Bipin G. Nair

Proteogenomic Analysis of Mycobacterium tuberculosis By High Resolution Mass Spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ∼80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ∼250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

Quantitative tissue proteomics of esophageal squamous cell carcinoma for novel biomarker discovery

Esophageal squamous cell carcinoma (ESCC) is among the top ten most frequent malignancies worldwide. In this study, our objective was to identify potential biomarkers for ESCC through a quantitative proteomic approach using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. We compared the protein expression profiles of ESCC tumor tissues with the corresponding adjacent normal tissue from ten patients. LC-MS/MS analysis of strong cation exchange chromatography fractions was carried out on an Accurate Mass QTOF mass spectrometer, which led to the identification of 687 proteins. In all, 257 proteins were identified as differentially expressed in ESCC as compared to normal. We found several previously known protein biomarkers to be upregulated in ESCC including thrombospondin 1 (THBS1), periostin 1 (POSTN) and heat shock 70 kDa protein 9 (HSPA9) confirming the validity of our approach. In addition, several novel proteins that had not been reported previously were identified in our screen. These novel biomarker candidates included prosaposin (PSAP), plectin 1 (PLEC1) and protein disulfide isomerase A 4 (PDIA4) that were further validated to be overexpressed by immunohistochemical labeling using tissue microarrays. The success of our study shows that this mass spectrometric strategy can be applied to cancers in general to develop a panel of candidate biomarkers, which can then be validated by other techniques.

Proteomic analysis of human vitreous humor

BackgroundThe vitreous humor is a transparent, gelatinous mass whose main constituent is water. It plays an important role in providing metabolic nutrient requirements of the lens, coordinating eye growth and providing support to the retina. It is in close proximity to the retina and reflects many of the changes occurring in this tissue. The biochemical changes occurring in the vitreous could provide a better understanding about the pathophysiological processes that occur in vitreoretinopathy. In this study, we investigated the proteome of normal human vitreous humor using high resolution Fourier transform mass spectrometry.ResultsThe vitreous humor was subjected to multiple fractionation techniques followed by LC-MS/MS analysis. We identified 1,205 proteins, 682 of which have not been described previously in the vitreous humor. Most proteins were localized to the extracellular space (24%), cytoplasm (20%) or plasma membrane (14%). Classification based on molecular function showed that 27% had catalytic activity, 10% structural activity, 10% binding activity, 4% cell and 4% transporter activity. Categorization for biological processes showed 28% participate in metabolism, 20% in cell communication and 13% in cell growth. The data have been deposited to the ProteomeXchange with identifier PXD000957.ConclusionThis large catalog of vitreous proteins should facilitate biomedical research into pathological conditions of the eye including diabetic retinopathy, retinal detachment and cataract.

Homology Modeling of GLUT4, an Insulin Regulated Facilitated Glucose Transporter and Docking Studies with ATP and its Inhibitors

Abstract GLUT4 is a 12 transmembrane (TM) protein belonging to the Class I facilitated glucose transporter family that transports glucose into the cells in an insulin regulated manner. GLUT4 plays a key role in the maintenance of blood glucose homeostasis and inhibition of glucose transporter activity may lead to insulin resistance, hallmark of type 2 diabetes. No crystal structure data is available for any members of the facilitated glucose transporter family. Here, in this paper, we have generated a homology model of GLUT4 based on experimental data available on GLUTI, a Class I facilitated glucose transporter and the crystal structure data obtained from the Glycerol 3-phosphate transporter. The model identified regions in GLUT4 that form a channel for the transport of glucose along with the substrate interacting residues. Docking and electrostatic potential data analysis of GLUT4 model has mapped an ATP binding region close to the binding site of cytochalasin B and genistein, two GLUT4 inhibitors, and this may explain the mechanism by which these inhibitors could potentially affect the GLUT4 function.

Epidermal Growth Factor Produces Inotropic and Chronotropic Effects in Rat Hearts by Increasing Cyclic AMP Accumulation

Previously we have shown that epidermal growth factor (EGF) stimulates cardiac adenylyl cyclase and increases cAMP accumulation in the rat heart (Nair et al., Biochem. J. 264, 563-571, 1989). Moreover, we have shown that the stimulation of adenylyl cyclase by EGF in heart is mediated via activation of the stimulatory GTP binding regulatory protein Gs alpha (Nair et al., J. Biol. Chem. 265, 21317-21322, 1990). Since cAMP increases the beating rate of hearts, studies were performed to investigate the effects of EGF on mechanical function of the heart and the role of cAMP in mediating the cardiac effects of EGF. In isolated perfused rat hearts EGF (15 nM) decreased perfusion pressure, increased ventricular contractility and heart rate in a manner similar to that observed with the beta-adrenergic receptor agonist isoproterenol (10 nM). In the presence of the adenosine A1 receptor agonist (-)-N6-(R-phenylisopropyl)-adenosine (PIA, 100 nM) which via activation of the inhibitory GTP binding protein Gi inhibits adenylyl cyclase, the effects of EGF on cAMP accumulation in the heart were markedly attenuated. PIA also decreased the ability of EGF and isoproterenol to alter cardiac contractility and beating rate. However, PIA did not attenuate the increase in heart rate and contractility induced by the alpha-adrenergic agonist phenylephrine which does not stimulate cAMP accumulation in the heart. These data suggest that EGF alters cardiac function by increasing cellular cAMP accumulation.

