17 results on '"Divyendu Singh"'
Search Results
2. Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor
- Author
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Siddhartha S. Saha, Ernest L. Raymond, Joseph R. Woska, Christine Grimaldi, Gary O. Caviness, Su-Ellen Brown, Detlev Mennerich, M. Lamine Mbow, Divyendu Singh, Rajkumar Ganesan, and C. Cheng Kao
- Subjects
LAMP1 ,TOLLIP ,Immunology ,Intracellular Signaling Peptides and Proteins ,Receptors, Interleukin ,Cell Biology ,Receptor-mediated endocytosis ,Biology ,Endocytosis ,Biochemistry ,Cell Line ,Transport protein ,Cell biology ,Protein Transport ,Humans ,Signal transduction ,Lysosomes ,Receptor ,Molecular Biology ,Intracellular ,Interleukin-1 ,Signal Transduction - Abstract
Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.
- Published
- 2015
3. Auxiliary Ligand-Assisted Structural Variation of Cd(II) Metal–Organic Frameworks Showing 2D → 3D Polycatenation and Interpenetration: Synthesis, Structure, Luminescence Properties, and Selective Sensing of Trinitrophenol
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C. M. Nagaraja and Divyendu Singh
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Ethylene ,Hexagonal crystal system ,Ligand ,Stereochemistry ,General Chemistry ,Mixed ligand ,Condensed Matter Physics ,chemistry.chemical_compound ,Crystallography ,chemistry ,General Materials Science ,Metal-organic framework ,Luminescence ,Single crystal ,Topology (chemistry) - Abstract
Four new metal–organic frameworks (MOFs) of Cd(II) ion, [Cd(fdc)(bipy)1.5](1), [{Cd(fdc)(bpee)1.5}.3(H2O)](2), [Cd(fdc)(3bpdb)(H2O)](3), and [{Cd2(fdc)2(4bpdb)3}.1.5(4bpdb).2(H2O)](4) (where, fdc = 2,5-furandicarboxylate dianion, bipy = 4,4′-bipyridine, bpee = 1,2-bis(4-pyridyl)ethylene, 3bpdb = 1,4-bis(3-pyridyl)-2,3-diaza-1,3-butadiene, and 4bpdb = 1,4-bis(4-pyridyl)-2,3-diaza-1,3-butadiene) have been successfully synthesized using mixed ligand systems and characterized by single crystal X-ray analysis and other physicochemical studies. Structural determination revealed that compounds 1 and 4 possess a rare 2D → 3D polycatenated pillared-bilayer structure with {48.62} SP 2-periodic net topology. Compound 2 has a novel threefold interpenetrating 3D hexagonal framework structure with {46.64} sqc-net topology, whereas compound 3 features a 2D Zig-Zag network with {44.62} sql-net topology. Interestingly, compounds 1 and 4 are the first examples of Cd(II) MOFs based on fdc ligand and bipy/4bpdb spacers exhib...
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- 2015
4. Temperature dependent structural variation from 2D supramolecular network to 3D interpenetrated metal–organic framework: In situ cleavage of S–S and C–S bonds
- Author
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Bharat Ugale, Divyendu Singh, and C. M. Nagaraja
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Chemistry ,Ligand ,Inorganic chemistry ,Supramolecular chemistry ,Infrared spectroscopy ,Condensed Matter Physics ,Electronic, Optical and Magnetic Materials ,Inorganic Chemistry ,Crystallography ,Chemical bond ,X-ray crystallography ,Materials Chemistry ,Ceramics and Composites ,Molecule ,Metal-organic framework ,Physical and Theoretical Chemistry ,Thermal analysis - Abstract
Two new Zn(II)–organic compounds, [Zn(muco)(dbds)2(H2O)2] (1) and [Zn(muco)(dbs)] (2) (where, muco=trans, trans-muconate dianion, dbds=4,4′-dipyridyldisulfide and dbs=4,4′-dipyridylsulfide) have been synthesized from same precursors but at two different temperatures. Both the compounds have been characterized by single-crystal X-ray diffraction, powder X-ray diffraction, elemental analysis, IR spectroscopy, thermal analysis and photoluminescence studies. Compound 1 prepared at room temperature possesses a molecular structure extended to 2D supramolecular network through (H–O…H) hydrogen-bonding interactions. Compound 2, obtained at high temperature (100 °C) shows a 3-fold interpenetrating 3D framework constituted by an in situ generated dbs linker by the cleavage of S–S and C–S bonds of dbds linker. Thus, the influence of reaction temperature on the formation of two structural phases has been demonstrated. Both 1 and 2 exhibit ligand based luminescence emission owing to n→π⁎ and π→π⁎ transitions and also high thermal stabilities.
