Papers by Narayanan Manoj
Journal of Cell Science, 2020
Global and endothelial loss of PTP-PEST is associated with impaired cardiovascular development an... more Global and endothelial loss of PTP-PEST is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1 % oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry (LC/MS/MS) revealed that alpha subunits of AMPK (α1 and α2) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation) which was rescued by AMPK activator, metformin (500 µM). Since endothelial autophagy is a pre...
S, the INSL3/relaxin receptor-like and the Snail and Human LGRs. The tree is rooted using a human... more S, the INSL3/relaxin receptor-like and the Snail and Human LGRs. The tree is rooted using a human LHCGR and a orphan LGR (ci0100151424) that are distantly related to these sequences. Support values are indicated in percentages. Taxons represented in numerals refer to GPCRs as per information in Additional data file . GPCRs that deviate from the predicted 7TM structure are marked using a '#' symbol. B) Schematic diagram representing the modular domain organization corresponding to the clusters identified in the phylogenetic tree.<b>Copyright information:</b>Taken from "The repertoire of G protein-coupled receptors in the sea squirt "http://www.biomedcentral.com/1471-2148/8/129BMC Evolutionary Biology 2008;8():129-129.Published online 1 May 2008PMCID:PMC2396169.
Molecular Phylogenetics and Evolution, 2021
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biochimica et biophysica acta. Proteins and proteomics, 2020
Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptati... more Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free ene...
Biochimica et Biophysica Acta (BBA) - General Subjects, 2019
Introduction: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and... more Introduction: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined. Methods: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α 2 β 1 γ 1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α 2 subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. Results: AG interacts with the α 2 subunit of AMPK and activates the trimeric kinase with an EC 50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. Conclusion: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. General significance: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.
FEBS Letters, 2019
Eukaryotes employ a subset of dynamins to mediate mitochondrial fusion and fission dynamics. Here... more Eukaryotes employ a subset of dynamins to mediate mitochondrial fusion and fission dynamics. Here we report the molecular evolution and diversification of the dynamin-related mitochondrial proteins that drive the fission (Drp1) and the fusion processes (mitofusin and OPA1). We demonstrate that the three paralogs emerged concurrently in an early mitochondriate eukaryotic ancestor. Furthermore, multiple independent duplication events from an ancestral bifunctional fission protein gave rise to specialized fission proteins. The evolutionary history of these proteins is marked by transformations that include independent gain and loss events occurring at the levels of entire genes, specific functional domains, and intronic regions. The domain level variations primarily comprise loss-gain of lineage specific domains that are present in the terminal regions of the sequences.
Protein Engineering, Design and Selection, 2017
The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a... more The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical α/β hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of~30-40°C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.
Biochimie, Jan 18, 2017
Imposition of different biotic and abiotic stress conditions results in an increase in intracellu... more Imposition of different biotic and abiotic stress conditions results in an increase in intracellular levels of Ca(2+) which is sensed by various sensor proteins. Calmodulin (CaM) is one of the best studied transducers of Ca(2+) signals. CaM undergoes conformational changes upon binding to Ca(2+) and interacts with different types of proteins, thereby, regulating their activities. The present study reports the cloning and characterization of a sorghum cDNA encoding a protein (SbGRBP) that shows homology to glycine-rich RNA-binding proteins. The expression of SbGRBP in the sorghum seedlings is modulated by heat stress. The SbGRBP protein is localized in the nucleus as well as in cytosol, and shows interaction with CaM that requires the presence of Ca(2+). SbGRBP depicts binding to single and also double stranded DNA. Fluorescence spectroscopic analyses suggest that interaction of SbGRBP with nucleic acids may be modulated after binding with CaM. To our knowledge, this is the first stu...
Journal of Biosciences
In 1986 Rumelhart et al., investigated a wide variety of network designs, they announced the disc... more In 1986 Rumelhart et al., investigated a wide variety of network designs, they announced the discovery of a method of enabling a network to learn to discriminate between classes of patterns that are not linearly separable, they called the method backward propagation of errors. The two types, motor and sensory patterns being distinguished by the network are identified to the learning network by the desired output fed to the network in the learning process. Many data records representing the two types were processed by different mathematical modeling approaches. The models coefficients are considered to be the features which would be utilized for discrimination between motor and sensory responses. Then the results were compared to the desired output for each record and the percentage of correct estimations was calculated for each one. Non-linear model results have proven to be the best single model in application on neural networks, which confirms the non-linear behavior of the nervous system.
