Biomedical subjects

Roderick A Capaldi

Publications and source records attributed to Roderick A Capaldi.

At least 19 recordsLinked to original sources

PubMed2006

The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study.

F(1)F(0) ATP synthase is ectopically expressed on the surface of several cell types, including endothelium and cancer cells. This study uses immunocytochemical detection methods via highly specific monoclonal antibodies to explore the possibility of plasma membrane localization of other mitochondrial proteins using an osteosarcoma cell line in which the location of the mitochondrial reticulum can be clearly traced by green fluorescent protein tagging of the organelle. We found that subunits of three of the four respiratory chain complexes were present on the surface of these cells. Additionally, we show for the first time that F(0) subunits d and OSCP of the ATP synthase are ectopically expressed. In all cases the OXPHOS proteins show a punctate distribution, consistent with data from proteome analysis of isolated lipid rafts that place the various mitochondrial proteins in plasma membrane microdomains. We also examined the cell surface for marker membrane proteins from several other intracellular organelles including ER, golgi and nuclear envelope. They were not found on the surface of the osteosarcoma cells. We conclude that mitochondrial membrane proteins are ectopically expressed, but not proteins from other cellular organelles. A specific mechanism by which the mitochondrion and plasma membrane fuse to deliver organellar proteins is suggested.

Cell Line, Tumor↗

PubMed2006

Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled.

Loss of mitochondrial complex I catalytic activity in the electron transport chain (ETC) is found in multiple tissues from individuals with sporadic Parkinson's disease (PD) and is a property of some PD model neurotoxins. Using special ETC subunit-specific and complex I immunocapture antibodies directed against the entire complex I macroassembly, we quantified ETC proteins and protein oxidation of complex I subunits in brain mitochondria from 10 PD and 12 age-matched control (CTL) samples. We measured nicotinamide adenine dinucleotide (NADH)-driven electron transfer rates through complex I and correlated these with complex I subunit oxidation levels and reductions of its 8 kDa subunit. PD brain complex I shows 11% increase in ND6, 34% decrease in its 8 kDa subunit and contains 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes We found no changes in levels of ETC proteins from complexes II-V. Oxidative damage patterns to PD complex I are reproduced by incubation of CTL brain mitochondria with NADH in the presence of rotenone but not by exogenous oxidant. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and positively correlate with reduction in PD 8 kDa subunit. Reduced complex I function in PD brain mitochondria appears to arise from oxidation of its catalytic subunits from internal processes, not from external oxidative stress, and correlates with complex I misassembly. This complex I auto-oxidation may derive from abnormalities in mitochondrial or nuclear encoded subunits, complex I assembly factors, rotenone-like complex I toxins, or some combination.

Cells, Cultured↗

PubMed2005

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria.

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.

Amino Acid Sequence↗

PubMed2005

Mass spectrometric identification of a novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I.

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.

Alkaline Phosphatase↗

PubMed2004

Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line.

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.

Animals↗

PubMed2004

Focused proteomics: towards a high throughput monoclonal antibody-based resolution of proteins for diagnosis of mitochondrial diseases.

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.

Animals↗

PubMed2004

An Inhibitor of the F1 subunit of ATP synthase (IF1) modulates the activity of angiostatin on the endothelial cell surface.

Angiostatin binds to endothelial cell (EC) surface F(1)-F(0) ATP synthase, leading to inhibition of EC migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F(1) ATP synthase and inhibited F(1)-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. Although exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared with angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.

Angiostatins↗

PubMed2004

Immunological approaches to the characterization and diagnosis of mitochondrial disease.

Monoclonal antibodies (mAbs) are important tools in the diagnosis and characterization of mitochondrial diseases. They can be used in immunohistochemical and/or Western blotting approaches to identify misassembled OXPHOS complexes or pyruvate dehydrogenase deficiencies where the intact complex is not formed which is the great majority of cases. The advantage of antibody based approaches is that they can be quantitative, require very small amounts of tissue sample and are fast, simple and relatively cheap to perform. Here we provide details of the mAbs currently available and describe optimized protocols for both immunohistochemistry using patient fibroblasts as well as Western blotting using either cell culture or biopsy material.

Journal Article↗

PubMed2004

Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells.

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.

Biosensing Techniques↗

PubMed2004

Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators.

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.

Cell Line↗

PubMed2004

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.

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.

Animals↗

PubMed2004

Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry.

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.