- Published
- 2015
5. LL-37 Peptide Enhancement of Signal Transduction by Toll-like Receptor 3 Is Regulated by pH
- Author
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C. Cheng Kao, Robert C. Vaughan, and Divyendu Singh
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chemistry.chemical_classification ,Toll-like receptor ,Endosome ,viruses ,RNA-binding protein ,Peptide ,Cell Biology ,Biology ,Biochemistry ,Cell biology ,chemistry ,TLR3 ,Signal transduction ,Binding site ,Molecular Biology ,Peptide sequence - Abstract
LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of ∼1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37·poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling.
- Published
- 2014
6. Absence of Grail promotes CD8+ T cell anti-tumour activity
- Author
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Yi Yang, Anupama Sahoo, Oanh N. Hoang, Roza Nurieva, Andrei Alekseev, Cara Haymaker, Hong Qin, Krit Ritthipichai, Shao Cong Sun, Yared Hailemichael, Qiang Zou, Sattva S. Neelapu, Willem W. Overwijk, Chantale Bernatchez, Tiejun Wang, Kimberly S. Schluns, Divyendu Singh, Larry W. Kwak, and Junmei Wang
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0301 basic medicine ,Lymphoma ,Science ,Receptor expression ,medicine.medical_treatment ,Ubiquitin-Protein Ligases ,Cell ,General Physics and Astronomy ,Biology ,CD8-Positive T-Lymphocytes ,General Biochemistry, Genetics and Molecular Biology ,Article ,Immune tolerance ,03 medical and health sciences ,Mice ,Cancer immunotherapy ,hemic and lymphatic diseases ,medicine ,Immune Tolerance ,Cytotoxic T cell ,Animals ,Humans ,Receptor ,Mice, Knockout ,Multidisciplinary ,Interleukins ,General Chemistry ,3. Good health ,Ubiquitin ligase ,030104 developmental biology ,medicine.anatomical_structure ,Neutrophil Infiltration ,Immunology ,biology.protein ,Cancer research ,Receptors, Interleukin-21 ,CD8 - Abstract
T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity., Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.
- Published
- 2017
7. Targeting the Src Homology 2 (SH2) Domain of Signal Transducer and Activator of Transcription 6 (STAT6) with Cell-Permeable, Phosphatase-Stable Phosphopeptide Mimics Potently Inhibits Tyr641 Phosphorylation and Transcriptional Activity
- Author
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Gilbert R. Lee, Pietro Morlacchi, J. Morgan Knight, John E. Ladbury, John S. McMurray, Todd M. Link, Lydia E. Kavraki, Ankur Dhanik, Pijus K. Mandal, Divyendu Singh, David B. Corry, and Roza Nurieva
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CD4-Positive T-Lymphocytes ,Models, Molecular ,Phosphopeptides ,Transcriptional Activation ,Phosphatase ,SH2 domain ,Article ,Cell Line ,src Homology Domains ,Mice ,Structure-Activity Relationship ,Drug Discovery ,Animals ,Prodrugs ,Phosphorylation ,Receptor ,STAT6 ,Interleukin-13 ,Dose-Response Relationship, Drug ,Chemistry ,Phosphopeptide ,respiratory system ,Asthma ,Phosphoric Monoester Hydrolases ,Receptors, Interleukin-3 ,Rats ,Receptors, Interleukin-4 ,Mice, Inbred C57BL ,Biochemistry ,Gene Expression Regulation ,STAT protein ,Molecular Medicine ,Tyrosine ,Interleukin-4 ,STAT6 Transcription Factor ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.
- Published
- 2015
8. Molecular analysis of a UDP-GlcNAc:polypeptide α-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi
- Author
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Lucia Mendonça-Previato, Norton Heise, Slim Sassi, José O. Previato, Divyendu Singh, Christa L. Feasley, Jennifer M. Johnson, Hanke van der Wel, Carolina M. Koeller, and Christopher M. West
- Subjects
Glycosylation ,CAZy ,Trypanosoma cruzi ,Protozoan Proteins ,N-Acetylglucosaminyltransferases ,Biochemistry ,Uridine Diphosphate ,Dictyostelium discoideum ,chemistry.chemical_compound ,parasitic diseases ,Glycosyltransferase ,Animals ,Dictyostelium ,Threonine ,Leishmania ,biology ,Mucins ,biology.organism_classification ,Molecular biology ,carbohydrates (lipids) ,chemistry ,Ectodomain ,biology.protein ,Original Article ,Genome, Protozoan ,Protein Processing, Post-Translational - Abstract
Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, alpha-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide alpha-GlcNAc-transferase (pp-alphaGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[(3)H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[(3)H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that (3)H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-alphaGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-alphaGalNAcTs that initiate mucin-type O-glycosylation in animals.