Proceedings of the International Symposium on Biocomputing - ISB '10, 2010
Abstract The amino sugar metabolic pathway is an essential pathway that is conserved in all three... more Abstract The amino sugar metabolic pathway is an essential pathway that is conserved in all three kingdoms of life. NagA, a highly conserved enzyme, carries out the first committed step in the biosynthetic pathway to amino sugar nucleotides. To better understand the ...
BMC Evolutionary Biology, 2014
Background: G protein-coupled receptors (GPCRs) play a central role in eukaryotic signal transduc... more Background: G protein-coupled receptors (GPCRs) play a central role in eukaryotic signal transduction. However, the GPCR component of this signalling system, at the early origins of metazoans is not fully understood. Here we aim to identify and classify GPCRs in Amphimedon queenslandica (sponge), a member of an earliest diverging metazoan lineage (Porifera). Furthermore, phylogenetic comparisons of sponge GPCRs with eumetazoan and bilaterian GPCRs will be essential to our understanding of the GPCR system at the roots of metazoan evolution. Results: We present a curated list of 220 GPCRs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of 282 predicted GPCR sequences obtained using Pfam search. Phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major GRAFS families including Glutamate (33), Rhodopsin (126), Adhesion (40) and Frizzled (3). Interestingly, the sponge Rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. This suggests that sponge Rhodopsins diverged considerably from that found in other basal metazoans. A few sponge Adhesions clustered basal to Adhesion subfamilies commonly found in most vertebrates, suggesting some Adhesion subfamilies may have diverged prior to the emergence of Bilateria. Furthermore, at least eight of the sponge Adhesion members have a hormone binding motif (HRM domain) in their N-termini, although hormones have yet to be identified in sponges. We also phylogenetically clarified that sponge has homologs of metabotropic glutamate (mGluRs) and GABA receptors. Conclusion: Our phylogenetic comparisons of sponge GPCRs with other metazoan genomes suggest that sponge contains a significantly diversified set of GPCRs. This is evident at the family/subfamily level comparisons for most GPCR families, in particular for the Rhodopsin family of GPCRs. In summary, this study provides a framework to perform future experimental and comparative studies to further verify and understand the roles of GPCRs that predates the divergence of bilaterian and eumetazoan lineages.
Structure, 2003
(Figure 2). The human enzyme shows low sequence similarity to the bacterial enzyme, suggesting th... more (Figure 2). The human enzyme shows low sequence similarity to the bacterial enzyme, suggesting the possi-Ithaca, New York 14853 bility of developing selective inhibitors of the bacterial enzyme. Because bacteria do not have a PPC transport system, this enzyme is potentially a good target for the development of new antibacterials. Summary Serine misincorporation in coenzyme A biosynthesis would result in the formation of oxo-coenzyme A in The structure of human phosphopantothenoylcysteine which the cofactor thiol is replaced by an alcohol. Be-(PPC) synthetase was determined at 2.3 Å resolution. cause the thiol group plays a key role in the biochemistry PPC synthetase is a dimer with identical monomers. of this cofactor, oxo-coenzyme A is likely to inhibit cell Some features of the monomer fold resemble a group growth. PPC synthetase shows high selectivity for cysof NAD-dependent enzymes, while other features reteine over serine (our unpublished results). The mechasemble the ribokinase fold. The ATP, phosphopantonistic basis for this selectivity is a fundamental problem thenate, and cysteine binding sites were deduced from in enzymology and is generally poorly understood. The modeling studies. Highly conserved ATP binding resionly relevant structural study was carried out on the cysteidues include Gly43, Ser61, Gly63, Gly66, Phe230, and nyl-tRNA synthetase, where the selectivity results primarily Asn258. Highly conserved phosphopantothenate bindfrom the coordination of the cysteine thiol to an active site ing residues include Asn59, Ala179, Ala180, and Asp183 zinc ion (Newberry et al., 2002). The structural studies from one monomer and Arg55 from the adjacent monodescribed here allow us to examine some of the possible mer. The structure predicts a ping pong mechanism mechanisms for this