Biomedical subjects

Soumitra S Ghosh

Publications and source records attributed to Soumitra S Ghosh.

12 recordsLinked to original sources

PubMed2006

Efficient high-throughput discovery of large peptidic hormones and biomarkers.

A novel approach is presented for the simultaneous identification and relative quantification of secreted peptides, particularly those that have been historically difficult to analyze in a concerted manner. Peptides exceeding 60 residues with various degrees of post-translational modification were identified on a liquid chromatographic time scale. The approach demonstrates high efficiency pattern-based recognition analysis of complex neuroendocrine peptide sets and enables rapid identification of biomarkers from biological material.

Amino Acid Sequence↗

PubMed2004

Mediation of spontaneous knee osteoarthritis by progressive chondrocyte ATP depletion in Hartley guinea pigs.

OBJECTIVE: Because articular chondrocytes reside in a hypoxic milieu, anaerobic glycolysis is central in generating ATP to support chondrocyte matrix synthesis and viability, with mitochondrial oxidative phosphorylation possibly providing physiologic reserve ATP generation. Nitric oxide (NO) potently suppresses mitochondrial oxidative phosphorylation. Because enhanced cartilage NO generation occurs in osteoarthritis (OA), we systematically tested for mitochondrial dysfunction in the pathogenesis of OA. METHODS: We assessed chondrocytes for ATP depletion and for in situ changes in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee OA in male Hartley guinea pigs, a model in which chondrocalcinosis also supervenes. RESULTS: Spontaneous NO release from knee cartilage samples in organ culture doubled between ages 2 months and 8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels declined by approximately 50%, despite a lack of mitochondrial ultrastructure abnormalities in knee chondrocytes. As ATP depletion progressed with aging in knee chondrocytes, an increased ratio of lactate to pyruvate was observed, consistent with an adaptive augmentation of glycolysis to mitochondrial dysfunction. Furthermore, we observed progressive elevation of chondrocyte ATP-scavenging nucleotide pyrophosphatase/phosphodiesterase (NPP) activity and extracellular levels of the NPP enzymatic end product inorganic pyrophosphate (PPi), which stimulate chondrocalcinosis. CONCLUSION: Profound chondrocyte ATP depletion develops in association with heightened NO generation in guinea pig knee OA. Increased NPP activity and concordant increases in extracellular PPi, which are strongly associated with human aging-associated degenerative arthropathy and directly stimulate chondrocalcinosis, may be primarily driven by chondrocyte ATP depletion. Our findings implicate a decreased mitochondrial bioenergetic reserve as a pathogenic factor in both degenerative arthropathy and chondrocalcinosis in aging.

Adenosine Triphosphate↗

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

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

The subunit composition of the human NADH dehydrogenase obtained by rapid one-step immunopurification.

Defects of the NADH dehydrogenase complex are predominantly manifested in mitochondrial diseases and are significantly associated with the development of many late onset neurological disorders such as Parkinson's disease. Here we describe an immunocapture procedure for isolating this multisubunit membrane-bound complex from human tissue. Using small amounts of immunoisolated protein, one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass finger printing (PMF), and nanoflow liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), we can resolve and identify the human homologues of 42 polypeptides detected so far in the more extensively studied beef heart complex I. These polypeptides include the GRIM-19 protein, which is claimed to be involved in apoptosis, a polypeptide first identified by gene screening as a neuronal protein, as well as a protein thought to be in differentiation linked processes. The concordance of data from human and bovine complex I isolated by different procedures adds to the certainty that these novel proteins of seemingly diverse function are a part of complex I.

Animals↗

PubMed2003

Characterization of the human heart mitochondrial proteome.

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

Adolescent↗

PubMed2003

Efficient syntheses of benzothiazepines as antagonists for the mitochondrial sodium-calcium exchanger: potential therapeutics for type II diabetes.

Type II diabetes mellitus is a chronic metabolic disorder that can lead to serious cardiovascular, renal, neurologic, and retinal complications. While several drugs are currently prescribed to treat type II diabetes, their efficacy is limited by mechanism-related side effects (weight gain, hypoglycemia, gastrointestinal distress), inadequate efficacy for use as monotherapy, and the development of tolerance to the agents. Consequently, combination therapies are frequently employed to effectively regulate blood glucose levels. We have focused on the mitochondrial sodium-calcium exchanger (mNCE) as a novel target for diabetes drug discovery. We have proposed that inhibition of the mNCE can be used to regulate calcium flux across the mitochondrial membrane, thereby enhancing mitochondrial oxidative metabolism, which in turn enhances glucose-stimulated insulin secretion (GSIS) in the pancreatic beta-cell. In this paper, we report the facile synthesis of benzothiazepines and derivatives by S-alkylation using 2-aminobenzhydrols. The syntheses of other bicyclic analogues based on benzothiazepine, benzothiazecine, benzodiazecine, and benzodiazepine templates are also described. These compounds have been evaluated for their inhibition of mNCE activity, and the results from the structure-activity relationship (SAR) studies are discussed.

Animals↗

PubMed2003

Global organellar proteomics.

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.

Amino Acid Sequence↗

PubMed2002

A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies.

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).

Antibodies, Monoclonal↗

PubMed2002

Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups.

The evolution of the human mitochondrial genome is characterized by the emergence of ethnically distinct lineages or haplogroups. Nine European, seven Asian (including Native American), and three African mitochondrial DNA (mtDNA) haplogroups have been identified previously on the basis of the presence or absence of a relatively small number of restriction-enzyme recognition sites or on the basis of nucleotide sequences of the D-loop region. We have used reduced-median-network approaches to analyze 560 complete European, Asian, and African mtDNA coding-region sequences from unrelated individuals to develop a more complete understanding of sequence diversity both within and between haplogroups. A total of 497 haplogroup-associated polymorphisms were identified, 323 (65%) of which were associated with one haplogroup and 174 (35%) of which were associated with two or more haplogroups. Approximately one-half of these polymorphisms are reported for the first time here. Our results confirm and substantially extend the phylogenetic relationships among mitochondrial genomes described elsewhere from the major human ethnic groups. Another important result is that there were numerous instances both of parallel mutations at the same site and of reversion (i.e., homoplasy). It is likely that homoplasy in the coding region will confound evolutionary analysis of small sequence sets. By a linkage-disequilibrium approach, additional evidence for the absence of human mtDNA recombination is presented here.

Africa↗

PubMedDate not supplied

An alternative strategy to determine the mitochondrial proteome using sucrose gradient fractionation and 1D PAGE on highly purified human heart mitochondria.

An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-beta-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family.

Blotting, Western↗

PubMedDate not supplied

Expanded coverage of the human heart mitochondrial proteome using multidimensional liquid chromatography coupled with tandem mass spectrometry.

Recent evidence suggests that mitochondria are closely linked with the aging process and degenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, there has been increasing interest in cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We have previously reported results of a one-dimensional electrophoresis/liquid chromatography MS/MS study to characterize the proteome of normal human heart mitochondria (Taylor et al. Nat. Biotechnol. 2003, 21, 281-286). We now report two subsequent studies where multidimensional liquid chromatography MS/MS was investigated as an alternative means for characterizing the same sample.

Chromatography, Liquid↗