mitochondrial proteins such as the OXPHOS chain.
Assuntos
Anticorpos Monoclonais , Doenças Mitocondriais/diagnóstico , Proteínas/análise , Proteômica/métodos , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Imuno-Histoquímica/métodos , Translocases Mitocondriais de ADP e ATP/análise , Translocases Mitocondriais de ADP e ATP/imunologia , Doenças Mitocondriais/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/imunologia , Complexo Piruvato Desidrogenase/análise , Complexo Piruvato Desidrogenase/imunologiaRESUMO
The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.8-microm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.
Assuntos
Citoesqueleto/fisiologia , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Técnicas Biossensoriais , Citoesqueleto/química , DNA Mitocondrial/química , Proteínas de Fluorescência Verde , Humanos , Membranas Intracelulares/química , Proteínas Luminescentes , Proteínas de Membrana/química , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Mitose , Complexo Piruvato Desidrogenase/química , Fase S , Células Tumorais CultivadasRESUMO
Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) consists of at least 46 subunits. Phosphorylation of the 42-kDa subunit NDUFA10 was recently reported using a novel phosphoprotein stain [Schulenberg et al. (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J. Biol. Chem. 278, 27251]. Two smaller Complex I phosphoproteins, ESSS and MWFE, and their sites of modification, have since been determined [Chen et al. (2004) The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. 279, 26036]. Here we identify the site of phosphorylation in NDUFA10 from bovine heart mitochondria by tandem mass spectrometry. A single phosphopeptide spanning residues 47-60 was identified and confirmed by synthesis to be (47)LITVDGNICSGKpSK(60), establishing serine-59 as the site of phosphorylation.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The phosphorylation of mitochondrial proteins is pivotal to the regulation of respiratory activity in the cell and to signaling pathways leading to apoptosis, as well as for other vital mitochondrial processes. A number of protein kinases have been identified in mitochondria but the physiological substrates for many of these remain unknown or poorly understood. By necessity, most studies of mitochondrial phosphoproteins to date have been conducted using in vitro incorporation of 32P. However, proteins that are highly phosphorylated from in situ reactions are not necessarily detected by this approach. In this study, a new small molecule fluorophore has been employed to characterize steady-state levels of mitochondrial phosphoproteins. The dye is capable of sensitive detection of phosphorylated amino acid residues in proteins separated by gel electrophoresis. When the fluorescent dye is combined with a total protein stain in a sequential gel staining procedure, the phosphorylated proteins can be visualized in the same gel as the total proteins. To optimize resolution of the proteins in mitochondria, a previously described sucrose gradient fractionation method was employed prior to gel electrophoresis. Phosphorylated proteins, as defined by the fluorescence of the phosphosensor, were excised from the gels and identified by peptide mass fingerprinting. One novel and prominent phosphoprotein identified in this manner was determined to be the 42-kDa subunit of mitochondrial complex I.
Assuntos
Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Animais , Bovinos , Complexo I de Transporte de Elétrons , Corantes Fluorescentes , Técnicas In Vitro , Cinética , Mitocôndrias Cardíacas/química , Mitocôndrias Cardíacas/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Mapeamento de Peptídeos , Fosforilação , Subunidades Proteicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.