selectivity in PPC synthetase. with initial formation of an acyladenylate intermediate, Most of the structures from the pantothenate and cofollowed by release of pyrophosphate and attack by enzyme A biosynthetic pathways have been determined cysteine to form the final products PPC and AMP. by X-ray crystallography. These include ketopantoate hydroxymethyltransferase (Protein Data Bank ID code Introduction 1M3U), ketopantoate reductase (Matak-Vinkovic et al., 2001; PDB 1KS9), aspartate-1-decarboxylase (Albert et Coenzyme A and phosphopantetheine function as acyl al., 1998; PDB 1AW8), pantothenate synthetase (von carriers and as carbonyl activating groups for Claisen Delft et al., 2001; PDB 1IHO), pantothenate kinase (Yun condensations, as well as for amide, ester, and thioester et al., 2000; PDB 1ESN), PPC decarboxylase (Albert et forming reactions. These cofactors play a key role in al., 2000; Kupke et al., 2001; PDB 1E20), phosphopanthe biosynthesis and breakdown of fatty acids, in the tetheine adenylyltransferase (Izard and Geerlof, 1999; biosynthesis of nonribosomal peptides and polyketides, PDB 1QJC), and dephosphocoenzyme A kinase (Obmoand in many other biochemical pathways. lova et al., 2001; PDB 1JJV). Here we report the structure Coenzyme A is biosynthesized from phosphopantoof human PPC synthetase, the remaining unknown thenate in all organisms using four enzymatic steps structure in the biosynthesis of coenzyme A, provide (Begley et al., 2001; Figure 1). Phosphopantothenoylcysmodels for the substrate binding sites, and identify key teine (PPC) synthetase catalyzes the first step. Although active site residues. the coenzyme A biosynthetic pathway is well established, the gene for PPC synthetase was only recently identified (Daugherty et al., 2002; Kupke, 2002; Strauss Results et al., 2001). In humans, PPC synthetase and PPC decarboxylase, the next enzyme in the pathway, are encoded Description of the Final Model by separate genes, coaB and coaC, respectively, while in The crystal structure of human PPC synthetase was some bacteria (e.g., Escherichia coli) these genes are fused. determined by single wavelength anomalous dispersion Human PPC synthetase contains 311 amino acid resimethods using selenomethionine (SeMet)-labeled produes and functions as a dimer (Daugherty et al., 2002). tein. The final model, refined at 2.3 Å resolution, includes The bifunctional bacterial enzymes contain about 400 two monomers designated A and B, which are related residues of which about 250 at the C terminus align with by 2-fold noncrystallographic symmetry. The model inhuman PPC synthetase. The PPC synthetase from the cludes 549 amino acids, two sulfate anions, and 218 bifunctional E. coli enzyme is also a dimer when exwater molecules. Residues 1-17, 111, 188-201, 260pressed separately (Kupke, 2002). The human enzyme 265, and 308-311 of monomer A and residues 1-6, 108requires ATP and produces AMP as one of the products, 112, 192-202, 260-264, and 308-311 of monomer B are while the E. coli enzyme utilizes CTP and produces CMP disordered in the electron density maps and hence not built into the model. The crystal structure confirms that PPC synthetase is
PLoS ONE, 2009
Background: Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are ke... more Background: Heterotrimeric G proteins and regulators of G protein signaling (RGS) proteins are key downstream interacting partners in the G protein coupled receptor (GPCR) signaling pathway. The highly versatile GPCR transmembrane signaling system is a consequence of the coupling of a diverse set of receptors to downstream partners that include multiple subforms of G proteins and regulatory proteins including RGS proteins, among others. While the GPCR repertoire of Ciona intestinalis, representing the basal chordate is known, the repertoire of the heterotrimeric G proteins and RGS proteins is unknown. Methodology/Principal Findings: In the present study, we performed an in-silico genome-wide search of C. intestinalis for its complement of G proteins and RGS proteins. The identification of several one-to-one orthologs of human G proteins at the levels of families, subfamilies and types and of homologs of the human RGS proteins suggests an evolutionarily conserved structure function relationship of the GPCR signaling mechanism in the chordates. Conclusions: The C. intestinalis genome encodes a highly conserved, albeit, limited repertoire of the heterotrimeric G protein complexes with the size of subunit types comparable with that in lower eukaryotes.