- Published
- 2009
9. MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption
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Divyendu Singh, Kelly L. McCann, and Farhad Imani
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Pulmonary and Respiratory Medicine ,MAPK/ERK pathway ,Membrane permeability ,MAP Kinase Signaling System ,Physiology ,p38 mitogen-activated protein kinases ,MAP Kinase Kinase 2 ,HSP27 Heat-Shock Proteins ,MAP Kinase Kinase 1 ,Apoptosis ,Bronchi ,Vascular permeability ,Respiratory Mucosa ,Respiratory Syncytial Virus Infections ,p38 Mitogen-Activated Protein Kinases ,Article ,Capillary Permeability ,Hsp27 ,Physiology (medical) ,Heat shock protein ,Humans ,Cells, Cultured ,Cytoskeleton ,Heat-Shock Proteins ,A549 cell ,biology ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,Neoplasm Proteins ,Cell biology ,Paracellular transport ,Extravascular Lung Water ,Immunology ,biology.protein ,Molecular Chaperones - Abstract
Singh D, McCann KL, Imani F. MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption. Am J Physiol Lung Cell Mol Physiol 293: L436–L445, 2007. First published June 8, 2007; doi:10.1152/ajplung.00097.2007.—Respiratory syncytial virus (RSV) is the major cause of bronchiolitis in infants, and a common feature of RSV infections is increased lung permeability. The accumulation of fluid in the infected lungs is caused by changes in the endothelial and epithelial membrane integrity. However, the exact mechanisms of viral-induced fluid extravasation remain unclear. Here, we report that infection of human epithelial cells with RSV results in significant epithelial membrane barrier disruption as assessed by a decrease in transepithelial electrical resistance (TEpR). This decrease in TEpR, which indicates changes in paracellular permeability, was mediated by marked cellular cytoskeletal rearrangement. Importantly, the decrease in TEpR was attenuated by using p38 MAPK inhibitors (SB-203580) but was partially affected by JNK inhibitor SP-600125. Interestingly, treatment of A549 cells with MEK1/2 inhibitor (U-0126) led to a decrease in TEpR in the absence of RSV infection. The changes in TEpR were concomitant with an increase in heat shock protein 27 (Hsp27) phosphorylation and with actin microfilament rearrangement. Thus our data suggest that p38 MAPK and Hsp27 are required for RSV induction of human epithelial membrane permeability.
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- 2007
10. A luminescent 3D interpenetrating metal-organic framework for highly selective sensing of nitrobenzene
- Author
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C. M. Nagaraja and Divyendu Singh
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Ethane ,Materials science ,Pyridines ,Molecular Conformation ,Photochemistry ,Highly selective ,Crystallography, X-Ray ,Fluorescence ,Molecular conformation ,Inorganic Chemistry ,Nitrobenzene ,chemistry.chemical_compound ,Zinc ,Spectrometry, Fluorescence ,chemistry ,Coordination Complexes ,Organometallic Compounds ,Quantum Theory ,Metal-organic framework ,Luminescence ,Furans ,Nitrobenzenes - Abstract
A three-dimensional (3D), luminescent, 5-fold interpenetrating metal–organic framework (MOF), [Zn2(fdc)2(bpee)2(H2O)·2H2O] (1) exhibiting highly selective sensing of nitrobenzene (NB) via a fluorescence quenching mechanism has been demonstrated.
- Published
- 2014
11. The human antimicrobial peptide LL-37, but not the mouse ortholog, mCRAMP, can stimulate signaling by poly(I:C) through a FPRL1-dependent pathway
- Author
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Divyendu Singh, C. Cheng Kao, Rongsu Qi, Jarrat Jordan, and Lani San Mateo
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Lipopolysaccharides ,Endosome ,Molecular Sequence Data ,Immunology ,Endosomes ,Biology ,Endocytosis ,Biochemistry ,Cell Line ,Mice ,Cathelicidins ,Animals ,Humans ,Amino Acid Sequence ,Receptors, Lipoxin ,Receptor ,Molecular Biology ,Innate immune system ,Interleukin-6 ,beta-Cyclodextrins ,Colocalization ,Cell Biology ,Molecular biology ,Receptors, Formyl Peptide ,Toll-Like Receptor 3 ,Protein Transport ,Poly I-C ,Cell culture ,TLR3 ,lipids (amino acids, peptides, and proteins) ,Signal transduction ,Antimicrobial Cationic Peptides ,Protein Binding ,Signal Transduction - Abstract
LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.