Regulation of cardiac adenylyl cyclase by Epidermal Growth Factor (EGF): Role of EGF receptor protein tyrosine kinase activity

We have shown previously that the alpha subunit of the stimulatory GTP binding regulatory component of adenylyl cyclase (Gs alpha) mediates epidermal growth factor (EGF)-elicited stimulation of rat cardiac adenylyl cyclase (Nair et al., J Biol Chem 265: 21317-21322, 1990). Employing purified protein phosphotyrosine phosphatase, and benzylidene derivatives (tyrphostins: compounds 11 and 12) that selectively inhibit EGF receptor protein tyrosine kinase (EGFRK) activity, the role of EGFRK in EGF-mediated stimulation of cardiac adenylyl cyclase was investigated. The ability of the tyrphostins to inhibit the EGFRK activity in cardiac membranes was determined by monitoring tyrosine phosphorylation of either the 170 kDa protein or immunoprecipitated EGF receptor at 0 degrees and room temperature, respectively. Compounds 11 and 12, in a concentration-dependent manner, inhibited EGF receptor tyrosine kinase activity. In assays of adenylyl cyclase activity neither compound 11 nor compound 12 altered Gpp(NH)p- or isoproterenol-stimulated activity. However, both compounds, in a concentration-dependent manner, attenuated the ability of EGF to stimulate adenylyl cyclase activity without altering specific binding of [125I]EGF to cardiac membranes. Similarly, protein phosphotyrosine phosphatase obliterated the ability of EGF, but not isoproterenol, to stimulate adenylyl cyclase. Thus, we conclude that protein tyrosine kinase activity of the EGF receptor is essential for the stimulation of cardiac adenylyl cyclase by EGF.

Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9.

Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1′ pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dose-dependent manner, with an IC50 of 11.11 μM. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.

Pancreatic Cancer Database: An integrative resource for pancreatic cancer

Pancreatic cancer is the fourth leading cause of cancer-related death in the world. The etiology of pancreatic cancer is heterogeneous with a wide range of alterations that have already been reported at the level of the genome, transcriptome, and proteome. The past decade has witnessed a large number of experimental studies using high-throughput technology platforms to identify genes whose expression at the transcript or protein levels is altered in pancreatic cancer. Based on expression studies, a number of molecules have also been proposed as potential biomarkers for diagnosis and prognosis of this deadly cancer. Currently, there are no repositories which provide an integrative view of multiple Omics data sets from published research on pancreatic cancer. Here, we describe the development of a web-based resource, Pancreatic Cancer Database (http://www.pancreaticcancerdatabase.org), as a unified platform for pancreatic cancer research. PCD contains manually curated information pertaining to quantitative alterations in miRNA, mRNA, and proteins obtained from small-scale as well as high-throughput studies of pancreatic cancer tissues and cell lines. We believe that PCD will serve as an integrative platform for scientific community involved in pancreatic cancer research.

Modeling spike-train processing in the cerebellum granular layer and changes in plasticity reveal single neuron effects in neural ensembles

The cerebellum input stage has been known to perform combinatorial operations on input signals. In this paper, two types of mathematical models were used to reproduce the role of feed-forward inhibition and computation in the granular layer microcircuitry to investigate spike train processing. A simple spiking model and a biophysically-detailed model of the network were used to study signal recoding in the granular layer and to test observations like center-surround organization and time-window hypothesis in addition to effects of induced plasticity. Simulations suggest that simple neuron models may be used to abstract timing phenomenon in large networks, however detailed models were needed to reconstruct population coding via evoked local field potentials (LFP) and for simulating changes in synaptic plasticity. Our results also indicated that spatio-temporal code of the granular network is mainly controlled by the feed-forward inhibition from the Golgi cell synapses. Spike amplitude and total number of spikes were modulated by LTP and LTD. Reconstructing granular layer evoked-LFP suggests that granular layer propagates the nonlinearities of individual neurons. Simulations indicate that granular layer network operates a robust population code for a wide range of intervals, controlled by the Golgi cell inhibition and is regulated by the post-synaptic excitability.

Tantalum oxide honeycomb architectures for the development of a non-enzymatic glucose sensor with wide detection range

Tantalum oxide honeycomb nanostructures (THNS) were fabricated by electrochemical anodisation of tantalum in H2SO4-HF medium. XRD analysis showed that annealing of THNS at 400 °C improves the crystallinity. HRSEM and AFM results illustrated that nanopores with an average diameter of 30 nm were uniformly distributed and the pore size reduced to 24 nm and 18 nm during subsequent electrodeposition of Pt and CuO. Electrodeposited Pt and CuO exhibited face centered cubic (fcc) and monoclinic crystal structure respectively. Cyclic voltammetric studies revealed that, on the hybrid material electrooxidation of glucose occurs at a lower potential (0.45 V). The sensor exhibited linear response to glucose up to 31 mM, fast response time (<3 s) and a low detection limit of 1 μM (S/N=3). The sensor is free of interference from ascorbic acid, uric acid, dopamine and acetaminophen. Sensor was used to analyze glucose in blood serum samples.