Gene, 2011
The epidermal growth factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, characterized by the highl... more The epidermal growth factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, characterized by the highly conserved EGF and CFC domains, are extracellular membrane associated growth factor-like glycoproteins. These proteins are essential components of the Nodal signaling pathway during early vertebrate embryogenesis. Homologs of the EGF-CFC family have also been implicated in tumorigenesis in humans. Yet, little is known about the mode of molecular evolution in this family. Here we investigate the origin, extent of conservation and evolutionary relationships of EGF-CFC proteins across the metazoa. The results suggest that the first appearance of the EGF-CFC gene occurred in the ancestor of the deuterostomes. Phylogenetic analysis supports the classification of the family into distinct subfamilies that appear to have evolved through lineage-specific duplication and divergence. Site-specific analyses of evolutionary rate shifts between the two major mammalian paralogous subfamilies, Cripto and Cryptic, reveal critical amino acid sites that may account for the observed functional divergence. Furthermore, estimates of functional divergence suggest that rapid change of evolutionary rates at sites located mainly in the CFC domain may contribute towards distinct functional properties of the two paralogs.
BMC Evolutionary Biology, 2008
Background G protein-coupled receptors (GPCRs) constitute a large family of integral transmembran... more Background G protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates. Results We have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as define...
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2009
Alr1529, a serine hydrolase from the cyanobacteria Anabaena sp. strain PCC 7120 is a member of th... more Alr1529, a serine hydrolase from the cyanobacteria Anabaena sp. strain PCC 7120 is a member of the SGNH hydrolase superfamily. Biochemical characterization of the purified enzyme revealed that the protein is a dimer in solution and is specific for aryl esters of short chain carboxylic acids. The enzyme was regioselective for α-naphthyl esters with maximum activity at pH 7.5 and has a broad optimal temperature range (25-45°C). A structure based comparison of Alr1529 with other superfamily members confirmed the presence of the catalytic triad (Ser17-Asp179-His182) and oxyanion hole (Ser17-Arg54-Asn87) residues. Alr1529 exhibits a previously undescribed variation in the active site wherein a conserved Gly, a proton donor making up the oxyanion hole in the SGNH hydrolases, is substituted by Arg54. Site-directed mutagenesis studies suggest that Arg54 is crucial for substrate binding and catalytic activity. Ser17 plays a very crucial role in catalysis as evident from the 50-fold lower activity of the S17A mutant.
Archives of Biochemistry and Biophysics, 2007
Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic ... more Phospholipid scramblases are a group of homologous proteins that are conserved in all eukaryotic organisms. They are believed to be involved in destroying plasma membrane phospholipid asymmetry at critical cellular events like cell activation, injury and apoptosis. However, a detailed mechanism of phospholipid scrambling still awaits a proper understanding. The most studied member of this family, phospholipid scramblase 1 (PLSCR1) (a 37 kDa protein), is involved in rapid Ca 2+ dependent transbilayer redistribution of plasma membrane phospholipids. Recently the function of PLSCR1 as a phospholipids translocator has been challenged and evidences suggest that PLSCR1 acts as signaling molecule. It has been shown to be involved in protein phosphorylation and as a potential activator of genes in response to interferon and other cytokines. Interferon induced rapid biosynthesis of PLSCR1 targets some of the protein into the nucleus, where it binds to the promoter region of inositol 1,4,5-triphosphate (IP3) receptor type 1 (IP3R1) gene and induces its expression. Palmitoylation of PLSCR1 acts as a switch, controlling its localization either to the PM or inside the nucleus. In the present review, we discuss the current understanding of PLSCR1 in relation to its trafficking, localization and signaling functions.
Acta Crystallographica Section D Biological Crystallography, 2003
Phosphopantothenoylcysteine (PPC) decarboxylase is an essential enzyme in the biosynthesis of coe... more Phosphopantothenoylcysteine (PPC) decarboxylase is an essential enzyme in the biosynthesis of coenzyme A and catalyzes the decarboxylation of PPC to phosphopantetheine. Human PPC decarboxylase has been expressed in Escherichia coli, purified and crystallized. The Laue class of the diffraction data appears to be 3m, suggesting space group R32 with two monomers per asymmetric unit. However, the crystals belong to the space group R3 and the asymmetric unit contains four monomers. The structure has been solved using molecular replacement and refined to a current R factor of 29%. The crystal packing can be considered as two interlaced lattices, each consistent with space group R32 and with the corresponding twofold axes parallel to each other but separated along the threefold axis. Thus, the true space group is R3 with four monomers per asymmetric unit.
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Papers by Narayanan Manoj