Assuntos
Anticorpos Monoclonais/imunologia , Mitocôndrias Cardíacas/metabolismo , Fosforilação Oxidativa , Fosfoproteínas/metabolismo , Proteômica , Animais , Bovinos , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Corantes Fluorescentes/químicaRESUMO
Protein phosphorylation plays a vital role in the regulation of most aspects of cellular activity, being key to propagating messages within signal transduction pathways and to modulating protein function. Pro-Q Diamond phosphoprotein gel stain is suitable for the fluorescence detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The technology is especially appropriate for profiling steady-state and dynamic phosphorylation on a proteome-wide scale, as demonstrated through detection of the native phosphorylation of cardiac mitochondrial phosphoproteins and changes in this profile arising from the activity of a protein kinase. For example, Pro-Q Diamond phosphoprotein gel stain was employed to demonstrate that among the 46 subunits of the mitochondrial respiratory chain complex, NADH:ubiquinone oxidoreductase (complex I), a 42 kDa subunit is phosphorylated in the steady-state. However, exposure of mitochondria to cAMP-dependent protein kinase increases phosphorylation of this 42 kDa subunit and results in de novo phosphorylation of an 18 kDa subunit as well. Since Pro-Q Diamond dye binds to phosphorylated residues noncovalently, the staining technology is fully compatible with modern microchemical analysis procedures, such as peptide mass profiling by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and post-source decay analysis of peptide phosphorylation.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Corantes Fluorescentes/química , Mitocôndrias Cardíacas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Animais , Bovinos , Feminino , Células HeLa , Humanos , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Current methods for determining ambient redox potential in cells are labor-intensive and generally require destruction of tissue. This precludes single cell or real time studies of changes in redox poise that result from metabolic processes or environmental influences. By substitution of surface-exposed residues on the Aequorea victoria green fluorescent protein (GFP) with cysteines in appropriate positions to form disulfide bonds, reduction-oxidation-sensitive GFPs (roGFPs) have been created. roGFPs have two fluorescence excitation maxima at about 400 and 490 nm and display rapid and reversible ratiometric changes in fluorescence in response to changes in ambient redox potential in vitro and in vivo. Crystal structure analyses of reduced and oxidized crystals of roGFP2 at 2.0- and 1.9-A resolution, respectively, reveal in the oxidized state a highly strained disulfide and localized main chain structural changes that presumably account for the state-dependent spectral changes. roGFP1 has been targeted to the mitochondria in HeLa cells. Fluorometric measurements on these cells using a fluorescence microscope or in cell suspension using a fluorometer reveal that the roGFP1 probe is in dynamic equilibrium with the mitochondrial redox status and responds to membrane-permeable reductants and oxidants. The roGFP1 probe reports that the matrix space in HeLa cell mitochondria is highly reducing, with a midpoint potential near -360 mV (assuming mitochondrial pH approximately 8.0 at 37 degrees C). In other work (C. T. Dooley, T. M. Dore, G. Hanson, W. C. Jackson, S. J. Remington, and R. Y. Tsien, submitted for publication), it is shown that the cytosol of HeLa cells is also unusually reducing but somewhat less so than the mitochondrial matrix.
Assuntos
Proteínas Luminescentes/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Linhagem Celular , Cristalografia por Raios X , Cisteína/química , Dissulfetos , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Fluorescência , Modelos Químicos , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Conformação Proteica , Temperatura , Fatores de Tempo , Transfecção , Difração de Raios XRESUMO
Human mitochondrial F(1)F(0) ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F(1) in a 96-well microplate activity assay system, to establish a method for fast high throughput screening of inhibitors, toxins, and drugs with very small amounts of enzyme. For preparative purification, mitochondria from human heart tissue as well as cultured fibroblasts were solubilized with dodecyl-beta-d-maltoside, and the F(1)F(0) was isolated with anti-F(1) monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F(1)F(0) contained a full complement of subunits that were identified with specific antibodies against five of the subunits (alpha, beta, OSCP, d, and IF(1)) and by MALDI-TOF and/or LC/MS/MS for all subunits except subunit c, which could not be resolved by these methods because of the limits of detection. Microscale immunocapture of F(1)F(0) from detergent-solubilized mitochondria or whole cell fibroblast extracts was performed using anti-F(1) monoclonal antibody immobilized on 96-well microplates. The captured complex V displayed ATP hydrolysis activity that was fully oligomycin and inhibitor protein IF(1)-sensitive. Moreover, IF(1) could be co-isolated with F(1)F(0) when the immunocapture procedure was carried out at pH 6.5 but was absent when the ATP synthase was isolated at pH 8.0. Immunocaptured F(1)F(0) lacking IF(1) could be inhibited by more than 90% by addition of recombinant inhibitor protein, and conversely, F(1)F(0) containing IF(1) could be activated more than 10-fold by brief exposure to pH 8.0, inducing the release of inhibitor protein. With this microplate system an ATP hydrolysis assay of complex V could be carried out with as little as 10 ng of heart mitochondria/well and as few as 3 x 10(4) cells/well from fibroblast cultures. The system is therefore suitable to screen patient-derived samples for alterations in amount or functionality of both the F(1)F(0) ATPase and IF(1).