- Published
- 2013
12. Proteolytic processing regulates Toll-like receptor 3 stability and endosomal localization
- Author
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Rongsu Qi, Divyendu Singh, and C. Cheng Kao
- Subjects
Proteases ,Interferon Inducers ,Endosome ,viruses ,Immunology ,chemical and pharmacologic phenomena ,Endosomes ,Biology ,Biochemistry ,Protein Structure, Secondary ,Cell Line ,symbols.namesake ,Mice ,Animals ,Humans ,Molecular Biology ,Toll-like receptor ,Innate immune system ,Protein Stability ,virus diseases ,Membrane Transport Proteins ,hemic and immune systems ,Cell Biology ,Golgi apparatus ,Cell biology ,Protein Structure, Tertiary ,Toll-Like Receptor 3 ,HEK293 Cells ,Poly I-C ,Ectodomain ,TLR3 ,Mutation ,Proteolysis ,symbols ,Signal transduction ,Lysosomes ,Half-Life ,Protein Binding ,Signal Transduction - Abstract
Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.
- Published
- 2012
13. Prolyl hydroxylation- and glycosylation-dependent functions of Skp1 in O2-regulated development of Dictyostelium
- Author
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Divyendu Singh, Zhuo A. Wang, Christopher M. West, and Hanke van der Wel
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Glycosylation ,Proline ,Protein subunit ,Ubiquitin-Protein Ligases ,Blotting, Western ,Procollagen-Proline Dioxygenase ,Hydroxylation ,Mass Spectrometry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Skp1 ,Glycosyltransferase ,Dictyostelium ,Hypoxia ,Molecular Biology ,S-Phase Kinase-Associated Proteins ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Prolyl hydroxylation ,biology ,030302 biochemistry & molecular biology ,Gene Expression Regulation, Developmental ,Cell Biology ,biology.organism_classification ,Oxygen ,Luminescent Proteins ,Biochemistry ,chemistry ,Mutation ,biology.protein ,Procollagen-proline dioxygenase ,Developmental Biology - Abstract
O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.
- Published
- 2010
14. HSV ICP0 recruits USP7 to modulate TLR-mediated innate response
- Author
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Divyendu Singh, William J. Bowers, Howard J. Federoff, Hongiu Liu, Khaled A. Tolba, Sandrine Daubeuf, and Yaohong Tan
- Subjects
viruses ,Ubiquitin-Protein Ligases ,Immunology ,Biology ,medicine.disease_cause ,Biochemistry ,Immediate early protein ,Immediate-Early Proteins ,Ubiquitin-Specific Peptidase 7 ,Immune system ,medicine ,Humans ,Receptor ,Cells, Cultured ,Immunobiology ,TNF Receptor-Associated Factor 6 ,Toll-like receptor ,Ubiquitin ,Toll-Like Receptors ,Pattern recognition receptor ,NF-kappa B ,Herpes Simplex ,Cell Biology ,Hematology ,NFKB1 ,Acquired immune system ,Immunity, Innate ,I-kappa B Kinase ,Protein Transport ,Herpes simplex virus ,Protein Processing, Post-Translational ,Ubiquitin Thiolesterase ,Protein Binding - Abstract
Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-κB and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKKγ, thus terminating TLR-mediated NF-κB and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits this physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence.
- Published
- 2008
15. Contact of Entamoeba histolytica with baby hamster kidney-21 (BHK-21) cell line on cysteine proteinase activity
- Author
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Divyendu, Singh, S R, Naik, and Sita, Naik
- Subjects
Chromium ,Electrophoresis ,Cysteine Endopeptidases ,Hemoglobins ,Acetylgalactosamine ,Entamoebiasis ,Liver ,Cricetinae ,Lectins ,Entamoeba histolytica ,Animals ,Caseins ,Cell Line - Abstract
Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells.Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay.Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor.The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.
- Published
- 2004
16. Targeting the SrcHomology 2 (SH2) Domain of SignalTransducer and Activator of Transcription 6 (STAT6) with Cell-Permeable,Phosphatase-Stable Phosphopeptide Mimics Potently Inhibits Tyr641Phosphorylation and Transcriptional Activity.
- Author
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PijusK. Mandal, Pietro Morlacchi, J. Morgan Knight, Todd M. Link, Gilbert R. Lee, Roza Nurieva, Divyendu Singh, Ankur Dhanik, Lydia Kavraki, DavidB. Corry, John E. Ladbury, and John S. McMurray
- Published
- 2015
- Full Text
- View/download PDF
17. Molecular analysis of a UDP-GlcNAc:polypeptide {alpha}-N-acetylglucosaminyltransferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi.
- Author
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Norton Heise, Divyendu Singh, Hanke van der Wel, Slim O Sassi, Jennifer M Johnson, Christa L Feasley, Carolina M Koeller, Jose O Previato, Lucia Mendonça-Previato, and Christopher M West
- Subjects
- *
MOLECULAR biology , *POLYPEPTIDES , *TRANSFERASES , *MUCINS , *GLYCOSYLATION , *TRYPANOSOMA cruzi , *CHAGAS' disease , *MICROBIAL genetics - Abstract
Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, α-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide α-GlcNAc-transferase (pp-αGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[3H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[3H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that 3H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-αGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-αGalNAcTs that initiate mucin-type O-glycosylation in animals. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
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