Animals↗

PubMed2004

Rapid analysis of mitochondrial DNA depletion by fluorescence in situ hybridization and immunocytochemistry: potential strategies for HIV therapeutic monitoring.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been a mainstay in the treatment of human immunodeficiency virus since the introduction of azidothymidine (AZT) in 1987. However, none of the current therapies can completely eradicate the virus, necessitating long-term use of anti-retroviral drugs to prevent viral re-growth. One of the side effects associated with long-term use of NRTIs is mitochondrial toxicity stemming from inhibition of the mitochondrial DNA (mtDNA) polymerase gamma, which leads to mtDNA depletion and consequently to mitochondrial dysfunction. Here we report the use of fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI 2',3'-dideoxycytidine (ddC). These techniques are amenable to both microscopy and flow cytometry, allowing analysis of populations of cells on a single-cell basis. We show that, as mtDNA depletion progresses, a mosaic population develops, with some cells being depleted of and others retaining mtDNA. These techniques could be useful as potential therapeutic monitors to indicate when NRTI therapy should be interrupted to prevent mitochondrial toxicity and could aid in the development of less toxic NRTIs by providing an assay suitable for pharmacodynamic evaluation of candidate molecules.

Cell Line↗

PubMed2003

Oxidative damage to mitochondrial complex I due to peroxynitrite: identification of reactive tyrosines by mass spectrometry.

There is growing evidence that oxidative phosphorylation (OXPHOS) generates reactive oxygen and nitrogen species within mitochondria as unwanted byproducts that can damage OXPHOS enzymes with subsequent enhancement of free radical production. The accumulation of this oxidative damage to mitochondria in brain is thought to lead to neuronal cell death resulting in neurodegeneration. The predominant reactive nitrogen species in mitochondria are nitric oxide and peroxynitrite. Here we show that peroxynitrite reacts with mitochondrial membranes from bovine heart to significantly inhibit the activities of complexes I, II, and V (50-80%) but with less effect upon complex IV and no significant inhibition of complex III. Because inhibition of complex I activity has been a reported feature of Parkinson's disease, we undertook a detailed analysis of peroxynitrite-induced modifications to proteins from an enriched complex I preparation. Immunological and mass spectrometric approaches coupled with two-dimensional PAGE have been used to show that peroxynitrite modification resulting in a 3-nitrotyrosine signature is predominantly associated with the complex I subunits, 49-kDa subunit (NDUFS2), TYKY (NDUFS8), B17.2 (17.2-kDa differentiation associated protein), B15 (NDUFB4), and B14 (NDUFA6). Nitration sites and estimates of modification yields were deduced from MS/MS fragmentograms and extracted ion chromatograms, respectively, for the last three of these subunits as well as for two co-purifying proteins, the beta and the d subunits of the F1F0-ATP synthase. Subunits B15 (NDUFB4) and B14 (NDUFA6) contained the highest degree of nitration. The most reactive site in subunit B14 was Tyr122, while the most reactive region in B15 contained 3 closely spaced tyrosines Tyr46, Tyr50, and Tyr51. In addition, a site of oxidation of tryptophan was detected in subunit B17.2 adding to the number of post-translationally modified tryptophans we have detected in complex I subunits (Taylor, S. W., Fahy, E., Murray, J., Capaldi, R. A., and Ghosh, S. S. (2003) J. Biol. Chem. 278, 19587-19590). These sites of oxidation and nitration may be useful biomarkers for assessing oxidative stress in neurodegenerative disorders.

Amino Acid Sequence↗

PubMed2003

The cristal membrane of mitochondria is the principal site of oxidative phosphorylation.

The inner membrane system of mitochondria us known to consist of two contiguous but distinct membranes: the inner boundary membrane, which apposes the outer membrane, and the cristal membrane, which forms tubules or lamellae in the interior. Using immunolabeling and transmission electron microscopy of bovine heart tissue, we have calculated that around 94% of both Complex III of the respiratory chain and the ATP synthase are located in the cristal membrane, and only around 6% of either is in the inner boundary membrane. When accounting for the topographical ratio of cristal membrane versus inner boundary membrane, we find that both complexes exist at a 2.2-2.6-fold higher concentration in the cristal membrane. The residual protein in the inner boundary membrane may be newly assembled complexes destined for cristal membranes. Our results argue for restricted diffusion of complexes through the cristal junctions and indicate that the mitochondrial cristae comprise a regulated submitochondrial compartment specialized for ATP production.

ATP Synthetase Complexes↗

PubMed2003

Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye.

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.

Animals↗

PubMed2003

Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins.

We examined the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, we suggest that the susceptible tryptophan residues in this group of proteins are "hot spots" for oxidation in close proximity to a source of reactive oxygen species in respiring mitochondria.

Amino Acid Sequence↗

PubMed2003

Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex.

Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.

Adenosine Triphosphate↗